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Reveleris® PREP Purification SystemOperation Manual

11593898B en

ImprintProduct Identification: Operation Manual (Original), Reveleris® PREP

11593898B en

Publication date: 06.2016

BÜCHI Labortechnik AG Meierseggstrasse 40 Postfach CH-9230 Flawil 1

E-Mail: [email protected]

BUCHI reserves the right to make changes to the manual as deemed necessary in the light of experience; especial-ly in respect to structure, illustrations and technical detail.

This manual is copyright. Information from it may not be reproduced, distributed, or used for competitive purposes, nor made available to third parties. The manufacture of any component with the aid of this manual without prior written agreement is also prohibited.

BÜCHI Labortechnik AG

www.buchi.com Reveleris® PREP Operation Manual, Version B v

Safety Symbols used in Manual

Symboles de sécurité utilisés dans ce manuel

The Danger symbol indicates a hazardous situation that, if not avoided, will result in death or serious injury .

Le symbole DANGER signale une situation dangereuse qui, si elle ne peut être évitée, entraînera de graves blessures, voire la mort.

The Warning symbol indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury.

Le symbole AVERTISSEMENT signale une situation dangereuse qui, si elle ne peut être évitée, peut entraîner de graves blessures, voire la mort.

The Caution symbol indicates a potentially hazardous situation that, if not avoided, could result in minor or moderate injury.

Le symbole ATTENTION accompagné du symbole d’alerte de sécurité signale une situation dangereuse qui, si elle ne peut être évitée, peut entraîner des blessures mineures ou légères.

The Notice symbol is used to highlight information that will optimize the use and reliability of the system.

Le symbole REMARQUE est utilisé pour mettre en évidence des informations destinées à optimiser l’utilisation et la fiabilité du système.


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Important Safety Guidelines for the Reveleris ® PREP Purification System

Consignes de sécurité importantes pour le système de purification Reveleris® PREP

Please read the following warnings and cautions carefully before using the Reveleris® PREP Flash Chromatography System:

Veuillez lire les mises en garde et les avertissements suivants avant toute utilisation du système de chromatographie Reveleris® PREP:

This equipment is to be operated by trained personnel only.

Cet équipement doit être opéré seulement par des utilisateurs formés.

This equipment must be used as specified by the manufacturer; or overall safety will be impaired.

Cet équipement doit être utilisé comme spécifié par le fabricant; faute de quoi, la sécurité générale sera mise en péril.

To prevent spills, and to both minimize potential for personnel exposure to hazardous chemicals and the risk of fire do not fill re-fill solvent bottles when they are on top of the unit.

Afin d'éviter des fuites et de réduire l'exposition des utilisatuers aux produit chimiques toxiques et le risque de feu c'est fortement recommendé de ne pas remplir les récipients de solvants en haut de l'appareil.

The operation of the instrument involves the use and the separation of potentially hazardous and/or toxic materials. The operator should obtain and review MSDS for all solvents and modifiers employed in the system and follow the use, handling and disposal recommendations.

Le fonctionnement de l’instrument implique l’utilisation de solvants potentiellement dangereux et toxiques. L’opérateur doit obtenir une FDS pour tous les solvants et modificateurs employés dans le système et suivre les recommandations d'utilisation, de manipulation et de l'élimination.

It is strongly recommended that the system be operated in an appropriate laboratory fume hood. If the instrument is not operated in a fume hood, the user must maintain worker exposure levels below both your organization’s and applicable governmental occupational exposure limits.

Il est vivement recommandé d’utiliser le système dans une hotte de laboratoire appropriée. Si l’instrument n’est pas utilisé dans une hotte, l’utilisateur doit veiller à assurer une ventilation adéquate afin de garantir l’exposition aux vapeurs de solvant en dessous les limites officielles.

Only use the power supply cord supplied by the manufacturer. Disconnect the power cord from the power supply before changing the fuse(s). Make certain that you replace the fuse with one of the same rating (e.g. replace a 5A fuse with a 5A fuse).

Utilisez uniquement le cordon d’alimentation recommandé par le fabricant. Débranchez le cordon d’alimentation avant de procéder au remplacement des fusibles. Veillez à remplacer les fusibles par des fusibles de même intensité (par exemple., remplacez un fusible de 5 A par un autre fusible de 5 A).


www.buchi.com Reveleris® PREP Operation Manual, Version B vii

Make certain that the Reveleris ® PREP purification system is powered down and disconnect the power cor d from the power supply before the left side panel or front door is removed. These should only be opened by a trained technician. There are no user serviceable parts accessible from the right side or rear panel. These panels should only be removed by a qualified service technician.

Assurez-vous que le système purification Reveleris® PREP est hors tension et que le cordon d’alimentation reliant l’appareil au secteur est débranché avant d’enlever le panneau latéral gauche ou le panneau frontal. Aucune pièce pouvant être entretenue par l’utilisateur n’est accessible via les panneaux latéral droit et arrière. Ces panneaux peuvent uniquement être enlevés par un technicien de service qualifié.

Any item that is connected to the Reveleris ® PREP system via a data port must meet CE (or equivalent) certification requirements.

Tout élément relié au système Reveleris® PREP via un port de données doit satisfaire aux exigences de certification CE (ou équivalentes).

The quantity of flammable solvent that is kept in the immediate vicinity of the system should be minimized. All local and institutional regulations regarding the care and handling of flammable solvents as well as Good Laboratory Practices should be strictly obeyed .

La quantité de solvant inflammable conservée à proximité immédiate du système doit être réduite à un minimum afin d’éviter la propagation d’un incendie. Toutes les réglementations locales et institutionnelles relatives à la manipulation et à l’utilisation des solvants inflammables ainsi que les bonnes pratiques de laboratoire doivent être rigourement observées.

All solvents used in the instrument must have a minimum auto ignition temperature of greater than 85ºC.

Tous les solvants utilisés sur l'instrument doivent avoir un point de feu inférieur à 85°C.

Users must be aware of the hazards associated with use of the equipment and need to wear appropriate personal protective equipment and employ proper engineering controls.

Les utilisateurs doivent être conscients des risques associés à l'utilisation de l'équipement et la nécessité de porter un équipement de protection individuelle approprié et utiliser des contrôles techniques appropriés.


www.buchi.com Reveleris® PREP Operation Manual, Version B viii

Table of Contents

1. Introduction .................................................... ..................................................................... 11.1 About the Reveleris ® PREP Purification system ................................................................................... 1

1.2 Features of the Reveleris ® PREP purification System ......................................................................... 1

1.3 System Design ....................................................................................................................................... 1

1.3.1 Solvent Delivery System ................................................................................................................................... 2

1.3.2 Mode Switching Valve ....................................................................................................................................... 2

1.3.3 Sample Injection Systems ................................................................................................................................ 3

1.3.4 Cartridge/Column Assembly ............................................................................................................................ 3

1.3.5 Detection System ............................................................................................................................................... 3

1.3.6 Fraction Collector .............................................................................................................................................. 4

1.4 System Control and Data Storage ......................................................................................................... 4

1.5 Where to Go for Further Information ..................................................................................................... 4

2. Installation ...................................................... ..................................................................... 52.1 What You Will Need ............................................................................................................................... 5

2.1.1 Facility Requirements ....................................................................................................................................... 5

2.1.2 Space Requirements ......................................................................................................................................... 5

2.1.3 Electrical Requirements .................................................................................................................................... 5

2.1.4 Cartridge, column, and Mobile Phase ............................................................................................................. 5

2.1.5 External Gas Source (Air or Nitrogen) ............................................................................................................. 5

2.2 Unpacking .............................................................................................................................................. 5

2.3 Controls and Features ........................................................................................................................... 7

2.3.1 Front Panel and Controls .................................................................................................................................. 7

2.3.2 Rear Panel .......................................................................................................................................................... 8

2.4 Installing the Instrument ........................................................................................................................ 9

2.4.1 Unpack the Instrument ...................................................................................................................................... 9

2.4.2 Locating the Instrument in the Laboratory ...................................................................................................... 9

2.5 Fluid Connections .................................................................................................................................. 9

2.5.1 Solvent and Sensor Lines ................................................................................................................................. 9

2.5.2 Drain Setup ........................................................................................................................................................ 9

2.5.3 Prep injection valve and bulkhead Connections ......................................................................................... 10

2.6 ELSD Exhaust Connections ................................................................................................................ 11

2.7 External Air/Gas Supply Connection .................................................................................................. 11

2.8 Moving the Instrument ......................................................................................................................... 11

3. The User Interaction Program ....................................... ................................................... 123.1 Overview ............................................................................................................................................... 12

3.2 The Setup Screen ................................................................................................................................. 13

3.2.1 Chromatographic Conditions ......................................................................................................................... 13


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3.2.1.1 Flash Mode (Figure 3.1) ................................................................................................................................. 13

3.2.1.2 Prep Mode (FIgure 3.2) ........................ .......................................................................................................... 14

3.2.2 Detector Selection ........................................................................................................................................... 15

3.2.3 Collection Criteria ............................................................................................................................................ 15

3.2.4 Fraction Collection .......................................................................................................................................... 15

3.2.5 Solvent Selection & Priming .......................................................................................................................... 15

3.2.6 Mobile Phase Composition ............................................................................................................................. 17

3.2.6.1 Tabular Method ............................... ............................................................................................................... 17

3.2.6.2 Graphical Method .............................. ............................................................................................................. 17

3.2.7 Reveleris® Navigator™ Software ................................................................................................................... 18

3.2.8 Collection Vial Display .................................................................................................................................... 18

3.2.9 Saving and Retrieving Methods ..................................................................................................................... 18

3.3 Tools Menu ........................................................................................................................................... 19

3.3.1 Solvent Loading ............................................................................................................................................... 19

3.3.2 Solvent Definition ............................................................................................................................................ 19

3.3.3 Vapor Sensors and Limits .............................................................................................................................. 20

3.3.4 Configuration ................................................................................................................................................... 21

3.3.5 Calibration and Defaults ................................................................................................................................. 21

3.3.6 Manual Control................................................................................................................................................. 21

3.3.6.1 Air Purging (Flash Mode Only) ................. .................................................................................................... 21

3.3.6.2 Injection Port Rinsing (Flash Mode Only) ...... .............................................................................................. 22

3.3.6.3 Solid Loader Flushing (Flash Mode Only) ....... ............................................................................................ 22

3.3.6.4 Fraction Collector Arm Reset .................. ..................................................................................................... 22

3.3.6.5 Column Flushing .............................. .............................................................................................................. 22

3.3.6.6 Solvent Line Flushing ......................... ........................................................................................................... 23

3.3.6.7 Prep Sample Loop Rinsing (Prep LC Mode Only) .. ..................................................................................... 23

3.3.7 NP <> RP .......................................................................................................................................................... 23

3.3.8 Product Services ............................................................................................................................................. 24

3.3.9 UV-VIS Detection ............................................................................................................................................. 24

3.3.10 Debug diagnostics ........................................................................................................................................... 24

3.4 The Run Screen .................................................................................................................................... 24

3.4.1 Accessing the Run Screen ............................................................................................................................. 24

3.4.2 Components of the Run Screen ..................................................................................................................... 26

3.5 The Past Run Screen ........................................................................................................................... 28

3.5.1 Accessing the Past Run .................................................................................................................................. 28

3.5.2 Components of the Past Run Screen ............................................................................................................. 29

4. Separating a Sample .......................................... ............................................................... 304.1 Overview ............................................................................................................................................... 30

4.2 Powering up the System ...................................................................................................................... 30

4.3 Preparing the System for Operation ................................................................................................... 31


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4.3.1 Selecting Solvents and Priming the System ................................................................................................. 31

4.3.1.1 Assigning Solvents To Solvent Lines ........... ............................................................................................... 31

4.3.2 Calibrating (Zeroing) the Solvent and Waste Safety Sensors...................................................................... 32

4.4 Installing a Column in the System ...................................................................................................... 33

4.4.1 Flash Columns/Cartridges .............................................................................................................................. 33

4.4.2 PREP Columns................................................................................................................................................. 33

4.5 Placing Collection Tubes in Fraction Collection Trays and Installing the Trays onto the System .. 33

4.6 Introducing a Sample into the System ................................................................................................ 34

4.6.1 Flash Mode ....................................................................................................................................................... 34

4.6.1.1 Liquid Samples ................................ .............................................................................................................. 34

4.6.1.2 Solid/Dry Sample............................... ............................................................................................................. 34

4.6.2 PREP Mode ...................................................................................................................................................... 35

4.7 Selecting or Developing a Method ...................................................................................................... 35

4.7.1 Selecting an Existing Method ......................................................................................................................... 35

4.7.2 Generating a New Method ............................................................................................................................... 36

4.8 Starting the Separation ........................................................................................................................ 37

4.9 Performing Additional Separations ..................................................................................................... 40

4.10 Printing ................................................................................................................................................. 41

4.11 Saving Run Report In PDF Format To USB Storage/Flash Drive ....................................................... 41

4.12 Powering Down the System ................................................................................................................ 42

5. Reveleris® Navigator™ Software ............................. ....................................................... 435.1 TLC-Silica ............................................................................................................................................. 43

5.1.1 Input Data ......................................................................................................................................................... 43

5.1.2 Flash Conditions .............................................................................................................................................. 44

5.1.3 Input Screen Control Buttons ......................................................................................................................... 44

5.1.4 Output Screen .................................................................................................................................................. 44

5.1.5 Output Screen Control Buttons ...................................................................................................................... 44

5.1.6 Accept Gradient ............................................................................................................................................... 44

5.1.7 Gradient Issues ................................................................................................................................................ 45

5.2 LC-C18 .................................................................................................................................................. 46

5.2.1 Input Data ......................................................................................................................................................... 46



5.2.4 Output Screen .................................................................................................................................................. 47


5.2.6 Accept Gradient ............................................................................................................................................... 47

5.2.7 Gradient Issues ................................................................................................................................................ 47

5.3 LC-Transfer .......................................................................................................................................... 49

5.3.1 Input Data ......................................................................................................................................................... 49



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5.3.4 Output Screen .................................................................................................................................................. 49


5.3.6 Accept Gradient ............................................................................................................................................... 50

6. Troubleshooting ............................................. ................................................................... 516.1 Introduction .......................................................................................................................................... 51

6.2 Software and Firmware Errors ............................................................................................................ 51

6.3 Troubleshooting Strategies ................................................................................................................. 51

6.3.1 Troubleshooting Checklists ............................................................................................................................. 51

6.3.2 General Instrumentation Issues ................. .................................................................................................... 52

6.3.3 Flash Column/Cartridge Recognition Issues ................................................................................................ 52

6.3.4 Solvent Delivery Issues ........................ ........................................................................................................... 53

6.3.5 Sample Injection Issues ........................ .......................................................................................................... 54

6.3.6 Fraction Collector Issues ................................................................................................................................ 54

6.3.7 Detection Issues .............................................................................................................................................. 55

6.3.8 Error Messages ................................. ............................................................................................................... 56

7. Maintenance .................................................... ................................................................... 587.1 Overview ............................................................................................................................................... 58

7.2 Cleaning the System ............................................................................................................................ 58

7.2.1 Routine Inspection .......................................................................................................................................... 58

7.3 Accessing System Components ......................................................................................................... 58

7.4 Solvent Pump Maintenance ................................................................................................................. 59

7.4.1 Replacing Piston Seals (Solvent Pump) ........................................................................................................ 59

7.4.2 Cleaning the Piston ......................................................................................................................................... 60

7.4.3 Replacing the Seal ........................................................................................................................................... 60

7.4.4 Check Valve Cleaning and Replacement ....................................................................................................... 60

7.5 UV Detector .......................................................................................................................................... 61

7.5.1 Cleaning the Flow Cell .................................................................................................................................... 61

7.5.2 Replacing the Lamp ......................................................................................................................................... 61

7.6 ELSD Detector ...................................................................................................................................... 61

7.6.1 Cleaning the Nebulizer .................................................................................................................................... 61

7.6.2 Replacing the Nebulizer Tubing ..................................................................................................................... 62

7.6.3 Cleaning the Drift Tube ................................................................................................................................... 62

7.7 Replacing the Fuse ................................................................................................................................ 63

7.8 Cleaning the Disk Space ........................... ............................................................................................ 63

7.8.1 Deleting Older Version of Reveleris® PREP Purification system Software ................................................. 63

7.8.2 Deleting the Log Files from the Current Software Version ........................................................................... 63

7.8.3 Transferring and Deleting the Run and Method File s .................................................................................... 63

7.8.4 Deleting the Files from the Recycle Bin ........ .................................................................................................. 64


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8. Appendices ........................................................................................................................ 658.1 Specifications ...................................................................................................................................... 65

8.2 Spare Parts and Optional Accessories ............................................................................................... 65

8.3 BUCHI Reveleris® Flash Cartridges ......................................................................................... ............. 66

8.4 BUCHI Pre-packed Preparative Columns ................................................................................ .............. 67

8.5 Common Solvents for Flash Chromatography ...................................................................... .............68

8.6 Volatile mobile phase modifiers ............................................................................................... ............73

8.7 Polarity Order of Common organic solvent Solvents ............................................................ .............738.8 Solid/Dry Sample Preparation .................................................................................. ............................ 74

9. Introduction to the Reveleris ® Dry Air Supply ................................................................. 769.1 About the Reveleris ® Dry Air Supply ..................................................................................................76

9.1.1 Principle of Operation ................................................................................................................................... 76

9.1.2 System Control ..............................................................................................................................................76

9.1.3 Where to Go for Further Information .......................................................................................................... 76

9.2 Installation and Operation ....................................................................................................................769.2.1 Location/System Requirements ................................................................................................................... 76

9.2.2 Unpacking ....................................................................................................................................................... 76

9.2.3 Installing the Instrument ................................................................................................................................ 78

9.2.4 Operating the Dry Air Supply ........................................................................................................................78

9.3 Maintenance and Troubleshooting ......................................................................................................789.3.1 Maintenance ....................................................................................................................................................7 8

9.3.2 Troubleshooting .............................................................................................................................................78

9.3.3 Software and Firmware Errors.......................................................................................................................7 8

9.3.4 Troubleshooting Strategies ........................................................................................................................... 78

9.3.5 Troubleshooting Checklists .......................................................................................................................... 78

9.4 General Instrumentation Issues/Error messages ...............................................................................79

9.5 Appendices .................................................................................................................................. ..........809.5.1 Specifications ................................................................................................................................................ 80

9.5.2 Spare Parts .................................................................................................................................................... ..80


www.buchi.com Reveleris® PREP Operation Manual, Version B 1

1. INTRODUCTION

1.1 ABOUT THE REVELERIS ® PREP PURIFICATION SYSTEM

The Reveleris® PREP Purification System is an advanced purification system designed to purify complex samples by both Flash Chromatography and Preparative Liquid Chromatography (Prep LC). The dual-mode system can be switched between flash chromatography and Prep LC with one software click. No hardware change is needed. The purification runs can be done at flow rates of up to 200mL/min with pressures up to 200psig for Flash Chromatography and 1700 psig for Prep LC.

The flash chromatographic separation is typically performed using a cartridge packed with 40-63µm silica and a mobile phase such as hexane: ethyl acetate (70:30) at a flow rate of 40mL/min at a moderate pressure (typically less than 100 psig). Although the resolution of flash chromatography is less than that of prep LC, the technique is extremely useful as it provides the chromatographer with the ability to separate gram size samples in a short period. For complex samples that require higher resolution, Prep LC is a better choice. Its separation is typically performed using a 10 to 22mm internal diameter column packed with 10-20um silica or C18 bonded silica particles. The pressure is dependent on the flow rates, column diameter and length, typically in the range of 500 to 1000psig. The Reveleris® PREP purification system allows you to perform both methods on one simple, intuitive system.

The Reveleris® PREP purification system includes a UV detector that can monitor three wavelengths simultaneously as well as an evaporative light scattering detector (ELSD). The ELSD provides the ability to monitor all non-volatile compounds in the mobile phase and allows the user to detect for compounds that do not absorb UV radiation. An additional benefit of the ELSD is the ability to employ mobile phases that absorb light at the same wavelength as the compound(s) of interest. Both detectors offer adjustable sensitivity settings to detect and collect small and large quantity of compounds.

1.2 FEATURES OF THE REVELERIS® PREP PURIFICATION SYSTEM

The Reveleris® PREP Purification system includes a number of advanced features that are provided to enhance the utility of flash chromatography and prep LC, including:

• A simple software click to switch mode between flashchromatography and prep LC. No hardware change isrequired.

• Fraction collection can be based on any detector signal(UV1, UV2, UV3, or ELSD); this provides a superbcontrol of the fractionation process and helps lead to ahigher degree of purity of the collected compound(s).

• The fraction collector is configured with swappablefraction collection trays, extending collection capabilitiesand providing a more flexible system.

• The auto prime feature simplifies pump priming andsolvent switching

• The four solvent bottles with large capacity andextended solvent capabilities minimize down time due torefilling of mobile phases. The solvent reservoir tray islocated on top of the system, thereby saving valuablelaboratory space.

• An exceedingly broad range of cartridges and prep-LCcolumns from BUCHI and other manufacturers can beemployed. If you have developed a method on a legacysystem, it can be carried over to the Reveleris® PREPpurification instrument with a minimum of effort.

1.3 SYSTEM DESIGN

The Reveleris® PREP Purification system is a computer controlled integrated system that includes the following:

• Solvent Delivery System (Section 1.3.1 )

• Mode Switching Valve (Section 1.3.2)

• Sample Injection Systems (Section 1.3.3 )

• Cartridge/Column Assembly (Section 1.3.4 )

• Detection System (Section 1.3.5 )

• Fraction Collector (Section 1.3.6 )

A schematic diagram is presented in Figure 1-1.

UVDetector

FractionCollector

SamplingValve

ELSDDetector

SolventReservoirs

PumpSystem

ModeValve

MultiportValve

Sample

FlashCartridge

InjectionValve

PrepColumn

Figure 1-1: Schematic Design of Reveleris ® PREP Purifi cation System



1.3.1 SOLVENT DELIVERY SYSTEM

A detailed diagram of the solvent delivery system is presented in Figure 1-2 . It consists of four solvent reservoirs. The solvent manifold valves control the delivery of solvents to the pumps and system. The pumps can deliver an isocratic mobile phase or a binary gradient mobile phase at a flow rate of 1-200mL/min (accuracy of +/-3% is not achieved until a minimum of 4mL/min) with a maximum pressure of 200psig for Flash Chromatography and up to 1700psig for prep LC (flow rates dependent). An Auto Prime Assembly is included in the solvent delivery system to allow for easy priming and rapid delivery of solvent as required (e.g. to remove all air when starting up). The back pressure regulator (BPR) is used after the pumps to ensure accurate mobile phase composition is delivered to the cartridge or column for separation.

ManifoldValves

SolventReservoirs

Pumps

PrimingSystem

PressureSensor

BPR ModeValve

Figu re 1-2: Solvent Delivery System (BPR=Back Pressure Regulator)

Several safety sensors are used in the Reveleris® PREP purification system. Vapor sensors are used to monitor the vapor levels in and around the instrument to protect against solvent leaks. When a leak is detected, the pump is stopped and the run is paused.

A solvent safety sensor is included with each solvent line and reservoir. When the sensor indicates it is necessary to add solvent, the system pause the separation and alerts user to add more solvent. The system can also be set up to automatically withdraw solvent from another reservoir.

A solvent safety sensor is also included in the waste line and reservoir and will alert user when it is necessary to empty the waste reservoir.

The waste solvent safety sensor and vapor sensors are safety devices designed to detect/prevent solvent spills and fire hazards. Do not disable or bypass these sensors.

1.3.2 MODE SWITCHING VALVE

The mode switching valve is a two position electrically actuated 6-port valve that directs the solvent to either flash or prep LC flow path (Figure 1-3 ). The flash and prep modes share a common fluidic path for solvent delivery, detection system, and fraction collection. The sample injection and cartridge/column paths are different and controlled by the mode switching valve. The software automatically set the mode valve to the proper position depending on the separation mode the user selected.

ModeValve

MultiportValve

Sample

FlashCartridge

InjectionValve

PrepColumn

Figure 1-3: Mode Selection Valve



1.3.3 SAMPLE INJECTION SYSTEMS

Flash Sample Injection System - The flash sample injection system (Figure 1-4 ) includes an interface to allow dry or liquid sample loading of the cartridge.

For samples that are readily soluble in the mobile phase, the sample is dissolved in the appropriate solvent and is injected on top of the cartridge using a syringe (3mL to 140mL) while the flow is off. After the injection, the flow of mobile phase is used to transport the sample through the cartridge. A check valve is added to the fluidic path between the multiport valve and the cartridge to prevent back flow of sample/solvent.

For samples that are not readily soluble in the mobile phase, the sample is adsorbed onto a solid phase (e.g. silica or alumina). The adsorbent with the sample is placed in a solid sample loader (3-60g capacity) in series before the cartridge. The mobile phase flow begins and the sample is desorbed from the loader to the top of the cartridge and through the cartridge.

ModeValve

SampleSyringe

CheckValve

MultiportValve

CartridgeFROMPUMP

TO DETECTOR

Figur e 1-4: Flash Mode Sample Injection System

The multi-port valve is employed in a number of activities including equilibrating the column, delivering solvent for the separation, providing pre- and/or post- injection flush operations and air purging the column and sample loader. Additionally, a column and sample bypass connectors are included for flushing the system.

Prep LC Sample Injection System - The Prep LC sample injection system (Figure 1-5) consists of an electrically actuated 6-port valve, an injection port for manual liquid sample loading, and an injection loop. The system comes standard with 5 mL sample loop. Other sample loop sizes (1mL, 10mL) are available as accessories.

ModeValve

SampleLoop

PrepColumn

InjectionValve

FROMPUMP

TO DETECTOR

Figure 1-5. Prep Mode Sample Injection System

1.3.4 CARTRIDGE/COLUMN ASSEMBLY

The cartridge/column contains the stationary phase that performs the separation of the compounds in the sample. The flash cartridge assembly accepts a broad range of cartridges from BUCHI and other suppliers ranging in capacity from 4 to 330g. A leak proof universal mounting is used for rapid installation of the

cartridge. Auto-cartridge recognition technology is used in the Reveleris® PREP purification system to detect cartridges equipped with cartridge recognition tag and automatically populate the appropriate method fields. Cartridges without the cartridge recognition tag can also be used with the Reveleris® PREP purification system.

If the auto-cartridge recognition system is not used, the maximum operating pressure is defaulted to 75psig.

A column bypass and a sample bypass are provided for system flushing. An external gas source is required to purge the solvent from the cartridge after each run and prepare for the next separation.

For Prep LC, any column between 1” to 3” from BUCHI and other suppliers can be used. The column may be set on the bench next to the instrument or installed on any column stand. Fittings to connect either 1/16” OD or 1/8” OD tubing to and from the column are provided.

1.3.5 DETECTION SYSTEM

Two detectors are incorporated into the system, a UV detector and an Evaporative Light Scattering Detector (ELSD). A sampling valve is used to sample a small portion of the sample to the ELSD. The user can select the signal from either or both detectors to trigger fraction collection or diversion of the mobile phase to waste.

SamplingValve

ELSDDetector

UVDetector

BPR

Waste

FractionCollector

PressureSensor

FROM FLASH CARTRIDGEOR PREP COLUMN

Figur e 1-6: Detection/Fraction Collection System

The UV detector provides variable wavelength detection between 200 and 500nm (850nm for optional visible detection) and allows for simultaneous monitoring of three wavelengths for triggering of the fraction collector. The ELSD is a mass sensitive detector that evaporates the mobile phase and monitors the particulate matter that passes through a photometric detector. A major benefit of this detector is that non-volatile compounds can be detected, regardless of their spectroscopic properties. In addition, the ELSD detector allows for the use of mobile phases that absorb light at the same wavelength as the compound of interest (e.g. acetone).

A second pressure sensor is added to the system before the sampling valve to monitor downstream pressure. A



30psi back pressure regulator (BPR) is also added to the detection system to improve overall system performance.

An external air or nitrogen gas source is needed to operate the ELSD. To simplify the operation of the Reveleris® PREP purification system, use the Reveleris® Dry Air Supply (article no. 145168621). The Reveleris® Dry Air Supply is designed to be used with Reveleris® PREP purification system. It simplifies the operation of the Reveleris® PREP purification system by producing particle free dry air on demand without the need for operator attention. Refer to Section 9 for more information on the Reveleris® Dry Air Supply.

1.3.6 FRACTION COLLECTOR

The eluent will be diverted to the fraction collector if the signal from any detector meets the user-selected criteria. The fraction collector can employ multi-position trays for 12, 13, 16, 18, and 25mm tubes as well as 480mL bottles. The capacity of the trays is indicated in Table 1-1 . Auto-tray recognition technology is used in the Reveleris® PREP purification system to identify the vial size, number of tubes, tube position, and maximum acceptable vial volume. An optical sensor is also used to detect if the tray is installed properly or out of place (misaligned). Once the trays are placed into the system, this information will be used to auto-populate the appropriate method fields (system default or user saved values will be used).

TABLE 1 -1: TRAY CAPACITY

Size of tube Number of Tubes

12mm o.d. x 75mm H 252 (2 trays)

13mm o.d. x 100mm H 224 (2 trays)

16mm o.d. x 150mm H 168 (2 trays)

18mm o.d. x 150mm H 120 (2 trays)

25mm o.d. x 150mm H 72 (2 trays)

480mL French Square Bottles 9 (1 tray)

1.4 SYSTEM CONTROL AND DATA STORAGE

The primary user interface is a color touch screen panel with a stylus that can be used to control all aspects of the system. Two USB connections are provided to allow the use of external devices such as printers, a mouse, a keyboard, data storage devices, COM ports, and memory downloads as well as the uploading of software upgrades and transfer of application data.

The Reveleris® PREP purification system automatically save data for each run and save methods, which can be readily recalled.

1.5 WHERE TO GO FOR FURTHER INFORMATION

Additional information about Flash Chromatography, Reveleris® Dry Air Supply, and optimizing the use of your

Reveleris® PREP purification system can be obtained from your BUCHI Service Representative or at www.buchi.com.



2. INSTALLATION

2.1 WHAT YOU WILL NEED

2.1.1 FACILITY REQUIREMENTS

The facility should meet the following environmental conditions:

• The temperature range of the laboratory should bebetween 18°C and 30°C (64°F - 86°F). The systemshould be installed in an area in which thetemperature range is fairly constant (the suggestedtemperature variation is no more than +/- 2°C/hr (+ /-5°F/hr)). The relative humidity of the area shouldmeet the ASHRE standard for human comfort.

• The system should not be placed near a window, airconditioning duct, etc. or any device in whichsignificant temperature changes may occur (e.g. anoven).

• The system should be positioned so that it is easy toaccess the switch and the power plug.

It is strongly recommended that the system be operated in an appropriate laboratory fume hood. If the instrument is not operated in a fume hood, the user must maintain worker exposure levels below both your organization’s and applicable governmental occupational exposure limits.

Open flames must be prohibited in the laboratory

• Corrosive vapors, solvents and dust should not beallowed in the laboratory as these can affect systemperformance.

2.1.2 SPACE REQUIREMENTS

The dimensions of the Reveleris® PREP purification system are 16.75" (42.54cm) H x 21.5" (54.61cm) W x 20.50" (52.07cm) D.

A minimum of 6" (15cm) should be provided at the rear of the unit to allow for proper ventilation.

If flammable solvents will be employed, ensure that all sources of heat are kept at least 6' (2m) from the system.

2.1.3 ELECTRICAL REQUIREMENTS

The Reveleris® PREP purification instrument includes an automatic voltage selector (85-265V, 50/60Hz) and has a power consumption of 1200W. The system should be connected to the main power line and should be connected to a safety earth. The system should not be connected to a power line that is susceptible to significant changes in power requirements such as a power line that is connected to a compressor, oven or other source of heat generation. If significant changes in the power may occur, a constant voltage transformer may be required. The system should not be installed near equipment that generates strong electromagnetic fields. If the power line is noisy, it may be necessary to install a noise filter.

2.1.4 CARTRIDGE, COLUMN, AND MOBILE PHASE

A cartridge or column and HPLC-grade mobile phase solvents that are capable of separating the compounds of interest are required. Non volatile impurities, stabilizers, or modifiers in the mobile phase will produce baseline noise in ELSD. If you are uncertain about the cartridge, column, and/or mobile phase to use, please contact your BUCHI Service Representative for assistance. A listing of the available cartridges and prep LC columns is provided in Sections 8.3 and 8.4 and at www.buchi.com. Refer to Section 8.8 for a list of volatile mobile phase modifiers that are suitable for ELSD.

2.1.5 EXTERNAL GAS SOURCE (AIR OR NITROGEN)

An external gas supply is required to run the Reveleris® PREP purification system. This can be a gas cylinder (air or nitrogen); house supplied air, an air pump/generator, or nitrogen compressor. The recommended gas coming into the system must be dry, with pressure of around 50psi. The regulator inside the instrument will regulate the pressure to about 40psi to provide 2-2.5L/min of gas flow to the ELSD.

To simplify the operation of the Reveleris® PREP purification system, use the Reveleris® Dry Air Supply (article no. 145168621). The Reveleris® Dry Air Supply is designed to be used with Reveleris® PREP purification system. It simplifies the operation of the Reveleris® PREP purification system by producing particle free dry air on demand without the need for operator attention. Refer to Section 9 for more information on the Reveleris ® Dry Air Supply.

A minimum of 2L/min of gas flow is required for proper ELSD operation.

2.2 UNPACKING

The Reveleris® PREP purification system and its accessories are shipped in one container. Unpack components carefully, making sure all items in the list below have been included and are in good condition. Save the container and packing material for future use.

The shipping container for the Reveleris® PREP Purification System contains:

• The Reveleris® PREP Purification instrument

• 2 Fraction Collection Trays (18 x 150mm)

• An accessory kit, with the following items:



Part No. Item Qty

143123523 Flash Solvent Tubing Assembly 1 143123524 Flash Waste Tubing Assembly 1

142118956 Sample Bypass 1

142118955 Column Bypass 1 142126973 Flash Touch-Screen Stylus 1 145167302 Adjustable Solid Loader Plunger (3 gram) 1 145152371 Filled Solid Loader 3gram 1 142118959 3/16" x 1/8" Antistatic Tubing 6

143132390 Corrugated Tubing Assembly 20

145148885 Fraction Collector Tray Assembly 1

145148940 Flash Solvent 38-430 Bottle Caps (5pk) 1 145148941 Flash Solvent GL-45 Bottle Caps (5pk) 1 142112993 Power Cord 1

142118923 Flangeless Fitting for 1/8” Tubing 5 142118925 1/8” Flangeless Ferrule and SS ring 5

142118924 Flangeless Fitting for 3/16” Solvent Tubing 4 142118926 3/16” Flangeless Ferrule 4

142114604 1/8” PTFE Tubing 10

143135935 5mL Sample Loop 1

143135936 Prep Bypass Tubing 1

143135852 1/8” x .085” Stainless Steel tubing, 3 ft 2

143135853 1/8” x .085” Stainless Steel tubing, 5ft 2

143135850 1/16” x 0.04” Stainless Steel tubing, 3ft 2 143135851 1/16” x 0.04” Stainless Steel tubing, 5 ft 2

143135854 1/16" x 0.03"ID PTFE tubing (Prep Sample Waste line) 3

142110666 One piece 1/16" PEEK Hex fitting 1

142129496 1/8” SS Fitting for ¼-28 port (for prep injection valve with1/8” OD tubing) 4

142129495 1/8” SS Ferrules for ¼-28 port (for prep injection valve with 1/8” OD tubing) 8

142129492 1/8” SS fitting for 5/16-24 port (for bulkhead and column with 1/8” OD tubing) 8

142129493 1/8” SS ferrules for 5/16-24 port (for bulkhead and column with 1/8” OD tubing) 12

142110574 1/16” SS fitting (for column with 10-32 port) 12

142110573 1/16” SS ferrules (for column with 1/16” OD tubing) 16

148610223 1/8” to 1/16” Tube Reducer for 5/16-24 port (for bulkhead) 1

143135950 1/8” to 1/16” Tube Reducer for ¼-28 port (for valve) 2

142110329 Female Luer Adapter for ¼-28 port 2

142118928 1/8” Flangeless Ferrule 2

142107259 3/8-7/16” Wrench (for 1/8” tubing) 1

142106694 ¼-5/16” Wrench (for 1/16” tubing) 1

142129503 1/8” Ball Driver x 3 1/8” Long 1

142107268 3/32” Ball Driver x 5 1/4” Long 1 142118927 Fitting Tool 1

148623732 5 Amp Fuse 2

148623731 10 Amp Fuse 2

The system has been carefully packed and shipped to ensure that it is received in proper condition. Any damage to the container or its contents should be reported immediately to your BUCHI Sales Representative. Please refer to Section 8.6, Warranty, Returns, and Repairs, for more information.

If replacement parts are needed, please refer to Section 8.2, Replacement Parts for part numbers.



2.3 CONTROLS AND FEATURES

2.3.1 FRONT PANEL AND CONTROLS

Figure 2-1: Front Panel



2.3.2 REAR PANEL

Figure 2-2: Rear Panel



2.4 INSTALLING THE INSTRUMENT

2.4.1 UNPACK THE INSTRUMENT

Remove the Reveleris® PREP purification instrument from its shipping container and position it on a level surface near or in a fume hood. Make sure there is free flow of air to the bottom of the unit and to the cooling fan at the back panel. Allow the instrument to warm to ambient temperature if necessary.

The instrument is typically installed by a BUCHI trained person.

Save the shipping container for future use.

2.4.2 LOCATING THE INSTRUMENT IN THE LABORATORY

It is strongly recommended that the system be operated in an appropriate laboratory fume hood. If the instrument is not operated in a fume hood, the user must maintain worker exposure levels below both your organization's and applicable governmental occupational exposure limits.

It is recommended that static dissipative floor mats be used in the immediate vicinity of the instrument.

Electrical Connections 1. Plug the power cord provided with the unit into the

power module on the back panel of the Reveleris® PREP purification instrument (Figure 2-2 ) and then plug the power cord into the mains. The system automatically detects the input voltage (85-265V).

Only use the power supply cord recommended by the manufacturer.

2. Plug in any auxiliary devices such as a printer. Anydevice that is connected to the system must meet CE(or equivalent) requirements.

3. If the Reveleris® Dry Air Supply is used as theexternal air supply source, connect the power cableprovided in the Reveleris® Dry Air Supply accessorykit to the “Dry Air Supply Power Out” port at the rearpanel (Figure 2-2) of the Reveleris® PREPpurification system. This provides the DC powerneeded to operate the Dry Air Supply.

2.5 FLUID CONNECTIONS

2.5.1 SOLVENT AND SENSOR LINES

Connect all solvent lines and the solvent and waste sensor lines to the manifold on the rear panel (Figure 2-2 ).

Many solvents and samples that are used by the system may present a potential hazard to the user. Protection appropriate to the hazard must be provided for users and others working in the area. This protection should be in accordance with all applicable federal, state and local laws and regulations and in compliance with your organization’s chemical safety program.

Make certain that the solvent bottles and drain containers are chemically compatible with the solvents that are used.

To prevent spills, and to both minimize potential for personnel exposure to hazardous chemicals and the risk of fire do not fill re-fill solvent bottles when they are on top of the unit.

Fill the solvent reservoirs with the appropriate solvent and place them on top of the instrument. Insert a delivery line with a filter and sensor line into each bottle and firmly attach the bottle cap to each bottle. Place the waste sensor line in the waste container toward the top of the container opening to prevent overfilling.

2.5.2 DRAIN SETUP

Attach all drain/waste tubing to the drain/waste outlets on the rear of the instrument (Figure 2-3 ). Extend the tubing to a waste container (not included) either at bench or at floor level and make certain that the container is sealed to prevent solvent fumes from escaping. The waste container will contain solvents from your mobile phase and should be disposed of properly.

SD

WASTE/DRAINS

VW V

AIR

PW

VW - FLASH MULTIPORT VALVE WASTEV - FRACTION COLLECTOR WASTEPW- PRIMING WASTESD - SOLVENT DRAINAIR - EXTERNAL AIR

Figure 2-3: Waste/Drain/Air Connections

If you place the waste container on the bench next to your system or at any other location that is higher than the bench top, the end of the waste tubing should be above the level of the liquid in the waste container to prevent siphoning.



The waste container should be large enough to hold the combined contents of all solvent reservoirs. It may be necessary to use a secondary containment vessel. All waste containers should be shatterproof.

2.5.3 PREP INJECTION VALVE AND BULKHEAD CONNECTIONS

Tubing, fittings (nuts and ferrules), sample loop, and injection syringe port adapter needed for connections to the prep injection valve and the bulkhead on the instrument are provided in the accessory kit. The valve ports are ¼-28 and the bulkhead port is 5/16-24. The correct fitting must be used in order to make proper connections. Two different diameters (1/16” and 1/8” OD) and lengths (3ft and 5 ft) of stainless steel tubing are provided to connect to/from the prep column. Choose the tubing length and OD that is appropriate for your prep column.

1. Install the 5mL sample loop (article no. 143135935)onto the prep injection valve ports labeled “LOOP” asshown in Figure 2-4. Push the tubing all the wayinto the ¼-28 port until it seats firmly. This isessential for a proper Zero Dead Volume connection.Manually turn the nut until it is finger tight. Use anopen end wrench to turn the nut 1/4 turn past fingertight.

Figure 2-4: Sample connection to the Prep Injection Valve

2. Install the syringe female luer adapter (P/N142110329) to the prep injection valve port labeled“SAMPLE IN” using the 1/8” flangeless ferrule (P/N142118928) as shown in Figure 2.5.

3. Connect the 1/16" x 0.03"ID PTFE tubing (P/N143135854) to the prep injection valve “WASTE”port using the one piece 1/16” PEEK fitting (P/N142110666) and the 1/8” to 1/16” Reducer for¼-28 port (P/N 143135950). Place the other endof the tubing in a beaker or small waste bottle tocollect sample loop waste.

4. Choose the stainless steel tubing appropriate foryour column and connect the tubing to the “TOCOLUMN” port on the valve. Follow the guides inTable 2-1 to select the proper fitting for theconnection. Connect the other end to your prepcolumn inlet (Section 4.4.2).

5. Connect the second piece of the stainless steeltubing to the bulk head fitting labeled “FROMCOLUMN”. Follow the guides in Table 2-1 to selectthe proper fitting for the connection. Connect theother end to your prep column outlet (Section4.4.2).

Figure 2-5: Prep Injection Valve Fluidic Connections



TABLE 2 -1: FITTING GUIDELINES FOR CONNECTIONS TO THE PREP INJECTION VALVE AND BULKHEAD Ports Tubing OD

1/8” OD 1/16” OD Description P/N Description P/N

Adapter N/A N/A 1/8” to 1/16” Reducer

for ¼-28 port 143135950 “TO COLUMN” on Valve

Fitting 1/8” SS Fitting for ¼-28 port

142129496 1/16” SS Fitting 142110574

Ferrule 1/8” SS Ferrule for ¼-28 port

142129495 1/16” SS Ferrule 142110573

Adapter N/A N/A 1/8” to 1/16” Reducer

for 5/16-24 port 148610223 “FROM COLUMN” on Bulkhead

Fitting 5/16-24 SS Fitting 142129492 1/16” SS Fitting 142110574

Ferrule 1/8” SS Ferrule 142129493 1/16” SS Ferrule 142110573

2.6 ELSD EXHAUST CONNECTIONS

If the instrument is not installed in a hood:

• Connect the ELSD exhaust on the rear panel (Figure 2-2) to the laboratory exhaust air system using thecorrugated tubing provided in the accessory kit. If any airin this system is re-circulated, appropriate traps shouldbe employed to prevent restrainment of toxic orhazardous material. A source of exhaust to remove anyvapors from the solvent bottles and waste container isalso recommended.

Please make sure that the ELSD exhaust tubing is not connected directly to a vacuum source.

• An exhaust hose should be placed over the solventbottles to remove vapors.

2.7 EXTERNAL AIR/GAS SUPPLY CONNECTION

Connect gas supply line to the Air I n port on the rear panel (Figure 2-2).

The pressure inside the instrument is regulated to deliver 2-2.5L/min of gas flow to the ELSD. The regulator is normally set to between 37-45psi depending on the back pressure of the nebulizer. The gas coming into the system must have sufficient pressure (50psi is recommended) to provide the required gas flow to the system.

The internal regulator can accept a maximum pressure of 300psi coming into the system.

2.8 MOVING THE INSTRUMENT

If the instrument is to be moved, remove the solvent bottles from the top of the chromatograph and disconnect all solvent, sensor, and waste lines from the rear panel. Ensure that the waste line is empty and secure it. Unplug the system from the power outlet and disconnect all auxiliary devices (e.g. printer, mouse and keyboard).

The system weighs 92lb (41.73kg). At least two people should be used to move the instrument. Never attempt to move the unit with solvent reservoirs installed or with tubes in the fraction collector.



3. THE USER INTERACTION PROGRAM

3.1 OVERVIEW

When the unit is powered up, a number of self-test procedures are performed and the system is initialized. A Reveleris® PREP purification instrument screen is presented during the start-up period which is followed by a Windows® software start screen. The software automatically opens to the Setup window (Figures 3-1 and 3-2) at the conclusion of the initialization procedure.

The Setup window is used to choose operating mode (Flash or Prep LC) and to enter operating parameters and initiate a separation. When a run is started, the Run in Progress window (Section 3.4 ) is displayed. If desired, the operator can view previous runs via the Past Run window (Section 3.5 ). The active window can be selected via the View menu.

This chapter describes the various features of the user interaction scheme. It should be used in conjunction with

Chapter 4 , which describes how the operator separates a sample.

If Reveleris ® Dry Air Supply is used as the external air supply source, it’s operation is controlled by the Reveleris ® PREP purification system sof tware. Every time the Reveleris ® PREP purification sof tware is started it performs a pressure check to determine the type of air source connected to the system. If the Reveleris ® Dry Air Supply is used, the pressure will be low initially. The Reveleris ® PREP pur ification software will then turns on the air compressor pump inside the Reveleris® Dry Air Supply for a few seconds to check for pressure. If the pressure meets specification, this information is then stored in the software for its operation. During the operation of the Reveleris ® PREP purification system, the air pump is tur ned on only when needed. Refer to Section 9.0 for more information on the Reveleris® Dry Air Supply.

Figure 3-1: The Flash Mode Setup Screen



Figure 3-2: The Prep Mode Setup Screen

3.2 THE SETUP SCREEN

The Setup screen is used to select the operating mode and enter the conditions for the separation of a sample. To select either Flash or Preparative mode, open the Mode drop down on the menu bar at the top of the screen.

The conditions for the separation consist of the following sections:

• Chromatographic Conditions (Section 3.2.1 )

• Detector Selection (Section 3.2.2 )

• Collection Criteria (Section 3.2.3 )

• Fraction Collection Information (Section 3.2.4 )

• Solvent Selection (Section 3.2.5 )

• Mobile Phase Composition (Section 3.2.6 )

• Reveleris® Navigator™ Software Information (Section3.2.7)

• Collection Vial Display (Section 3.2.8 )

• Saving and Retrieving Methods (Section 3.2.9 )

3.2.1 CHROMATOGRAPHIC CONDITIONS

The chromatographic conditions section is different between flash chromatography and prep LC.

3.2.1.1 FLASH MODE (FIGURE 3.1)

Colu mn : The Column field indicates the column/cartridge installed onto the Reveleris® PREP purification system. The column type is determined by either the auto-column recognition technology for columns equipped with a recognition tag or manually selecting from the generic drop



down menu. The system will automatically populate default run conditions corresponding to that column size. The maximum column pressure is preset between 75psig to 200psig depending on the type of column detected. All generic columns default to 75psig as the maximum pressure.

Flow Rate : When the column is selected the suggested flow rate is automatically populated in the Flow Rate field and can be edited by the user (range is 1-200mL/min). To edit the flow rate, touch in the field to present a numeric keypad (Figure 3-3), enter the desired value and press OK.

Figure 3-3: Numeric Keypad

A red field indicates an invalid entry. The value must be edited before a separation can be initiated.

If desired, the operator can enter information via a standard Windows® software keyboard connected to the system via a USB port. If this option is used, the desired value is selected by moving the cursor to the appropriate field and typing the value in via the keyboard.

Duration Units : The runs can be setup in Duration Units of minutes or CV (column volumes). The default setting for the instrument is minutes. The Column field must be filled prior to changing the Duration Units to CV. All corresponding run setup units will simultaneously change to match the chosen duration unit.

Equilibration : The Equilibration field indicates the period of time or number of column volumes that the mobile phase flows through the column before the sample is injected and is edited in the same manner as the Flow Rate.

The minimum equilibration time is dependent on the flow rate and column size. The time should be selected to fully equilibrate the UV detector and flush the fraction collector arm.

Run Length : The Run Length field indicates the time for the separation or the number of column volumes required. It can be edited in the same way as the Flow Rate.

Air Purge : The Air Purge field indicates the period of time that air is passed through the column after the separation to remove mobile phase and can be edited in the same way as the Flow Rate.

3.2.1.2 PREP MODE (FIGURE 3.2)

Column : The Column field allows user to type the column name. To type the column name, touch in the field to present a QWERTY keyboard (Figure 3-4 ). Up to 24 characters can be typed. The column name will be saved for the run and will be printed on the report. The column name will also be saved in the method if the method is saved. The run can also be started if no column is entered and the run will continue as normal with “No column entered” printed on the run report.

Figure 3-4: QWERTY Keyboard Flow Rate : The flow rate needs to be entered by the user (range is 1-200mL/min). To edit the flow rate, touch in the field to present a numeric keypad (Figure 3-3 ), enter the desired value and press OK.

If desired, the operator can enter information via a standard Windows® keyboard connected to the system via a USB port. If this option is used, the desired value is selected by moving the cursor to the appropriate field and typing the value in via the keyboard.

Max Pressure : The maximum pressure is automatically populated based on the entered flow rate. The maximum pressure is inversely proportional to the flow rate. The user can override the default maximum pressure to a lower value if the column pressure limit is lower. To edit the max pressure, touch in the field to present a numeric keypad (Figure 3-5 ). The keypad will show min and max values depending on the flow rate selected. Enter the desired value and press OK.


www..com Reveleris® PREP Operation Manual, Version B 15

Figu re 3-5: Prep Mode Max Pressure Keyboard

Equilibration : The Equilibration field indicates the period of time the mobile phase flows through the column before the sample is injected and is edited in the same manner as the Flow Rate.

The minimum equilibration time is dependent on the flow rate and column size. The time should be selected to fully equilibrate the column and the detectors, and flush the fraction collector arm.

Run Length : The Run Length field indicates the time for the separation required. It can be edited in the same way as the Flow Rate.

Air Purge : This is grayed out as it is not applicable in the Prep mode.

3.2.2 DETECTOR SELECTION

The Reveleris® PREP purification system includes a three-channel UV detector and an evaporative light scattering detector (ELSD) to monitor the eluted sample and determine if/when a fraction should be collected. Any combination of detector channels can be employed.

To employ a UV detector channel, place a check mark in the appropriate box and enter the desired wavelength in the same way as the Flow Rate is entered.

To employ the ELSD, place a check mark in the ELSD box.

An external gas source must be provided to the Reveleris ® PREP system in order for the ELSD to fun ction.

3.2.3 COLLECTION CRITERIA

The collection of a sample can be started or terminated by either Slope Detection or by Threshold Detection.

• To use Slope Detection to trigger the onset ortermination of sample collection, place a check mark inthe Slope Detection check box. Make certain that one(or more) detector channels are selected (Section3.2.2).

The sensitivity selection refers to the degree of filtering used in the chromatogram.

– High sensitivity uses a small filter and presents anoisier chromatogram. This may lead to thepresence of a large number of small peaks in thechromatogram due to the detection of tracecompounds and collection of a number of smallfractions.

– Low sensitivity selection uses a large filter andpresents a less noisy chromatogram. This mayremove small peaks from the chromatogram andlead to the non-collection of small peaks.

– Medium sensitivity is a reasonable compromise formost cases.

• To use Threshold Detection to trigger the onset ortermination of sample collection, place a check mark inthe Threshold Detection check box and indicate thedesired threshold. Make certain that one (or more)detector channels are selected (Section 3.2.2 ).

If more than one criteria are selected for peak collection, it is possible that the different criteria will be met at slightly different times in the separation. As an example, assume that the absorbance threshold is set at 0.1AU and the wavelengths are set at 254nm and 280nm. In this example, let us assume that the absorbance at a given time at 254nm is 0.1AU and the absorbance at 280nm is 0.08AU. Peak collection will begin when the absorbance at 254nm is 0.11AU. About 10 seconds later, the absorbance at 254nm is 0.19AU and the absorbance at 280nm is 0.11AU, so another peak collection event will occur, moving the fraction collector to a new tube. (Note that if multiple detections occur within a couple of seconds of each other, the system may assume it is from the same peak and will not move to a new fraction collection tube.)

3.2.4 FRACTION COLLECTION

Select the desired option for fraction collection via the radio buttons. The Maximum field in the Per-Vial volume area is dependent on the tray(s) in the system as determined by the auto-tray recognition technology and is not editable by the user.

The Peak and Non-Peaks fields in the Per-Vial volume area default to the maximum volume of the vial detected. These values are editable by the user in the same manner as the Flow Rate. The range is zero to the maximum value for the size of tubes in the tray.

3.2.5 SOLVENT SELECTION & PRIMING

The Reveleris® PREP purification system can deliver isocratic and binary gradients that are generated from up to four solvent bottles. When the system is initially installed, the



solvent fields (lower left corner) will indicate <Empty> and it is necessary to select the solvents to be used in the separation.

To select a solvent for Solvent A:

1. Press <No solvent chosen> to present the drop downmenu shown in Figure 3-6 . In addition, the messageSolvent A must be chosen because it is used in thesolvent gradient sequence is presented.

Figure 3-6: Solvent Selection Menu

2. Select <Load a Solvent…> to present the SolventLoading dialog box (Figure 3-7 ).

Figure 3-7: Solvent Loading Dialog Box

It is important to calibrate the solvent safety sensors before starting a run (See Section 3.3.5).

3. Press Load to present the Load and Prime SolventLine dialog box (Figure 3-8 ).

Figure 3-8: Load Solvent Line Dialog Box

4. Press the Empty field to access a list of solvents thatare commonly used for flash chromatography andselect the solvent that is in the bottle associated with

line 1 (e.g. Hexane). If desired, you could add solvents to this list (Section 3.3.2 ).

5. Once a solvent is loaded, the Auto Prime button isactivated. Press the Auto Prime button to automaticallyprime the solvent line and pumps. The priming statusis displayed at the bottom of the screen (Figure 3-9 ).

Figure 3-9: Auto Prime Solvent Line Dialog Box

During the auto prime process, the system will pull solvent from line 1 through pump 1 and pump 2 consecutively. It will also verify that each pump is successfully primed. If priming of pump 1 is not successful, the process is automatically repeated 2 more times before proceeding to pump 2. If priming of pump 1 failed after the three trials, it will not proceed to pump 2 and the user needs to press the Auto prime button to repeat the auto prime process.

6. After the priming is completed, press Close to return tothe Solvent Loading screen (Figure 3-7) , which willnow display the name of the solvent that was justloaded (e.g. Line 1: Hexane).

7. Repeat steps 1-6 to load solvent in each of the othersolvent lines. An example of solvent region with foursolvents loaded is presented in Figure 3-10 .

Line 1-4 refers to the solvent that is withdrawn from line/solvent bottle 1-4 of the system, while A-D refers to its definition with respect to the gradient that is formed (Section 3.2.6). Any combination of solvents can be loaded so that Line 1 can be A, B, C or D.

Figure 3-10: Complete Solvent Field



A minimum of two solvents must be defined for operation of the system (this does not require that two different solvents are required for operation, you can set one solvent to 100% and the other to 0% for an isocratic separation). If desired, you can use two or more solvent lines to deliver the same solvent, thereby reducing the frequency of refilling bottles. When the same solvent is chosen for multiple lines, the fields turn yellow to alert the user and a message of “The same solvent type is being used multiple times” is displayed. This is by design so that when the first solvent safety sensor alerts, the system will automatically switch to the second reservoir.

3.2.6 MOBILE PHASE COMPOSITION

The composition of the mobile phase as a function of time can be indicated by entering the gradient in a table or graphically using the plot in the lower right corner of the Setup window.

3.2.6.1 TABULAR METHOD

To enter a gradient table:

1. Press the Table button to present the Gradient Table(Figure 3-11 ). The Gradient Table presentationdefaults to the duration units chosen in Section 3.2.1 .

Figure 3-11: Gradient Table

2. Click on the AB▼ field to access a list of the varioussolvent line combinations (AB, AC, AD, etc.) and selectthe two solvents that you want to use at Min. (time) =0.0. The letters A, B, C, D refer to the solvents loadedin the Solvent field.

3. Click on the % 2nd field and enter the fraction of thesecond solvent that you want to use. As an example, ifyou want the mobile phase at Min. 0 to be 20%Hexane/80% Ethyl Acetate, you would enter 80 in thisfield.

4. To add additional lines to the Gradient Table, press thenumber field (1►) to present a menu to add the nextline above or below the selected line. The inserted linewill be editable in the same manner as the line for Min.= 0. As you generate a gradient table, note that:

– there is a line for Min = 0,

– no time is duplicated

– Min/CV refers to the number of minutes or columnvolumes for the step and is not cumulative,

5. To delete lines to the Gradient Table, press thenumber field (1►) to present a menu to delete theselected line.

6. When you have completed the gradient table, pressClose. The gradient will be presented as a plot on thegraph (e.g. Figure 3-12 ).

Figure 3-12: Typical Gradient

3.2.6.2 GRAPHICAL METHOD

As an alternative, the gradient can be generated or edited directly via the plot. To employ this mode:

1. Verify the Edit box is chosen in the Graph area.

2. Click on the line at the time for which you want toadd/edit the gradient and drag it to the desired %B,then remove the stylus (as an alternative, you can usea mouse). As an example, the %B (Figure 3-12 ) canbe increased to 100% by pointing the mouse at the 8-minute mark and dragging the point to 100% B (Figure3-13).

Figure 3-13: Edited Gradient

3. To remove a step, drag the point to the baseline or toany gray area around the graph until a red X is visible,then release.

4. Solvent line combinations can be accessed by clickingon the AB▼ box to reveal a drop down menu.



The boxes that indicate AB in Figure 3-13 are used to select the solvents to be used in the gradient in the same manner as the Gradient table (Figure 3-11).

The estimated solvent that will be used for the separation is displayed below the solvent list in the lower left corner of the Setup window.

3.2.7 REVELERIS® NAVIGATOR™ SOFTWARE

Figure 3-14: Reveleris® Navigator™ Software Password Box

The Reveleris® Navigator™ software is an advanced method optimizer designed to save you time and money with your purification challenges. It provides optimum gradient conditions based on input from other scouting methods like TLC and LC. This feature is available in Flash mode only. See Section 5 for more information.

3.2.8 COLLECTION VIAL DISPLAY

The upper right hand corner of the Setup screen presents the collection vial matrix corresponding to the trays detected by the auto-tray recognition technology. The estimated number of vials required for the separation is displayed below the solvent usage list in the lower left corner of the Setup window.

Two different size trays may be used in the Reveleris ® PREP purification system simultaneously. The system will default collection volume to the smaller of the two trays.

Each individual tray may be used multiple times during a run after confirming that all vials are empty.

3.2.9 SAVING AND RETRIEVING METHODS

Once you have generated a method, it can be stored and retrieved.

New method can be saved in the Default folder or a new folder can be created.

To save a method in the Default folder, press Save Method As on the File menu to present the Save Method As dialog box (Figure 3-15 ), press the Enter Method Name field to present a QWERTY keyboard, enter the desired name and press OK.

Figure 3-15: Save Method As Dialog Box

To create a new method folder, press the Create New Folder button to present the Create New Folder dialog box (Figure 3-16).

Figure 3-16: Create New Folder Dialog Box

Press the field under the Create New Folder to present a QWERTY keyboard, enter the desired name and press OK. To save the method in the custom folder, use the drop down arrow under the Choose Folder to select the folder (Figure 3-17). Then, press the Enter Method Name field to present a QWERTY keyboard, enter the desired name and press OK. The new method will be saved into the newly created method folder.



Figu re 3-17: Set Method Name – Choose Folder Dialog Box

The Save Method entry on the File menu will save the method with the same name in the same folder.

There is no maximum limit of characters for a method folder or name, but only a limited number of characters can be displayed on the screen. If the combination of the folder and method names are too long, only the folder name will be displayed on the screen during run or when viewing past run. The complete folder and method names will be printed on the report.

To open a stored method, press Open Method on the File menu, which presents a list of existing method folders and methods. Click on the desired method folder and then the desired method and press OK.

If a method that was saved in a different mode than the instrument is currently in is selected, the Mode Change screen will be displayed for you to confirm that you want to switch mode.

If needed, you can annotate a method by pressing Edit Method Notes in the Save Method As menu or pressing the

button adjacent to the method name on top of the Setup Window to present a dialog box where you can add any relevant information.

If a stored method is opened and changes are made, an asterisk appears next to the name to denote alterations were made to the original method.

3.3 TOOLS MENU

The Tools drop down menu provides the user easy access to perform several functions on the Reveleris® PREP purification system and consist of the following sections:

• Solvent Loading (Section 3.3.1 )

• Solvent Definition (Section 3.3.2 )

• Vapor Sensors and Limits (Section 3.3.3 )

• Configuration (Section 3.3.4 )

• Calibration and Defaults (Section 3.3.5 )

• Manual Control (Section 3.3.6 )

• NP <> RP (Section 3.3.7 )

• Product Services (Section 3.3.8 )

• UV-VIS Detection (Section 3.3.9 )

• Debug Diagnostics (Section 3.3.10 )

3.3.1 SOLVENT LOADING

The Solvent Loading dialog box (Figure 3-18 ), which is accessed by selecting Solvent Loading on the Tools menu or via the Solvent Selection area of the Setup window, is used to introduce solvents into the system. Solvent loading was previously discussed in Section 3.2.5 .


3.3.2 SOLVENT DEFINITION

The Solvent Definition dialog box (Figure 3-19 ) is used to add, edit or delete solvents to the list of solvents that can be used with the system.



Figure 3-19: Solvent Definition Dialog Box

The Add Solvent button presents a dialog box (Figure 3-20 ) in which you can enter the name of the solvent and any descriptive information. If you press in either the Name or Info field, a QWERTY keyboard will be presented. Enter the solvent name in the Name field and any description or information about the solvent in the Info field. Once the custom solvent name has been entered, the Verify Solvents button becomes active (changes from gray to green). Press the Verify Solvents button to continue to the next step.

Figure 3-20: Solvent Information Dialog Box

Once the Verify Solvents button is pressed, the Solvent Verification box (Figure 3-21 ) will be presented. Place a check mark inside each box if any of those solvents are contained in the newly created custom solvent. You can check none or multiple boxes depending on the composition of the custom solvent, then press OK to finish.

Figure 3-21: Solvent Verification Box

The new custom solvents will automatically appear in the Solvent Types list after all steps above are completed. Nothing will be added to the list if Close or Cancel is pressed.

To edit an existing solvent, move the highlight to the solvent name and press Edit Solvent to present the solvent information dialog box, with name of the solvent and any descriptive information that has been entered.

To delete a solvent, move the highlight to the solvent name and press Delete Solvent. A message will be presented to confirm if you are certain that you want to delete the solvent.

Solvent that is currently loaded in a line must be unloaded first before it can be deleted from the Solvent Types list.

3.3.3 VAPOR SENSORS AND LIMITS

The waste solvent safety sensor and vapor sensors are safety devices designed to detect/prevent solvent spills and fire hazards. Do not disable or bypass these sensors.

There are two vapor sensors located in the Reveleris® PREP purification instrument. Sensor one is located near the back of the unit and sensor two is located near the front of the unit. The settings for these sensors are as follows:

– High sensitivity is generally used for non-volatilesolvents

– Low sensitivity is generally used for volatile orsemi-volatile solvents

– Medium sensitivity is a reasonable compromisebetween the low and high setting.



Figu re 3-22: Vapor Sensor Setting Box

If the system detects vapors above the set point (Limit), an alarm will sound. During a run, the system will pause to allow the user to check for any leak or spill. The run cannot be resumed until the detected vapors drop below the set point.

3.3.4 CONFIGURATION

The Configuration dialog box (Figure 3-23 ) provides an easy access to several systems’ settings for the Reveleris® PREP purification instrument. This menu can be used to view Windows® software properties, access saved files, connect to a printer, or set up a page for printing.

Figure 3-23: Configuration Dialog Box

3.3.5 CALIBRATION AND DEFAULTS

The Calibration and Defaults menu has several features to reset system’s settings:

– Touch Screen can be reset if needed by clicking onthe Calibrate button and following the on screeninstructions

– Date/Time can be set from this menu which opensthe standard Windows® application

– UV Lamp Lifetime should be reset by clicking theReset button each time the UV lamp is replaced.This will re-start the clock, and when the lamp isabout to expire, a warning message reminding theuser to replace the lamp will appear.

– Solvent Safety Sensors for all solvent lines andwaste can be recalibrated by lifting each individualline out of the reservoir into the air, pressing the

corresponding zero button and replacing the line back into the reservoir and waste container

– Default Settings can be used to return the solventlist to the original list installed on the unit

– The Audible Alarm can be deactivated by removingthe check from the box. This does not turn thesafety features off; only the audible portion of thealarm is turned off (not recommended)

Figure 3-24: Calibration and Default Dialog Box

3.3.6 MANUAL CONTROL

Manual Control opens a second drop down window to many features that would normally be used during a run. These features are available only during Setup and are not available during runs. Also, not all features are available in both Flash and Prep LC modes. Grayed out features indicate that they are not available in the mode the instrument is currently in.

Figure 3-25: Manual Control Menu

3.3.6.1 AIR PURGING (FLASH MODE ONLY)

Air Purging can be used to pass air through the column or dry sample solid loader to remove excess solvent from the column or sample loader. Enter the desired time into the Air Purge Time field and press the appropriate (Column Purge or Sample Purge) button. To stop purging earlier than the set time, press the same button again or Done.



Figu re 3-26: Air Purging Dialog Box

3.3.6.2 INJECTION PORT RINSING (FLASH MODE ONLY)

Injection Port Rinsing can be used to manually rinse the injection port with a syringe. After opening the dialog box, follow the on screen instructions to rinse port.

Figure 3-27: Injection Port Rinsing Dialog Box

3.3.6.3 SOLID LOADER FLUSHING (FLASH MODE ONLY)

Solid Loader Flushing can be used before or after a run to flush solvents through the dry sample solid loader. Install a column or bypass column as the solvent will flow through the entire system. Verify that the column or bypass column is recognized in the column field. Install the solid loader or solid loader bypass into the system. Click the column installed and solid loader installed boxes. Choose the lines, solvent mix and flow rate required to flush. Press Enable Solvent Flow to start and stop the mobile phase.

The same default column pressures settings described in Section 3.2.1.1 apply.

Figure 3-28: Solid Loader Flushing Dialog Box

3.3.6.4 FRACTION COLLECTOR ARM RESET

Fraction Collector Arm Reset can be used to reset the fraction collector arm when an error related to the fraction collector arm occurred or when the arm did not automatically reset after a run. Pressing Reset will move the arm back to the home position.

Figure 3-29: Fraction Collector Reset Dialog Box

3.3.6.5 COLUMN FLUSHING

Flash Mode: Column Flushing can be used to flush solvents through the column and/or system. To flush the column, install the column and verify that the column is recognized in the column field (for column equipped with auto-recognition tag) or pick the column from the drop down list. Click the column installed box. To flush the system, install the bypass column in place of the column. Choose the lines, solvent mix and flow rate required to flush. Press Enable Solvent Flow to start and stop the solvent flow.


Figure 3-30: Flash Mode Column Flushing Dialog Box

Prep Mode: As in the Flash mode, Column Flushing can be used to flush solvents through the column and/or system. To flush the column, install the column and verify that the inlet and outlet



tubing from the column is connected to the system. Click the column installed box. To flush the system, install bypass tubing in place of the column. Choose the lines, solvent mix and flow rate required to flush. Edit the default maximum pressure if the column pressure limit is lower than the default value. Press Enable Solvent Flow to start and stop the solvent flow.


Figure 3-31: Prep Mode Column Flushing Dialog Box

3.3.6.6 SOLVENT LINE FLUSHING

Solvent Line Flushing can be used to flush solvent through the lines up to the multi-port valve. This is useful when changing solvents in the same line to ensure the line is filled with the new solvent. Choose the solvent line to be flushed. Press Start to start solvent flow and press Stop to stop the flow.

This feature is NOT a replacement for priming lines as described in Section 3.2.5. Line Flushing can be used only if pumps are already primed with solvent.

Figure 3-32: Solvent Line Flushing Dialog Box

3.3.6.7 PREP SAMPLE LOOP RINSING (PREP LC MODE ONLY)

Prep Sample Loop Rinsing can be used to manually rinse the sample loop using a syringe. After opening the dialog box, follow the on screen instructions. Press Rinse Sample Loop button to open the valve and turn the loop to waste. When “Please inject now” appears, manually inject the solvent to flush the loop. Press Done when complete to close the feature and reset the valve.

Figure 3-33: Prep Sample Loop Rinsing Dialog Box

3.3.7 NP <> RP

NP <> RP can be used to flush the entire system when switching from normal phase to reverse phase solvents or vice versa. NP<>RP feature is available in both flash and prep modes to flush the corresponding fluidic flow path. Follow the onscreen instructions to flush the system.

The instruction is slightly different for Flash mode vs. Prep LC mode. Please read the on screen instructions carefully to ensure the proper bypass column or tubing is installed.

Figure 3-34: Flash Mode NP <> RP Dialog Box



3.3.8 PRODUCT SERVICES

Access to the Product Services screen is currently limited to BUCHI trained personnel.

3.3.9 UV-VIS DETECTION

UV-VIS Detection is used to expand the UV-VIS range from 500nm to 850nm. This feature can only be enabled if the UV-VIS hardware is installed in the instrument. To enable this feature, contact your BUCHI Sales Representative to obtain the instrument specific password. Once this feature is enabled on the instrument, the UV-VIS Detection will be removed from the Tools drop down menu and the UV-VIS range will be defaulted to 850nm.

Figure 3-35: UV-VIS Dialog Box

3.3.10 DEBUG DIAGNOSTICS

Debug Diagnostics is used to troubleshoot certain problems. A service technician may ask you to read the values during troubleshooting process.

3.4 THE RUN SCREEN

This section defines the basic setup procedure to start a run.

3.4.1 ACCESSING THE RUN SCREEN

When the button on the Setup screen is pressed, a Set Run Name dialog box (Figure 3-36 ) is presented to allow the user to assign a run name.

If a generic column is used for the Flash separation or you are in the prep mode starting a prep run, the Set Run Name dialog box will be displayed only after the user confirmed that the column is installed.

New run can be saved in the “Default” folder or a new folder can be created.

Figure 3-36: Set Run Name Dialog Box

To create a new folder, press the Create New Folder button to present the Create New Folder dialog box (Figure 3-37 ).

Figure 3-37: Create New Folder Dialog Box

Press the field under the Create New Folder to present a QWERTY keyboard, enter the desired name and press OK. To save the run in the custom folder, use the drop down arrow under the Choose Folder to select the folder.



Figu re 3-38: Set Run Name – Choose Folder Dialog Box

The default run name is yyyy-mo-da_hh-mm-ss (e.g. 2014-03-12_14-23-10) but the user can enter any desired run name.

There is no maximum limit of characters for folder or run names, but only a limited number of characters can be displayed on the screen. If the combination of the folder and run names are too long, only therun name will be displayed on the screen during run or when viewing past run. The complete folder and run names will be printed on the report.

After a file name has been entered, the Sample Loading box (Figures 3-39 and 3-40) will be presented.

Figure 3-39: Flash Mode Sample Loading Dialog Box

Figure 3-40: Prep Mode Sample Loading Dialog Box

The Lower flow rate for specified time after injection feature is used for certain separations when the pressure increased significantly at the start of the run after sample injection. This is more common in the Prep mode and occurs when the sample or sample solvent is not easily miscible with the mobile phase solvent. Under this situation, you can reduce the flow rate at the start of the separation after sample injection for a specified period of time to reduce the system’s back pressure. Once the specified time at the reduced flow rate expires, the separation will resume at the original flow rate set in the method.

The Reveleris® software also offers another feature when the system’s pressure limit is exceeded before the set flow rate is reached. The software will automatically reduce the flow rate until the system’s back pressure remains within the system’s pressure limit. If the system’s pressure later stabilized at the lowered flow rate, the software will automatically increase the flow rate back up until it reaches the original set flow rate or at the highest flow rate setting the system’s pressure limit allows.

Anytime the flow rate is lowered during the run (automatically or manually set using the Lower flow rate for specified time feature), the flow rate field will change color to orange. After the run is completed, a message “Flow Rate Altered” will be displayed on the past run screen (Figure 3-41) and also on the printed report.


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Figu re 3-41: Past Run screen showing theAltered” message

To use the Lower flow rate for a specified timethe box, enter the lower flow rate value, and the time durationyou want the system to run at this lower flow rate. Otherwise,leave the box uncheck.

The flow rate can be alterthe separation begins if the lower flow rate is notrequired for the full time duration.

Select the tray(s) that are available for the run bythe appropriate Left or Right Tray box. Collection can start inany available vial by pressing Override Start Vialselecting the desired start vial location (Figure 3

Figu re 3-42: Select a Vial Dialog Box

For Flash dry samples, install the solid sample loaderassembly in the system (Section 4.6.2 )wait until the end of equilibration to load the sample into thesyringe.

For Prep mode liquid injection, wait until the end ofequilibration to load sample into the sample loop. Leave thesyringe in place until after you press thebutton.

Reveleris® PREP Operation Manual, Version B

screen showing the “Flow Rate

Lower flow rate for a specified time feature, check the box, enter the lower flow rate value, and the time duration

to run at this lower flow rate. Otherwise,

The flow rate can be alter ed any time after the separation begins if the lower flow rate is not

Select the tray(s) that are available for the run by checking box. Collection can start in

Override Start Vial and Figure 3 -42).

: Select a Vial Dialog Box

nstall the solid sample loader ). For liquid sample,

wait until the end of equilibration to load the sample into the

For Prep mode liquid injection, wait until the end of he sample loop. Leave the

syringe in place until after you press the Liquid Injection

If the syringe is removed and yoursample waste tubing is placed lower tloop, you may lose some samples due to siphoning.

Press the appropriate button corresponding to the type ofinjection (Dry Sample or LiLiquid Injection for Prep Modeup the Run screen (Figures 3

When the dry sample button is pressedthe Flash mode , user must confirm “loader assembly installed with the tubing connected?” inorder to continue the run.

Figure 3-43: The Flash Mode

Figure 3-44: The Prep Mode Run Screen

3.4.2 COMPONENTS OF THE RU

The Run screen shows the pattern for the fraction collectortrays installed in the instrument in the upper right corner andthe status of the system

26

If the syringe is removed and your sample waste tubing is placed lower t han the sample

some samples due to siphoning.

te button corresponding to the type of e or Liquid Injection for Flash Mode;

for Prep Mode) to initiate the run. This brings 3-43 and 3-44 ).

When the dry sample button is pressed in , user must confirm “ Is the dry sample

loader assembly installed with the tubing connected?” in

Flash Mode Run Screen

The Prep Mode Run Screen

COMPONENTS OF THE RUN SCREEN

screen shows the pattern for the fraction collector trays installed in the instrument in the upper right corner and the status of the system in the lower half. During


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equilibration, a series of messages describing the currentoperation is presented at the bottom of the screen. When thesample is being separated, the chromatogram is displayed onthe graph (Figure 3-45 ) and the fraction collection depictionindicates which tubes are being filled in.

Some method and collection parameters can be edited dthe run. All parameters that can be edited are highlighted ingreen. Parameters that cannot be edited are grayed out.

Figure 3-45: Run Screen During a Run

The gradient condition can be edited anytime during the run.In order to do this on the graph, the Edit/Viewtop right of the graph must be set to Editpresents the gradient table. The edit can also be done via thetable. When the button is set to View, the retention timecolumn volume and the signal size can be viewed by placingthe cursor on the graph.

The Gradient Hold button (Figure 3-46 ) will hold any gradientat the current solvent ratio in an isocratic temporary holdWhen the button is pushed again, the gradient continues tothe original end-point. (Figure 3-46 )operates.

Figure 3-46 : The Gradient Hold Screen

Here the first peak has eluted from the column and a gradienthold is required to form an isocratic gradient at 46% solventB. Two gradient points are shown as the isocrbegins; one shows the start of the isocratic hold and the othershows the moving end point. To end the i


equilibration, a series of messages describing the current the bottom of the screen. When the

sample is being separated, the chromatogram is displayed on ) and the fraction collection depiction

Some method and collection parameters can be edited during the run. All parameters that can be edited are highlighted in green. Parameters that cannot be edited are grayed out.

: Run Screen During a Run

The gradient condition can be edited anytime during the run. Edit/View button on the Edit. The Table button

can also be done via the , the retention time or

e can be viewed by placing

will hold any gradient at the current solvent ratio in an isocratic temporary hold.

the gradient continues to ) shows how this

: The Gradient Hold Screen

Here the first peak has eluted from the column and a gradient an isocratic gradient at 46% solvent

as the isocratic portion the start of the isocratic hold and the other

the moving end point. To end the isocratic hold simply

press the Gradient Hold button again and the run will resumethe original gradient slope (Figure 3

Figure 3-47: Gradient resumed after required isocratichold time

The gradient hold time will be added to the table and the totalrun time will be updated automatically.steps will not be saved in preonly applicable to the current run.press Save Method or Save Method As from the

The button to the left of thepressed to present the list of loaded solvents (and a smaller chromatogram. When the solvent lpresent, a larger arrow will appear which can be used toreturn to the full-scale chromatogram.

The y scale of the detector display can be changed via theOptions button, which presents a dialog box (For smaller peaks, it is necessathe peaks can be seen on the chromatogram.also be used to display/remove the ELSD and UV thresholdsetting on the chromatogram.

Figure 3-48: Graph Options

When the Edit/View button on the top rto Zoom, a region of the chromatogram can be expanded byplacing the cursor at one of the cornersand dragging the stylus/mouse to the far corner of the desired

region. To return to the full plot, press the

When a fraction is being collected, the stripe at the bottom ofthe chromatogram corresponds to the color in the traydepiction for the active tube. If the fraction is collected in twoor more tubes, the color will remain the same, if a differenfraction is collected, the color will change (white indicates thatno fraction is being collected).

27

button again and the run will resume (Figure 3 -47).

: Gradient resumed after required isocratic hold time

The gradient hold time will be added to the table and the total run time will be updated automatically. The isocratic hold

pre-programmed methods and are the current run. To save the new method,

press Save Method or Save Method As from the File menu.

button to the left of the chromatogram can be pressed to present the list of loaded solvents (Figure 3-1 ) and a smaller chromatogram. When the solvent list is present, a larger arrow will appear which can be used to

scale chromatogram.

The y scale of the detector display can be changed via the button, which presents a dialog box (Figure 3-48 ).

For smaller peaks, it is necessary to reduce the scale so that the peaks can be seen on the chromatogram. This box can also be used to display/remove the ELSD and UV threshold setting on the chromatogram.

: Graph Options Dialog Box

button on the top right of the graph is set region of the chromatogram can be expanded by

placing the cursor at one of the corners of the desired region and dragging the stylus/mouse to the far corner of the desired

region. To return to the full plot, press the button.

When a fraction is being collected, the stripe at the bottom of the chromatogram corresponds to the color in the tray depiction for the active tube. If the fraction is collected in two or more tubes, the color will remain the same, if a different fraction is collected, the color will change (white indicates that no fraction is being collected).



The button, which is adjacent to the method name and run name, accesses the information windows where you can annotate the method or run as desired.

If the method or run is saved in the custom folders, the custom folder names are displayed in the title bar at the top during separations or when past runs are viewed (Figure 3-49). The folder name is listed first followed by the corresponding method or run name. Only limited number of characters can be displayed on the screen. If the combination of the method folder name and the method name is too long, only the folder name will be listed for the method. If the combination of the folder name and the run name is too long, only the run name will be displayed on the screen. Both folder and method or run names will be printed completely on the printed report as more space are available on the report.

Figu re 3-49: Location of folder names for Methods and Runs

The Run panel at the lower right corner of the screen is used to control the operation of the system.

The START button is used to start the run or re-start the present operation if the system has been paused.

The PAUSE button stops the present operation. If the system is paused due to an error, this button will change to yellow.

The ADVANCE button advances to the next operation during equilibration. It is not available during a run.

The STOP button terminates the operation of the system.

3.5 THE PAST RUN SCREEN

After a run is complete, the system presents a Run is Complete dialog box (Figures 3-50 and 3-51) that gives the user options to continue the run, air purge the column (Flash mode only), and edit the run notes. Press Done when complete.

Figure 3-50: Flash Mode Run is Complete Dialog Box

Figure 3-51: Prep Mode Run is Complete Dialog Box

The Past Run screen (Figure 3-52 ) is then presented. Press Close to return to the Setup screen.

Figure 3-52: Flash Mode Past Run Screen

3.5.1 ACCESSING THE PAST RUN

To access a stored run, go to the File menu and select Open Run. The Open Run screen appears with runs stored in the Default folder presented (Figure 3-53 ).



Figu re 3-53: Open Run Screen

If the run is stored in a different folder, click on the desired folder and run name, and press OK. The Past Run screen (Figures 3-52 ) is then presented.

3.5.2 COMPONENTS OF THE PAST RUN SCREEN

On the Past Run screen, you can access a dialog box to annotate information about the method or the run by pressing

the button

Any previous run can be viewed via the Open Run command on the File drop down menu.

The past run files can be saved onto a USB Flash drive in a PDF format using the PDF to USB button. See Section 4.11 for more information. Some parameters of the run can be adjusted to improve viewing. For example, the chromatogram y scale of the detector display can be changed via the Options button (See Section 3.4.2 ) of the dialog box. This box can also be used to display/remove the ELSD and UV threshold setting on the chromatogram. A region of the chromatogram can be zoomed using the View button (see Section 3.4.2 ).

When printing or saving a past run in the PDF format, the report will show the chromatogram as seen on the display at the time you print or press the PDF to USB button.



4. SEPARATING A SAMPLE

4.1 OVERVIEW

This chapter is designed to lead the operator through the separation of a sample via the Reveleris® Prep Purification system and includes information about sample handling, preparation of the system for operation and related topics. A detailed discussion of the Reveleris® PREP operating software is presented in Chapter 3 .

4.2 POWERING UP THE SYSTEM

The system includes two power switches, one on the lower right corner of the rear panel and a push button on the display. It is recommended that the switch on the rear panel be left on at all times and the push button on the display is used to power up the system.

When the unit is powered up, a number of self-test procedures are performed and the system is initialized. A

Reveleris® PREP purification system screen is presented during the start-up period which is followed by a Window® software start screen. The software automatically opens to the Setup screen (Figure 4-1 ) and is presented at the conclusion of the initialization procedure. Section 3.2 describes the Setup screen in details. If the trays are installed on the unit during power up, the trays will be automatically detected and the collection vial matrix corresponding to the trays will be presented.

Figure 4-1: The Setup Screen (Flash Mode)



4.3 PREPARING THE SYSTEM FOR OPERATION

The discussion in this section assumes that the system has just been powered up and solvents, etc. have not been selected. If the system is powered up and solvents have been selected, it will not be necessary to perform all of the steps described below.

It is recommended that all operators wear static dissipative shoes while using the instrument. In addition, static dissipative floor mats should be placed in the vicinity of the instrument.

4.3.1 SELECTING SOLVENTS AND PRIMING THE SYSTEM

The Reveleris® PREP purification system includes four solvent lines that can be used to generate a binary gradient. The solvents used to define the gradient can be altered during the separation (e.g., you could use a gradient consisting of A and B for the first 10 minutes of a separation and a gradient consisting of A and C for the last 5 minutes). If desired, you could place the same solvent in two (or more) solvent bottles to reduce the frequency of refilling of solvent lines and/or develop a method with extended solvent capabilities.

The auto prime feature of the Reveleris® PREP purification system simplifies priming. During the auto prime process, the system will pull solvent through pump 1 and pump 2 consecutively, and also verify that each pump is successfully primed. If priming is not successful after the first trial, the process is automatically repeated 2 more times. The Auto Prime feature is accessible through the Solvent Loading screen.

The quantity of flammable solvent that is kept in the immediate vicinity of the system should be minimized to help prevent the spread of fire. All local and institutional regulations regarding the care and handling of flammable solvents, as well as Good Laboratory Practies, should be strictly obeyed.

4.3.1.1 ASSIGNING SOLVENTS TO SOLVENT LINES

The Solvent Loading dialog box (Figure 4-2 ), which is accessed by selecting Solvent Loading under the Tools menu or via the Solvent Selection area of the Setup screen, is used to indicate the solvent delivered by each solvent line and to prime the lines.

It is important to calibrate the solvent safety sensors before starting a run (See Section 4.3.2).


To load the solvent in Line 1 (the process is the same for all solvent lines):

1. Press Load to present the Load and Prime SolventLine 1 dialog box (Figure 4-3 ).

Figure 4-3: Load and Prime Solvent Line 1 Dialog Box

2. Press the Empty field to access a list of solvents thatare commonly used for flash chromatography andselect the solvent that is in the bottle reservoirassociated with line 1 (e.g. Hexane). If desired, youcan add solvents to this list (Section 3.3.2 ).

3. Press Auto Prime to automatically prime the solventline and pumps. The priming status will be displayedat the bottom of the box (Figure 4-4 ).



Figu re 4-4: Load and Prime Solvent Line 1 Dialog Box after a Solvent has been selected and primed

4. After the Auto Prime is completed press Close toreturn to Solvent Loading screen (Figure 4-2) to loadand prime the next solvent line.

5. Repeat steps 1-4 to load solvents in each of the othersolvent lines.

6. When solvent is successfully loaded and primed, thename of the solvent will be displayed in thecorresponding line. A typical solvent region with foursolvents loaded is presented in (Figure 4-5) .

Figure 4-5: Complete Solvent Field

7. To automatically withdraw solvent from anotherreservoir when the first reservoir has been depleted,load the same solvent in another reservoir.

If you are changing the solvent in a solvent line, it may be necessary to use an intermediate solvent if the old solvent and the new solvent are not miscible (iso-propanol is usually a good intermediate solvent). Also, when loading an intermediate solvent, it is necessary to peform auto prime to flush the auto prime fluidic paths with the intermediate solvent before introducing the new solvent.

Some buffers are not soluble in organic solvents. When using buffers, check their solubility in various solvents and use the approprite solvent (for example use water for inorganic buffers) to flush the buffer solution out of the system. Since there are shared fluidic paths for Flash and Prep modes, ensure all path are flushed properly.

Only solvents and mobile phases with volatile stabilizers or modifiers may be used with ELSD. See Section 8.8 for a list of suitable volatile mobile phase modifiers.

When using ethyl ether or Tetrahydrofuran (THF) as the solvent in combination with ELSD detection, nitrogen must be used as the gas source. This eliminates the formation of peroxide and provides stable ELSD baseline.

When using chlorinated solvents (such as chloroform or methylene chloride), flush the pump with IPA at the end of each day to avoid corrosion.

4.3.2 CALIBRATING (ZEROING) THE SOLVENT AND WASTE SAFETY SENSORS

The Solvent Safety Sensor for each solvent line used for the separation should be calibrated before starting a run to ensure that the system pauses the separation and alerts you to add more solvent when needed or if more than one reservoir contains the same solvent, the system will automatically withdraw solvent from the second reservoir. The Waste Safety Sensor should also be calibrated to ensure that the system will pause the separation and alerts you to empty the waste container when needed.

To calibrate the solvent and waste safety sensor, 1. Open the Calibration and Default dialog box (Figure

4-6) under the Tool menu. See Section 3.3.5 for more information.

2. Lift each line out of the reservoir into the air and pressthe Zero button.

3. Replace the line back into the reservoir.4. Repeat for all solvent lines and the Waste line.5. Press Close.

Figure 4-6: Calibration and Default Dialog Box



4.4 INSTALLING A COLUMN IN THE SYSTEM

To avoid leaks or pressure related failu res, follow all manufacturer’s directions to ensure all fittings and connections are tight and secure before using cartridges/columns.

4.4.1 FLASH COLUMNS/CARTRIDGES

The flash chromatography column/cartridge is installed in the column area. To install the column: 1. Lift the column arm head to release the lock

mechanism and pull the arm up. 2. Install the column by pushing the column inlet down

into the luer fitting at the bottom plate. 3. Lower the column arm down over the luer tip, align

it, and firmly press the arm in place to ensure a proper seal.

4. Once the column is installed properly, the correctcolumn name and size will be displayed in the Column field (for columns that are equipped with auto recognition tag) and the default run conditions will be automatically populated. If the column is not recognized, rotate the column until the tag is facing the back of the instrument to face the auto recognition tag reader.

5. For columns that are not equipped with the auto-recognition tag, manually select the column size from the drop down list. The system will then automatically populate default run conditions corresponding to that column size.

The column arm is fitted onto the column by gravity. The operator should make certain that the arm is locked in place to avoid solvent leaks or spills.

Installing or changing the column after the method has been loaded will cause the run conditions to change to the default conditions.

The column RFID reader does not read when in the prep mode. If a Flash RFID tagged column is installed on the system while in the prep mode and then the software is switched to flash mode, the column won’t be detected. The user needs to remove the column and reinstall on the system in order for the column to be detected and the fields to be automatically populated. Best practice is to install the Flash RFID column only when in the flash mode.

BUCHI provides a broad range of flash chromatography columns; these are listed in Section 8.3. As new columns are developed, they will be listed on the BUCHI website (www.buchi.com). Table 4-1 contains a listing of the recommended maximum sample size for each column.

TABLE 4 -1: MAXIMUM SAMPLE SIZE FOR FLASH CHROMATOGRAPHY COLUMNS

Column Size Sample

4g 0.8g

12g 2.4g

24g 4.8g

40g 8.0g

80g 16g

120g 24g

220g 44g

330g 66g

4.4.2 PREP COLUMNS

The prep column is installed in the prep flow path where the column can be set on the bench next to the instrument. Either 1/8” OD tubing or 1/16” OD tubing can be used to connect to and from the column. The tubing and appropriate fittings are included in the accessory kit. Refer to section 2.5.3 for tubing connections to the prep injection valve and bulkhead. To install the column:

1. Connect the tubing from the injection valve “TOCOLUMN” port to the column inlet. The type of fitting you use is dependent on your prep column hardware. In most cases, use the 1/8” SS fitting for 5/16-24 port (P/N 142129492) with 1/8” SS ferrule (P/N 142129493) if 1/8” OD tubing is used. If a1/16” OD tubing is used, use the 1/16” SS fitting (P/N 142110574) with 1/16” SS ferrule (P/N 142110573).

2. Connect the column outlet to the tubing coming outof the “FROM COLUMN” bulk head fitting. Use the same fitting as above to connect to the column outlet. Refer to section 2.5.3 for connections to the bulkhead fitting on the front panel of the instrument.

Once the column is installed properly, enter the column name and description in the Column field and other parameters (flow rate, maximum pressure and etc) as described in section 3.2.1.2.

BUCHI provides a broad range of prep LC columns; these are listed in Section 8.4.

4.5 PLACING COLLECTION TUBES IN FRACTION COLLECTION TRAYS AND INSTALLING THE TRAYS ONTO THE SYSTEM

Place sufficient collection tubes in the collection trays. The first available tube on each tray is always reserved for waste. After the method has been entered, an estimation of the number of tubes needed is displayed at the lower left corner of the Setup screen in the Solvent Selection area (Figure 4-1).

Place the trays in the collection area. There are two grooves that guide the trays during installation. The tab at the back of each tray must fit properly into the optical sensor slot to ensure proper tray detection. The tray recognition tag is used to identify the tube size, number of tubes, and the maximum acceptable volume per tube. Please make



sure that the correct tube size is used in the tray. Once the trays are loaded and recognized by the system, this information will be auto-populated on the Setup screen and the tray depiction will be presented on the display.

Do not manually move the collection arm when installing the trays. If the arm is forcibly moved, it is possible that the tracking of the arm will be disturbed and fractions will not be collected in the appropriate tubes. Use the Fraction Collector Arm Reset (Section 3.3.6.3) to move the arm.

4.6 INTRODUCING A SAMPLE INTO THE SYSTEM

4.6.1 FLASH MODE

4.6.1.1 LIQUID SAMPLES

Liquid samples are introduced into the system manually via a syringe through the luer port located on the right side of the column. The syringe can be filled with excess air to ensure complete delivery of all samples onto the column. The sample is injected into the system at the end of the equilibration, as described in Section 4.8 .

4.6.1.2 SOLID/DRY SAMPLE

For samples that are not readily soluble in the mobile phase, the sample is adsorbed onto a solid phase adsorbent (e.g. silica). Section 8.8 details the procedure to prepare the sample. The adsorbent with the sample is then placed in a solid loader (3-60g capacity) tube. BUCHI also offers pre-packed solid loader tubes. The sample is then introduced into the Reveleris® PREP purification system via the solid sample loader assembly shown in Figure 4-7. The complete assembly consists of a solid loader tube, sleeve and a plunger assembly made up of a plunger rod with o-ring and locking slots, cap plate with release tab and a round knob.

Figure 4-7: Solid Sample Loader Assembly

1. Press in the release tab on the cap plate and slide it allthe way up to the top round knob.

2. Insert the plunger assembly into the packed solid loadertube until the plunger rod rests on top of the media bed.

3. After inserting the plunger rod to the top of the media,press the release tab to slide the cap plate down to thetop of the loader tube until it is flush .

4. This step may create a small gap between media bedand plunger rod which is normal. Using two handsindividually hold the cap plate in one and the top roundknob in the other. Firmly pull the cap plate and topknob AWAY from each other (direction shown indiagram below) to lock the release tab into a lockingslot with an audible click as the tab pops up.



5. Remain holding the cap plate with one hand and useyour other hand to turn the top round knob hardclockwise until it stops to compress o-ring. Verify thatthe o-ring causes a slight bulge in the dry loader whentightened. This should be tight to prevent any leaking.

6. Insert the sleeve over the dry loader. Align the sleevetabs with the open slots on the bottom of the cap plate.

7. Turn the sleeve ¼ turn and ensure that the tabs areunder the lip of the cap plate. This locks the sleeve intothe dry loader assembly.

8. The completed dry loader assembly is now ready toattach to the instrument.

9. To attach the assembled dry loader to the Reveleris®

Prep purification system, simply place completedassembly onto the injection port on the Reveleris® Preppurification system and turn the sleeve clockwise to lockit in place. Next, connect the dry loader connectiontubing (attached to the left side panel of the instrument)to the top fitting of the dry loader assembly. The sampleis now ready for purification.

4.6.2 PREP MODE

In the prep mode, liquid samples are introduced into the sample loop manually via a syringe. The sample is injected into the system at the end of the equilibration, as described in Section 4.8.

4.7 SELECTING OR DEVELOPING A METHOD

4.7.1 SELECTING AN EXISTING METHOD

If a method exists that contains the conditions that you expect to use, select Open Method on the File menu to present a list of the methods (Figure 4-8 ). The software will automatically load the Default folder for methods. If the method is stored in a custom folder, press the drop down tab under the Choose folder to display previously created method folders. Click on the desired method folder, then select the method, and press OK to load.

Figure 4-8: Open Method Dialog Box

When opening a method that was saved in a different mode than the instrument is currently in, the



Mode Change screen will be displayed for you to confirm that you want to switch mode.

All parameters of the Prep method will be loaded. For Flash methods, everything will be loaded except for the Column selection. The column type is determined by either the auto-column recognition technology for columns equipped with a recognition tag or manually selected from the generic drop down menu.

If the existing Flash method is loaded/opened before a column is installed onto the instrument, the method parameters that are column dependent will change after the column is installed. Please review the method before starting the run as it may differ from the original method.

The column’s maximum pressure for prep runs is auto populated based on set flow rate. Once the max pressure value is manually altered, the auto-populate of maximum pressure will no longer functions when the flow rate is changed. The user will have to manually change the max pressure. This is also the case when loaded a saved method that has maximum pressure manually altered.

All method parameters can be edited and, if changes are made, it is suggested that the new method be saved via the Save Method As command on the File menu using a different file name.

If an existing method is edited, a “*” will be displayed at the end of the method name (e.g. Flash-RP U-P-BP*) to denote the original method has been modified. The “*” will be displayed until the method changes are saved.

The Per-Vial Volume parameters are dependent on the tray(s) in the system as determined by the auto-tray recognition technology. The Peak and Non-Peaks fields are defaulted to the maximum volume of the vial.

A typical set-up screen from a retrieved Flash method is shown in (Figure 4-9).

Figure 4-9: A Typical Set-up Screen

4.7.2 GENERATING A NEW METHOD

The discussion that follows presents the steps that must be performed if a new method is to be designed (i.e. the instrument has just been powered up with a column already installed and for flash mode, the column has already been detected/selected from the drop down list. For prep mode, the column name and description has already been entered).

To generate a new method:

1. Enter the chromatographic conditions (Section 3.2.1.1for Flash mode and 3.2.1.2 for Prep mode) if theydiffer from the default parameters.

Column equilibration is not required and the equilibration time can be set to zero if desired. The equilibration field will then turn yellow to warn the user that the fraction collector arm will not be flushed.

If zero equilibration is chosen, please make sure that the solvent type left in the fraction collector arm from the previous run will not affect the current run.

2. Select the detector(s) to be employed (Section 3.2.2 ).

If ELSD is employed, there is a 1 minute stabilization period required regardless of column equilibration time. The ELSD stabilization occurs in tandem with column equilibration. However, if the column equilibration time is less than 1 minute, the sample injection will not start until the ELSD stabilization is complete.

3. Select the detection option(s) to trigger fractioncollection (Section 3.2.3 ). Either Slope, Threshold, orboth can be chosen.

4. Select the fraction collection protocol (Section 3.2.4 ).The Per-Vial Volume is dependent on the tray that isemployed. The Peak and Non-Peaks vial volume willdefault to the maximum allowable volume for thevial/tray placed into the system.

5. Select the solvent for each of the solvent lines bypressing on the Solvent Line field adjacent to the letterthat you will use to define the solvent concentration inthe gradient (Section 3.2.5 ).

6. Generate the desired gradient (Section 3.2.6 ).



4.8 STARTING THE SEPARATION

Press the button to start the separation. The display will prompt for a run name (Figure 4-10 ).

If a generic column is used for the flash separation, or you are starting a prep run, the Set Run Name dialog box will be displayed only after the user confirmed that the column is installed.

New runs can be saved into the Default folder or a new folder can be created (Section 3.4.1)

Figure 4-10: Set Run Name Dialog Box

The default run name is yyyy-mo-da_hh-mm-ss (e.g. 2014-03-12_14-23-10)) and it will appear in the upper right hand corner of the screen during the run. Any desired run name can be entered (if you press on the Enter run name field, a QWERTY keyboard will be displayed). Press OK.

When the OK button is pressed, the Sample Loading dialog box will be displayed (Figures 4-11 and 4-12).

Figure 4-11: Flash mode Sample Loading Dialog Box

Figure 4-12: Prep Mode Sample Loading Dialog Box

To use the Lower flow rate for a specified time feature (Section 3.4.1 ), check the box, enter the lower flow rate


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value, and the time duration you want the system to run atthis lower flow rate. Otherwise, leave the box uncheck.

The flow rate can be altered any time afterthe separation begins if the lower flow rate is notrequired for the full time duration.

Anytime the flow rate is lowered duringthe run (automatically or manually setflow rate for specified time feature), the flow rate field willchange color to orange. After the run is completed, amessage “Flow Rate Altered” will be displayed onpast run screen (Figure 3- 41) and also on the printedreport.

Select the tray(s) that are available for the run by checkingthe appropriate Left or Right Tray box. Collection can start inany available vial by pressing Override Start Vialselecting the desired start vial location (Figure

Figure 4-13: Select a Vial Dialog Box

The first vial location will always be usedto collect waste solvent from the fraction collector armrinsing at the beginning of each run.

For Flash liquid sample, wait until the end of equilibration toload the sample into the syringe. For dry samples, install thesolid sample loader assembly in the system4.6.1.2).

For Prep mode liquid injection, wait until the end ofequilibration to load sample into the sample loop. Leave thesyringe in place until after you press thebutton.

If the syringe is removed and yoursample waste tubing is placed lower than the sampleloop, you may lose some samples due to siphoning.

Press the appropriate button corresponding to the type ofinjection to initiate the run.


value, and the time duration you want the system to run at te. Otherwise, leave the box uncheck.

The flow rate can be altered any time after the separation begins if the lower flow rate is not

Anytime the flow rate is lowered during the run (automatically or manually set using the Lower

feature), the flow rate field will change color to orange. After the run is completed, a message “Flow Rate Altered” will be displayed on the

41) and also on the printed

elect the tray(s) that are available for the run by checking box. Collection can start in

Override Start Vial and Figure 4-13).

Select a Vial Dialog Box

The first vial location will always be used to collect waste solvent from the fraction collector arm

liquid sample, wait until the end of equilibration to e syringe. For dry samples, install the

e loader assembly in the system (Section

For Prep mode liquid injection, wait until the end of equilibration to load sample into the sample loop. Leave the

ess the Liquid Injection

If the syringe is removed and your sample waste tubing is placed lower than the sample loop, you may lose some samples due to siphoning.

Press the appropriate button corresponding to the type of

Dry Samplestart automatically at the endLiquid Injection for flash or prep modeinject the sample after columnstart to resume the separation.

The display for Liquid Injection(Figures 4-14 and 4-15) . The system pauses at the end ofequilibration and prompts user to inject the sample.the start button to resume the separation.

Figu re 4-14: The Flash ModeInjection

Figure 4-15: The Prep Mode Run Screen for LiquidInjection

Once the separation starts, the chromatogram will bedisplayed (Figures 4-16 and 4will be displayed at the bottom of the screen and wupdated as relevant. A detailed discussion of thewas presented in Section 3.4.2

The maximum pressure for the system in the Flash mode is200psig (100psig for 330g Revelerisequipped with a recognition tag

38

Dry Sample injection in Flash mode will at the end of column equilibration.

for flash or prep mode prompts user to after column equilibration and press

to resume the separation.

Injection will appear as shown in The system pauses at the end of

equilibration and prompts user to inject the sample. Press the start button to resume the separation.

Flash Mode Run Screen for Liquid Injection

: The Prep Mode Run Screen for Liquid Injection

Once the separation starts, the chromatogram will be and 4-17). The status information

will be displayed at the bottom of the screen and will be A detailed discussion of the Run screen

Section 3.4.2 .

imum pressure for the system in the Flash mode is 200psig (100psig for 330g Reveleris® columns) for columns equipped with a recognition tag and 75psig for generic


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columns. In the prep mode, the maximum pressure isinversely proportional to the set flow rates, ranges from800psi at 200mL/min to 1700psi at flow rates of 75less.

The system will automatically reduce theflow rate incremen tally when the system pressurereaches the pressure limit. This will continue until thepressure remains below the limit or thebecomes too low to continue the run . If this occurs,pump will pause and a pressure warning appears.run is not paused and the pressure becomeslowered flow rate, the s oftware will automaticallyincrease the flow rate incrementally back to the originalsetting as long as the pressure remainsmaximum pressure limit.

If the p ressure build up is downstreamfrom the second pressure sensor exceeds 150psig) , the system will automatrun and issue a warning.

Figure 4-16: The Flash Mode Run Screen During a Run


In the prep mode, the maximum pressure is the set flow rates, ranges from

1700psi at flow rates of 75mL/min or

The system will automatically reduce the tally when the system pressure

reaches the pressure limit. This will continue until the pressure remains below the limit or the reduced flow rate

. If this occurs, the and a pressure warning appears. If the

run is not paused and the pressure becomes stable at the oftware will automatically

the flow rate incrementally back to the original remains below the

ressure build up is downstream pressure sensor P2 (the pressure

, the system will automat ically pause the

Screen During a Run

Figure 4-17 : The Prep Mode R

The Run panel at the lower right corner of the screen is usedto control the operation of the system.

The START buttonstart the present operation if the system has been paused

The PAUSE buttonthe system is paused due to an error or by request, thisbutton will change to yellow.

The ADVANCE buttonoperation during equilibration. It is not available during a run.

The STOP buttonsystem.

During a run, any parameter indicated in green (e.gdetection threshold(s)) and the fields that are not grayedcan be edited except for gradient steps that have alreadyoccurred. Any detection trace can be turned on/off bychecking/unchecking the box next to the detectors on top ofthe chromatogram plot as long as they were initially turned onin the Method Setup.

Pressing the Options button will open a Graph Options box(Figure 3-48). Using the ELSD or UV field, the maximumviewable scale can be set independently. Threshold settingscan be viewed on the chromatogram by pressing thecorresponding buttons. To remove the threshthe chromatogram, press the button again.

If all vials have been filled during a separation, the separationwill PAUSE and the button will turn yellow. To continueseparation, place a new tray filled with empty vials into thesystem. Press START to resume the run.

Each individual tray may be used multipletimes during a run after confirming that all vials are empty.

The separation is complete after the setvolume (for Flash mode) is met. If the system detects sign(compound eluting out of the column) at the end ofthe run will be extended automatically for an additional 5minutes.

After the separation is complete,Run is Complete dialog box will be presentedand 4-19 ). The Continue Rcontinue the run if the compound of interest has not yeteluted out of the column. additional 5 minutes at the last solvent condition.manually ended by pressing the STOP button, theRun button is grayed out.

During the run, the conditions can be edited.the run in the flash mode, the user can

39

: The Prep Mode R un Screen During a Run

panel at the lower right corner of the screen is used to control the operation of the system.

The START button is used to start the run or re-start the present operation if the system has been paused.

The PAUSE button stops the present operation. If the system is paused due to an error or by request, this

The ADVANCE button advances to the next on. It is not available during a run.

The STOP button terminates the operation of the

During a run, any parameter indicated in green (e.g., the and the fields that are not grayed-out

cept for gradient steps that have already trace can be turned on/off by

checking/unchecking the box next to the detectors on top of the chromatogram plot as long as they were initially turned on

button will open a Graph Options box the ELSD or UV field, the maximum

viewable scale can be set independently. Threshold settings can be viewed on the chromatogram by pressing the corresponding buttons. To remove the threshold setting from the chromatogram, press the button again.

If all vials have been filled during a separation, the separation will PAUSE and the button will turn yellow. To continue separation, place a new tray filled with empty vials into the

ess START to resume the run.

Each individual tray may be used multiple times during a run after confirming that all vials are empty.

The separation is complete after the set Run Length time or is met. If the system detects signal

(compound eluting out of the column) at the end of the run, automatically for an additional 5

After the separation is complete, the run will be paused and a dialog box will be presented (Figures 4-18 Continue Run button allows the user to if the compound of interest has not yet

eluted out of the column. The run will continue for an additional 5 minutes at the last solvent condition. If the run is

sing the STOP button, the Continue

During the run, the conditions can be edited. Before ending the user can initiate a column air



purge by entering the time and pressing the Column Purge button. This feature is grayed out in the prep mode (Figure 4-19). Run notes can be edited using the Edit Run Notes button.

Figure 4-18: Flash Mode Run is Complete Dialog Box

Figure 4-19: Prep Mode Run is Complete Dialog Box

To end the run, press Done to close the dialog box. This leads to the Post Run screen (Figure 4-20) on the display.

Figure 4-20: The Flash Mode Post Run Screen

To annotate the run or the method, press the button by the run name or method name while viewing a past run.

If desired, you can view previous runs (i.e. to compare separations) by selecting Open Run under the File menu.

4.9 PERFORMING ADDITIONAL SEPARATIONS

After a separation has been completed, additional separations can be performed by pressing the Close button on the Post Run screen, which will present the Setup screen again. To perform another separation, set up the column and

sample as described previously and press the

button. The system will use the existing method to separate the sample. Alternately, another method can be generated or recalled as described previously (Section 3.2.9 ).

The Solvent Loading dialog box (Section 4.3.1.1 ) assists the user in maintaining the system. If a solvent has been defined, the drop-down menu obtained by pressing the solvent name allows for changing the solvent or re-priming the solvent line.

Ensure that the new solvent is miscible with the old solvent. If the two solvents are not miscible, use an intermediate solvent such as iso-propanol to remove the original solvent. Also, when loading an intermediate solvent, it is necessary to peform auto prime to flush the auto prime fluidic paths with the intermediate solvent before introducing the new solvent.

Some buffers are not soluble in organic solvents. When using buffers, check their solubility in various solvents and use the approprite solvent (for example use water for inorganic buffers) to flush the buffer solution out of the system. Since there are shared fluidic paths for Flash and Prep modes, ensure all path are flushed properly.

If you change from a normal phase method to a reverse phase method, it is possible that the solvents are not miscible. The NP <> RP command under the Tools menu presents a dialog box that provides the instructions to properly flush the system. This feature can be used in either Flash or Prep Mode. Follow the instructions in the pop up box carefully as the steps are different for each mode (Figures 4-21 and 4-22 ).

Figure 4-21: Flash Mode NP<>RP Dialog Box


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Figure 4-22 : Prep Mode NP<>RP Dialog Box

NP<>RP feature is available in both flashand prep modes to flush the corresponding fluidic flowpath. Please follow the instructions in the pop upcarefully to ensure proper bypass column or tubing isinstalled.

4.10 PRINTING

Post Run files can be printed directly from the system to aconnected printer. (A printer must first be connected to theReveleris® Prep purification system in thescreen described in (Section 3.3.4 ). Open a past run(Section 3.5.1 ) and press Print Run Reportmenu. Select the desired printer and press OK to print report.

4.11 SAVING RUN REPORT IN PDF FORMAT TOUSB STORAGE/FLASH DRIVE

Post Run files can be saved onto a USB flash drive/memorystick using the “PDF to USB” button.

To save a chromatography run, insert a USB memory stickinto one of the convenient USB ports on the side of theReveleris® PREP purification system screen. Ensure thesystem recognizes the memory stick, either by a flashing lighton the stick or by going to “My Computer” on the RevelerisPREP purification system screen.

Once the memory stick has been recognized by the system,press the “PDF to USB” button, circled in the file will transfer as a PDF.


: Prep Mode NP<>RP Dialog Box

NP<>RP feature is available in both flash and prep modes to flush the corresponding fluidic flow path. Please follow the instructions in the pop up

proper bypass column or tubing is

files can be printed directly from the system to a connected printer. (A printer must first be connected to the

system in the Configuration ). Open a past run

Print Run Report under the File menu. Select the desired printer and press OK to print report.

PDF FORMAT TO

files can be saved onto a USB flash drive/memory

To save a chromatography run, insert a USB memory stick into one of the convenient USB ports on the side of the

system screen. Ensure the system recognizes the memory stick, either by a flashing light on the stick or by going to “My Computer” on the Reveleris®

Once the memory stick has been recognized by the system, circled in (Figure 4-23) , and

Figure 4-23: Prep Mode PDF

After the file has been transferred, a dialog box will open inthe screen stating the file has been successfully(Figure 4-24). The USB memory stick can now be removedand the files can be transferred to another computer andopened as a PDF file.

Figure 4-24: Prep Mode PDF to USB file transfer screen

When using the PDF to USB under the run name. If the same file name is already exist onthe USB Flash drive, the file name will increase incrementally.For example, if the run name is 3pk std mix run 1a, the filewill be saved as 3pk std mix run 1a.pdf. The second time thesame run is saved via PDF to USBwill be 3pk std mix run 1a (2).pdf.

After the file has been saved, press theDialog Box to get back to the Post Run screen. Then closethe Post Run screen to continue with a srun.

The PDF files appear on the memory stick as shownFigure 4-25 . Simply click on the icon to open and view therun report in PDF format.

41

Mode PDF to USB screen

After the file has been transferred, a dialog box will open in the screen stating the file has been successfully saved.

USB memory stick can now be removed and the files can be transferred to another computer and

Mode PDF to USB file transfer screen

button, the file will be saved under the run name. If the same file name is already exist on the USB Flash drive, the file name will increase incrementally. For example, if the run name is 3pk std mix run 1a, the file will be saved as 3pk std mix run 1a.pdf. The second time the

PDF to USB button, the new file name will be 3pk std mix run 1a (2).pdf.

After the file has been saved, press the Close button in the Dialog Box to get back to the Post Run screen. Then close the Post Run screen to continue with a second or subsequent

The PDF files appear on the memory stick as shown in click on the icon to open and view the



Figu re 4-25: PDF Files on Memory Stick

The open file will be viewed as shown in (Figure 4-26) . The run parameters, chromatogram, and tray mapping are displayed on the report. For easy identification of the fractions, vial numbers are printed on the tray mapping and the Vial Mapping Table listed the tray and vial location for the start and end of each peak.

Figure 4-26: Printed or PDF format Run Report

4.12 POWERING DOWN THE SYSTEM

From the Setup screen, go to File menu and choose Exit. This will take you out of the Reveleris® PREP software after you press Yes to the Exit screen (Figure 4-27). Then, press the Windows start button on the lower left corner to shut down the system.

Figure 4-27: Exit screen


www.buchi.com

5. REVELERIS® NAVIGATORSOFTWARE

The Reveleris® Navigator™ software is an advanced methodoptimizer designed to save you time and money with yourpurification challenges. It provides optimum gradientconditions based on input from other scouting methods suchas TLC and LC. To access the Revelerissoftware, press the Navigator™ button located above thegradient graph.

Figure 5-1: Reveleris® NAVIGATOR™

The Navigator ™ software is available in theflash mode only.

Contact your Grace

Figure 5-2: Reveleris® Navigator™ Software PBox

To enter the password, touch the password field and akeyboard will be displayed. Enter the password and pressOK. This brings up the Reveleris® Navigatorscreen.


NAVIGATOR™

is an advanced method optimizer designed to save you time and money with your purification challenges. It provides optimum gradient conditions based on input from other scouting methods such

Reveleris® Navigator™ button located above the

™ Software button

software is available in the

instrument specific password is requiredTo activate the Reveleris® Navigator™ ssoftware feature, an instrument specific password is required. Contact your BUCHI Service Representative to obtain the password.

Software P assword

To enter the password, touch the password field and a rd will be displayed. Enter the password and press

Navigator™ software

Three tabs are provided: i. TLC-Silica – uses data from TLC to provide gradient

conditions for normal phase separations of 2 or 3components

ii. LC-C18 – uses data from isocratic HPLC separationto provide a recommended gradientphase separations of 2 components.

iii. LC-Transfer – converts an HPLC gradient (smallscale) into a Reveleris

5.1 TLC-SILICA

The TLC-Silica Tool uses TLC separation data to provide arecommended gradient for silica separations of 2 or 3components.

Just below the tabs is a blue information bar that is presenton all the screens of the Revelerissection. Short instructions on use or notifications of issuesthat have been detected can be found in this bar. To seemore Information on the topic press the

5.1.1 INPUT DATA

To use this tool, separations of the sample componentsto be obtained using two Revelerisdifferent solvent concentrations. Calculate the Rf values ofeach spot by measuring the distance the spot moved anddividing this by the distance the solvent traveled.

Enter the information from these separations into theConditions section of the TLC3).

An exclusive feature of the Revelerisis the ability to optimize the separation of 3 components whenusing the TLC-Silica tool. Selection of 2 components or 3components using the radio buttons provides the correctnumber of input boxes for the Rf data.

Choose the solvents used froweaker solvent, B is the stronger solvent. Above the platedepictions, enter the solvent concentrations used in the TLCseparations. Along side of each plate depiction, enter the Rfvalues for each spot keeping the depicted othat on the actual plate.

Figure 5-3: TLC -

43

uses data from TLC to provide gradientconditions for normal phase separations of 2 or 3

uses data from isocratic HPLC separationto provide a recommended gradient for reversed phase separations of 2 components.

converts an HPLC gradient (smallReveleris® gradient (larger scale).

Tool uses TLC separation data to provide a recommended gradient for silica separations of 2 or 3

Just below the tabs is a blue information bar that is present Reveleris® Navigator™ software

section. Short instructions on use or notifications of issues that have been detected can be found in this bar. To see more Information on the topic press the Learn More button.

To use this tool, separations of the sample components need to be obtained using two Reveleris® silica TLC plates with two different solvent concentrations. Calculate the Rf values of each spot by measuring the distance the spot moved and dividing this by the distance the solvent traveled.

ion from these separations into the TLC section of the TLC-Silica Input Screen (Figure 5-

Reveleris® Navigator™ software is the ability to optimize the separation of 3 components when

Silica tool. Selection of 2 components or 3 components using the radio buttons provides the correct number of input boxes for the Rf data.

Choose the solvents used from the dropdown lists. A is the weaker solvent, B is the stronger solvent. Above the plate depictions, enter the solvent concentrations used in the TLC separations. Along side of each plate depiction, enter the Rf values for each spot keeping the depicted order the same as

-Silica Input Screen



For best results, use Reveleris ® silica TLC pla tes. The silica on these plates closely matches that in the Reveleris ® silica cartridges.

Rf values of 0 or 1 are not acceptable.

Rf1 must be greater than Rf2 for a given solvent concentration.

The Rf of a component in the stronger solvent system (higher %B) is larger than it is in the weaker solvent system (lower %B).

5.1.2 FLASH CONDITIONS

Select the silica column/cartridge to be used for the Flash separation from the dropdown list. The default flow rate recommended for the column will be displayed in the Flow Rate field however, it may be adjusted as desired.

Another unique feature of the Reveleris® Navigator™ software is the ability to select two separation modes. Selecting Speed optimizes the gradient to provide good separation with faster elution of the components. Selecting Purity optimizes the gradient to provide better resolution.

If the 3 components button is selected, Speed and Purity modes are not available.

Checking the box Continue gradient to 100% adds a ramp to 100% to the end of the recommended gradient.

5.1.3 INPUT SCREEN CONTROL BUTTONS

The Reset button clears all data entered into the screen. The Close button returns to the Setup Screen and preserves all data that was entered. The Calculate button initiates the Reveleris® Navigator™ software routine and leads to the output screen for the TLC-Silica Tool.

5.1.4 OUTPUT SCREEN

After pressing the Calculate button, if there are no elution issues, the output screen will be displayed (Figure 5-4 ).

The top portion of this screen shows the recommended gradient table and graph.

The bottom portion contains controls that can be adjusted without reentering the TLC data. Adjustments of these controls will change the table and graph.

Figure 5-4: TLC-Silica Output Screen

5.1.5 OUTPUT SCREEN CONTROL BUTTONS

The Close button returns to the Setup Screen. The data entered are not lost. Go Back returns to the previous screen. Accept downloads the gradient into the Setup Screen after a quick check to determine if the correct cartridge is loaded in the instrument.

5.1.6 ACCEPT GRADIENT

If the Reveleris® cartridge installed on the instrument matches the Reveleris® cartridge listed in the Column field, pressing the Accept button transfers the gradient condition into the Setup Screen. Otherwise, it will lead to a screen that requires the correct cartridge be installed before proceeding (Figure 5-5).

Figure 5-5: Check for Correct Reveleris ® Column

Insert the cartridge and press Continue to load the gradient into the Setup Screen.

If a Generic cartridge is chosen in the Column field, pressing the Accept button leads to a warning screen (Figure 5-6 ). Please confirm that the cartridge specified in the Column field is installed on the instrument. If not, install the specified



column. Then, press Continue to transfer the gradient conditions to the Setup Screen (Figure 5-7 ).

Figure 5-6: Check for Correct Generic Column

Figure 5-7: Setup Screen with ACCEPTED Gradient

5.1.7 GRADIENT ISSUES

After pressing the Calculate button, the Reveleris® Navigator™ software performs several checks before providing a gradient condition for optimum separation. If elution issues exist, several screens or messages will be displayed to describe the issue and recommended solution.

In cases where it may difficult to elute the compounds of interest, an Alert screen will be displayed (Figure 5-8 ). In this situation, it is strongly recommended that a new solvent pair is chosen and rerun the TLC separation

Figure 5-8: Alert for Difficult Separation

Press Close to remove the Alert or press Solvents to view a list of possible solvents that may be tried (Figure 5-9 ). Choosing a pair with higher solvent strength should allow elution of the components. New TLC data will need to be generated with the chosen pair.

Figure 5-9: Suggested Normal Phase SOLVENTS

The Solvents screen provides a list of possible solvent combinations. The user must decide which to try and then collect the appropriate TLC data.

Another issue is poor separation of components. If the Reveleris® Navigator™ software detects that there may be resolution problems, a message will be displayed in the blue information bar (Figure 5-10 ). In some cases the Speed and Purity buttons will also be disabled (Figure 5-11 ). Pressing Learn More will give an Alert (Figure 5-12 ). Press Accept to transfer the recommended gradient to set-up screen or chose a different solvent pair as above and rerun the TLC with these solvents.



Figu re 5-10: Poor Separation Message (Speed and Purity Allowed)

Figure 5-11: Poor Separation Message (Speed and Purity Blocked)

Figure 5-12: Alert for Poor Separation

5.2 LC-C18

The LC-C18 Tool uses isocratic HPLC separation data to provide a recommended gradient for reversed phase separations of 2 components.

Just below the tabs is a blue information bar that is present on all the screens of the Reveleris® Navigator™ software section. Short instructions on use or notifications of issues that have been detected can be found in this bar. To see more information on the topic press the Learn More button.

5.2.1 INPUT DATA

To use this tool, separations of the sample components need to be obtained in two isocratic runs on a Reveleris® C18 HPLC column with different mobile phase solvent concentrations. Obtain the retention times (t1 and t2) of the components from the chromatograms.

The void time (t0) may be measured using a void marker in the sample mix or a default value shown on the screen may be used.

Enter the information from these separations into the HPLC Conditions section of the screen.

Select the HPLC column from the dropdown list and enter the HPLC flow rate used. The default t0 value is calculated each time the HPLC column is selected from the dropdown list or each time the HPLC flow rate value is changed.

Choose the solvents used from the dropdown lists. A is the weaker solvent, B the stronger solvent. Above the chromatogram depictions, enter the solvent concentrations used in the HPLC separations.

Enter the retention time (t1 and t2) for each separation under each chromatogram. To override the default t0, enter a measured t0 into the corresponding boxes.

Figure 5-13: LC-C18 Input Screen



If different t0 values are entered only the smaller value is used.

t2 must be greater than t1 for a given solvent concentration.

The retention time of a component in the stronger solvent system (higher %B) is smaller than it is in the weaker solvent system (lower %B).


Select the C18 column/cartridge to be used for the Flash separation from the dropdown list. The default flow rate recommended for the column will be displayed in the Flow Rate field, however, it may be adjusted as desired.

Only Reveleris ® C18 HPLC columns and Reveleris ® C18 flash cartridges may be used. The phase in the HPLC column must match the phase in the flash cartridge to obtain accurate results.

A unique feature of the Reveleris® Navigator™ software is the ability to select two separation modes. Selecting Speed optimizes the gradient to provide good separation with faster elution of the components. Selecting Purity optimizes the gradient to provide better resolution.

Checking the box Continue gradient to 100% adds a ramp to 100% to the end of the recommended gradient.


The Reset button clears all data entered into the screen. The Close button returns to the Setup Screen and preserves all data that was entered. The Calculate button initiates the optimization routine and leads to the output screen for the LC-C18 Tool.

5.2.4 OUTPUT SCREEN

After pressing the Calculate button, if there are no elution issues, the output screen will be displayed (Figure 5-14 ).

The top portion of this screen shows the recommended gradient table and graph.

The bottom portion contains controls that can be changed without reentering the HPLC data. Adjustments of these controls will change the table and graph.

Figure 5-14: LC-C18 Output Screen


The Close button returns to the Setup Screen. The data entered is not lost. Go Back returns to the previous screen. Accept downloads the gradient into the Setup Screen after a quick check to determine if the correct cartridge is loaded in the instrument.


If the Reveleris® cartridge installed on the instrument matches the Reveleris® cartridge listed in the Column field, pressing the Accept button transfers the gradient condition into the Setup Screen. Otherwise, it will lead to a screen that requires the correct cartridge is installed before proceeding (Figure 5-15).



5.2.7 GRADIENT ISSUES

After pressing the Calculate button, the Reveleris® Navigator™ software performs several checks before providing a gradient condition for optimum separation. If



elution issues exist, several screens or messages will be displayed to describe the issue and recommended solution.

In cases where it may difficult to elute the compounds of interest, an Alert screen will be displayed (Figure 5-16 ). In this situation, it is strongly recommended that a new solvent pair is chosen and rerun the HPLC separation

Figure 5-16: Alert for Difficult Separation

Press Close to remove the Alert or press Solvents to view a list of possible solvents that may be tried (Figure 5-17 ). Choosing a pair with higher solvent strength should allow elution of the components. New HPLC data will need to be generated with the chosen pair.

Figure 5-17: Suggested Reversed Phase SOLVENTS

The Solvents screen provides a list of possible solvent combinations. The user must decide which to try and then collect the appropriate HPLC data.

Another issue is poor separation of components. If the Reveleris® Navigator™ software detects that there may be resolution problems, a message will be displayed in the blue information bar (Figure 5-18 ). In some cases the Speed and Purity buttons will also be disabled (Figure 5-19 ). Pressing Learn More will give an Alert (Figure 5-20 ). Accept the recommended gradient or chose a different solvent pair as above and rerun the HPLC with these solvents.

Figure 5-18 : Poor Separation Message (Speed and Purity Allo wed)

Figure 5-19: Poor Separation Message (Speed and Purity Blocked)



Figu re 5-20: Alert for Poor Separation

5.3 LC-TRANSFER

The LC-Transfer tool converts an HPLC gradient into a Reveleris® Flash Chromatography gradient.

Just below the tabs is a blue information bar that is present on all the screens of the Reveleris® Navigator™ software section. Short instructions on use or notifications of issues that have been detected can be found in this bar. To see more Information on the topic press the Learn More button.

5.3.1 INPUT DATA

To use this tool, input information from an HPLC gradient that is suitable to be transferred into a flash chromatography into the LC-Transfer Input Screen (Figure 5-21 ).

Figure 5-21: LC-Transfer Input Screen

Under the HPLC Conditions, select the HPLC column dimension from the dropdown list. Enter the flow rate used in the HPLC separation. This will generate an estimated HPLC t0 (void time). If there is a more accurate void time available, it can be entered in place of this estimated value.

If “other” is selected as the column dimension, the void time must be entered

Enter the HPLC gradient conditions into the entry boxes of the typical gradient diagram on the screen. Enter the lower and higher %B. Enter the values for t1, t2, and t3. These time (t1, t2, and t3) values are the times at which the events occurred, not the duration between events.

The values (t1, t2 and t3) are the times at which the events occur not the duration between events.


Select the column/cartridge to be used for the Flash separation from the dropdown list. The default flow rate recommended for the column will be displayed in the Flow Rate field; however, it may be adjusted as desired.

The cartridge selected should correspond to the type of HPLC column used so that the separations are similar.


The Reset button clears all data entered into the screen. The Close button returns to the Setup Screen and preserves all data that was entered. The Calculate button initiates calculations and leads to the output screen for the LC-Transfer Tool.

5.3.4 OUTPUT SCREEN

After pressing the Calculate button, the output screen will be displayed (Figure 5-22 ). The recommended gradient table and graph are shown on the top portion of the screen.

The bottom portion contains controls that can be changed without reentering the HPLC gradient. Adjustments of these controls will change the table and graph.

Figure 5-22: LC-Transfer Output Screen




The Close button returns to the Setup Screen. The data entered are not lost. Go Back returns to the previous screen. Accept downloads the gradient into the Setup Screen after a quick check to determine if the correct cartridge is loaded in the instrument.


If the Reveleris® cartridge installed on the instrument matches the Reveleris® cartridge listed in the Column field, pressing the Accept button transfers the gradient condition into the Setup Screen. Otherwise, it will lead to a screen that requires the correct cartridge is installed before proceeding (Figure 5-23).



If a Generic cartridge is chosen in the Column field, pressing the Accept button leads to a warning screen (Figure 5-24 ). Please confirm that the cartridge specified in the Column field is installed on the instrument. If not, install the specified column. Then, press Continue to transfer the gradient conditions to the Setup Screen.

Figure 5-24: Check for Correct Generic Column



6. TROUBLESHOOTING

Perform all trouble shooting acti vities safely, in accordance with all applicable federal, state and local laws and regulations and in compliance with your organization’s safety program.

Unit contains a class II laser and UV light sour ce. Troubleshooting and maintenance on these parts of the equipment must be performed by trained personnel only.

6.1 INTRODUCTION

Troubleshooting refers to the steps to determine the cause of a problem. The Reveleris® PREP purification system consists of several integrated components that include a solvent delivery system, injection system, cartridge/column assembly, UV detector, ELSD, and a fraction collector. An important aspect of troubleshooting is to determine which system is responsible for the problem. As an example, if the peaks from a sample are smaller than what is expected, the following problems might be the cause:

• The UV detector cell might be dirty.

• The syringe did not introduce the desired amount ofsample into the cartridge.

• The injector valve is not delivering the entire sample.

The most efficient method of determining the cause of the problem is to perform a systematic investigation into each potential cause.

While some problems are instrumental in nature, others are sample related. As part of a troubleshooting protocol, it is useful to develop a standard sample and separate it on a periodic basis. The results should be compared to previously obtained results; if small differences are obtained; it may be possible to highlight a problem before it becomes serious. As an example, a small decrease in the signal from what is expected might be due to the contamination of the cell in the UV detector. An additional benefit of the use of a standard sample is that it can help distinguish between instrumental problems and sample/solvent issues.

If any operating parameter appears to differ significantly from that which was obtained in a previous run, the cause for the difference should be looked into before additional runs are performed.

6.2 SOFTWARE AND FIRMWARE ERRORS

In the event of a suspected firmware or software error:

• Log any error messages

• Reboot the instrument and then attempt to operate thesystem

• Contact your BUCHI Service Representative ifrebooting the system does not correct the problem.

When you contact BUCHI, please provide as much information as possible relating to the problem, including what the system was doing before the problem was observed, detailed error messages (the exact message would be best), any abnormal events that were noted, anything which you feel may be of assistance in troubleshooting, the system serial number and software revision. If the error message or problem can be reproduced, please indicate all actions that were done to reach the message or problem.

6.3 TROUBLESHOOTING STRATEGIES

The Reveleris® PREP software provides simple troubleshooting steps. Press the info (i) button to obtain the troubleshooting information.

6.3.1 TROUBLESHOOTING CHECKLISTS

The following series of troubleshooting checklists are provided to assist the operator in determining the cause of a problem and suggests possible remedies. The problems described below are selected on the basis that they are likely occurrences in normal operation that can be readily resolved by the user. If you encounter problems that cannot be resolved using this troubleshooting section, please contact your BUCHI Service Representative.

The operating software of the Reveleris® PREP purification system includes a large number of internal procedures that automatically alert the user to any unacceptable situation and will not allow the system to start. As an example, if the user tries to start the system without having a column installed, the Start button at the bottom of the Set up Screen will change color to red and won’t be active.



6.3.2 GENERAL INSTRUMENTATION ISSUES

Problem Possible Causes Solution The system does not power up

Power is not being supplied to the system

• Verify that the power cord is plugged in• Make sure that the voltage, amperage and

frequency meet the instrument specifications.• Make sure that both power switches (at the rear

panel and on the display) are switched on• Verify that the fuse wire is not broken and fuses are

correctly installed in the instrument• If all of the above are OK and the instrument does

not power up then call BUCHI Customer Service

System shuts down automatically

Major fluctuations in line power are present

• Connect system to a Uninterrupted Power Supplyline

The display does not power up

The display cable may be disconnected.

• Check the back panel of the display and make surethe beige cable is connected

• Contact BUCHI Customer ServiceDefective switch on the display • Contact BUCHI Customer Service

The touch screen is not respo nsive

The touch screen is out of calibration

• Recalibrate the touch screen

The touch screen cable may be disconnected

• Check the back panel of the display and make surethe black cable is connected

• Contact BUCHI Customer ServiceCannot interface with printer

Printer cable is faulty • Replace the cable

Printer driver is not installed • Install the appropriate printer driverMonitor screen is black on power up

FPGA is bad • Contact BUCHI Customer Service

6.3.3 FLASH COLUMN/CARTRIDGE RECOGNITION ISSUES

Problem Possible Causes Solution Cartridge is not detected Cartridge recognition tag is not facing

the auto-cartridge recognition reader Turn cartridge so that label faces the auto-cartridge reader

Cartridge recognition tag is bad Use new cartridge

Auto-cartridge recognition reader is bad

Contact BUCHI Customer Service



6.3.4 SOLVENT DELIVERY ISSUES

Problem Possible Causes Solution

No solvent flow Empty solvent bottle • Refill the solvent bottle

Pump not primed • Prime the pump properlyCheck valve stuck • Remove the check valve and clean it by

sonicating the check valve in IPA• If sonication does not work replace the check valve

with a new check valveManifold valve stuck in closed position • Contact BUCHI Customer Service

Air bubbles in solvent line • Prime the pumpPump seals worn out • Replace the pump seals

Inconsistent solvent flow

Loose fitting/air leak into the pump • Find the loose fitting between mobile phasereservoir manifold and pump inlet fitting and tightenit up

Liquid leak/pump seals worn out • Fix the leak/replace the pump sealsPump head temperature reaches solvent boiling temperature, causing the pump to lose prime and stop flow (this is likely to occur when running methods with highly volatile solvents such as diethyl ether and methylene chloride)

• Premix the solvents to reduce solvent volatility• Place the highly volatile solvent bottle in an ice

bath to eliminate boiling• Contact BUCHI Customer Service

System p ump pressure reads zero or abnormally high (i.e. > 2000 psig)

Pressure sensor is defective • Contact BUCHI Customer Service

System pump pressure is higher than expected

Blocked solvent lines • Find the blocked lines and replace it

Over-tightened fitting • Loosen the fitting or replace it

Blocked columns or fluidic path • Locate the component that caused the blockage,repair, or replace the component

Leaks Fitting connection not tight • Find the loose fitting and tighten it up

Damaged solvent line • Find the damaged solvent line and replace it.

Pump not running Pump sensor is damaged • Contact BUCHI Customer Service

Pump sensor cable becomes disconnected

• Locate the cable and reconnect to the pumpsensor

• Contact BUCHI Customer Service

Pump power cable becomes disconnected

• Locate the power cable and reconnect to the mainboard or to the pump


Incorrect flow path Mode switching valve failed • Contact BUCHI Customer Service

Incorrect fluidic connections to/from the mode switching valve

• Check/correct the fluidic connections



6.3.5 SAMPLE INJECTION ISSUES


Leak around the injection port (Flash mode)

Dried sample or particulate matter interferes with syringe fitting

• Clean the injection port with appropriatesolvent or remove particulate matter

Defective injection port (Luer fitting) adapter

• Replace the injection port

Solid loader leaking

Loader hardware is not properly set up • Verify that the loader hardware is set upcorrectly. See section 4.6.1.2

Leak around the prep injection valve/sample loop

Loose fitting • Find the loose fitting and tighten it up

6.3.6 FRACTION COLLECTOR ISSUES

Problem Possible Causes Solution Liquid not being collected in fraction tubes

Incorrect fraction collection settings • Verify that fraction collectioninformation is set properly

Fraction collector valve is defective • Contact BUCHI CustomerService

Liquid not centered in fraction tube

Fraction collector is not calibrated • Recalibrate the fraction collector• Contact BUCHI Customer Service

Fraction collector (FC) arm does not move

FC arm did not home properly • Reset the FC arm

FC arm motor is slipping • Tighten the motor coupler• Contact BUCHI Customer Service

FC arm is obstructed • Check for cable or burr in the FCarm path and remove anyobstruction

Tray not detected Tray recognition tag is bad • Put another tray into the sameposition to see if it is recognizedto confirm the cause. Replacetag.

Optical sensor is bad • Contact BUCHI CustomerService

Liquid not being collected in fraction tubes

Incorrect fraction collection settings • Verify that fraction collectioninformation is set properly



6.3.7 DETECTION ISSUES

Problem Possible Causes Solution ELSD signal is low or disappears Sample is too volatile • Sample cannot be detected by

ELSD due to its volatility

No or Low ELSD Carrier gas flow • Verify there is gas supplied to theinstrument

• Verify that there is gas flow to thenebulizer from the ELSDsampling valve

• Verify that there is gas flow to theELSD sampling valve

• Check for leak in the gas linesSample stuck on column • Use stronger solvent or change

column chemistry

ELSD not equilibrated long enough • Restart run to re-stabilize and re-zero ELSD baseline

Blocked nebulizer • Sonicate nebulizer to clean orreplace the nebulizer

Blocked ELSD line • Trace the blockage and replacethe blocked line

Preamp is out of calibration • Contact BUCHI CustomerService

Rotor and/or stator in ELSD Sampling valve is worn, dirty, or clogged

• Replace the rotor and/or stator

Bad laser • Contact BUCHI CustomerService

Poor ELSD Peak shape Blocked nebulizer or nebulizer tubing • Clean the nebulizer or replacethe nebulizer tubing

Nebulizer tubing is not properly connected

• Reinstall the nebulizer tubingproperly (see section 7.6.2)

Noisy ELSD baseline Dirty or contaminated gas • Replace gas source• Replace filter

Gas is not dry/high humidity environment

• Use dryer or moisture removaldevice to remove moisture fromthe gas source

• Use the Reveleris® Dry AirSupply as the air source (seeSection 9 for more info)

• Use nitrogen in place of airMoisture trapped in the gas lines • Remove moisture from lines by

purging the air flow system withnitrogen for 5 minutes

Mobile phase is contaminated or contains non-volatile modifiers

• Use volatile modifiers in themobile phase (see section 8.8)

Solvent contains non volatile stabilizers

• Use solvent with volatilemodifiers

Dirty optics • Clean optics

Dirty drift tube • Clean drift tube



Electronics - Preamp not grounded properly

• Verify that preamp groundingcable is in place

Pre Amp board out of calibration • Contact BUCHI CustomerService

Nebulizer is partially blocked • Sonicate nebulizer to clean orreplace.

Silica or packing material leaked out of cartridge

• Replace cartridge. Flush system.

No UV signal UV light burned out • Replace UV light

UV hardware problem • Contact BUCHI CustomerService

Low UV signal Dirty flow cell • Clean flow cell

UV assembly misaligned • Realign UV assembly• Contact BUCHI Customer Service

6.3.8 ERROR MESSAGES


Confirm Prime error Pump is not primed • Auto prime the pump

Air trapped in pump head • Auto prime the pump

Hardware failure • Contact BUCHI Customer Service

Solvent P ressure error Blockage in the line • Locate the blockage. Clean or replace the line

Sample crashing/precipitate • Purge lines/system with a strong or appropriatesolvent that will dissolve the sample

Valve stuck in incorrect position • Reset valve position• Contact BUCHI Customer Service

Solvent Pressure P2 error

Blockage in line after the ELSD sampling valve

• Locate the blockage. Clean or replace the line

Watchdog or Server error

Timing/communication issue in software

• Reset. Exit out of application software if resetdoesn't work. Reboot if exiting applicationsoftware doesn't work.

Fraction collector error

Arm is obstructed • Check arm path to make sure there are noobstructions

FC arm did not home properly • Reset the FC armFC arm motor is slipping • Tighten the motor coupler


Home position sensor is bad • Contact BUCHI Customer Service

FC arm motor has no power • Contact BUCHI Customer Service

Vapor sensor error Vapor sensor settings are set too sensitive

• Change setting to least sensitive setting (low)

Leaks • Check for leaks inside the instrument and fix theleak

Solvent vapors in work area • Remove the solvents from the work area



Inlet gas pressure out of range - LOW

No gas or gas flow is low • Check the gas lines in the system for leaks• Make sure gas source/tank is available• If the Reveleris® Dry Air Supply is used, check

the gas line connection between the Reveleris®Dry Air Supply and the Flash system for leaks.Also, verify that DC power is supplied to the DryAir Supply

The pressure gauge is not set correctly

• Check pressure gauge setting to make sure thatit is set to deliver 2-2.5L/min gas flow (InletPressure with Air State off around 40psi)

Inlet gas pressure out of range- HIGH (Before run starts)

Blockage in the system • Check the gas lines in the system for blockages

The pressure gauge is not set correctly

• Check pressure gauge setting to make sure thatit is set to deliver 2L/min gas flow (Inlet Pressurewith Air State off around 40psi)

Inlet gas pressure out of range- HIGH (During run)

Blockage in the nebulizer or gas line to the nebulizer

• Check the nebulizer or tubing to the nebulizer forblockages

• Sonicate the nebulizer in a suitable solvent orreplace nebulizer

Solvent Safety Sensor alert

More solvent required • Add solvent to bottle

Solvent safety sensor was not calibrated

• Calibrate/zero the solvent safety sensorproperly

Waste Safety Sensor alert

Waste container is full • Empty waste container

Waste safety sensor was not calibrated

• Calibrate/zero the solvent safety sensorproperly



7. MAINTENANCE

Users must be aware of the hazards when per forming maintenance. Use appropriate PPE, power down and unplug the system when removing the front or left side panels and follow all applicable regulations and your organization’s safety program. There are no user serviceable parts accessible from the right side or rear panel. These panels should only be removed by a qualified service technician. Trained technician may access the front and left side panels for simple maintenance.

Unit contains a class II laser and UV light source. Trouble shooting and maintenance on these parts of the equipment must be performed by trained personnel only.

7.1 OVERVIEW

This chapter describes a series of activities that should be performed on a routine basis to optimize the performance and help ensure long term, safe operation of the Reveleris® PREP purification system. The suggested frequency for each activity indicated below is based on the “typical operation” of the system and is dependent on a variety of factors such as the purity of the solvent and the nature of the sample. Over time, it is likely that the user will need to determine an appropriate schedule that meets the specific application of the flash chromatography system.

7.2 CLEANING THE SYSTEM

The exterior surfaces of the system should be cleaned with a cloth, using water with a mild detergent on a periodic basis. If mobile phase or sample is accidentally spilled on the system, it should be removed immediately. If a mild detergent is not able to remove the foreign material, an organic solvent such as methanol or iso-propanol can be used.

Clean the racks and trays on a monthly basis with water containing a mild detergent.

7.2.1 ROUTINE INSPECTION

On a daily basis, the following activities should be performed:

• Check for leaks. If a leak is observed, resolve the issuebefore continuing.

• Inspect fittings; if solid material is deposited on a fitting,clean and tighten (replace) the fitting before continuing.

• Inspect all tubing. If defective tubing is found (e.g. kinks,bends, discoloration or deterioration), replace the tubing.When replacing the tubing on any valve, ensure that thereplacement tubing is placed in the proper port on thevalve.

• Empty the waste container

• Check all drain lines to ensure that liquid can flowthrough them to the waste container (i.e. there is noparticulate matter blocking the lines).

On a weekly basis, the following activities should be performed:

• Check the filters in the solvent bottles and clean ifnecessary. They can be cleaned by placing them in anultrasonic bath with a suitable solvent for a few minutes.

• Check and if necessary tighten the fitting that securesthe tubing from the mobile phase reservoir manifold tothe inlet check valve housing (Figure 7-3 ). Thisconnection can get loose over time and cause an airleak into the pumps.

7.3 ACCESSING SYSTEM COMPONENTS

Make certain that the Reveleris ® PREP purification system is powered down and disconnect the power cord from the power supply before the front or left side panel is removed. Failure to unplug the system may expose the user to dangerously high levels of electrical current.

The left side panel can be removed by a trained technician by removing the security screws to access the pump, UV Detector, and the air assembly components (Figure 7-1 ).

Figure 7-1: The Left Side View of the Reveleris ® PREP Purification System, Side Panel Removed)

The front door can be removed by a trained technician by removing the security screws to access the ELSD nebulizer and the sampling valve (Figure 7-2 ).



Figu re 7-2: The Front View of the Reveleris ® PREP Pur ification System, Front Panel Removed

7.4 SOLVENT PUMP MAINTENANCE

If lower than normal pressures, pressure var iations, or leaks in the pumping system are observed, it is likely that that the piston seal, piston, or check valves need to be cleaned or replaced. As a first approximation, the piston seals may need to be replaced after 1000 hours of running time. To prolong seal’s life, flush the pump with IPA at the end of each day or when not in use over an extended period.

When using chlorinated solvents (such as chloroform or methylene chloride), flush the pump with IPA at the end of each day to avoid corrosion.

The pumps are accessed via the left side panel of the instrument. Make certain that the Reveleris ® PREP system is powered down, and disconnect the power cord from the power supply before the left side panel is removed. Only trained technician may access the left side panel.

7.4.1 REPLACING PISTON SEALS (SOLVENT PUMP)

Materials Needed:

• Open-end wrench, 9/16" x 5/16"

• Allen wrench 5/32”

• Seal Insertion/Removal Tool (supplied with Seal Kit)

• Ultrasonic Bath

To replace the piston seals:

1. Disconnect the mobile phase tubing from the inlet(bottom) of the pump head using the 9/16” open-endwrench.

2. Disconnect the mobile phase outlet tubing from the topof the pump head using the 5/16” open end wrench.

3. Using the 5/32” Allen wrench, remove the four screwsholding the pump head.

Figure 7-3: Solvent Pumps

4. Carefully pull out the pump head from the pump.Remove the yellow seal from the piston if it did not stayin the pump head.

Be careful not to break the piston when removing the pump head. Twisting the pump head can cause the piston to break.

If you remove the piston seal, install a new seal after cleaning the head. It is not recommended that you reinstall the used piston seal since it is likely to be scratched and damaged during removal and will not provide a reliable seal if reused.

5. Remove the seal using the flanged end of the plasticseal removal tool supplied with the seal replacementkit as shown in Figure 7-4 . Avoid scratching thesealing surface in the pump head.

Inlet CheckValve Housing

Outlet CheckValve Housing

Screw (4) PerPump Head



Figure 7-4: Removing the Piston Seal

6. Inspect the piston seal cavity in the stainless steelpump head. Remove any foreign material using a cottonswab or equivalent. Avoid scratching the sealingsurfaces during the cleaning process and take care toensure that no fibers from the cleaning swab remain inthe components.

7. The pump head may be cleaned using a laboratorygrade detergent solution in an ultrasonic bath for atleast 30 minutes, followed by rinsing for at least 10minutes in distilled water. Be sure that all particlesloosened by the above procedures have beenremoved from the components before reassembly.

7.4.2 CLEANING THE PISTON

Materials Needed:

• Scouring pad (supplied with Seal Removal Kit)

• Lint free cloth saturated with Methanol

Use the scouring pad included in the seal replacement kit to clean the piston. Gently squeeze the piston within a folded section of the pad and rub the pad along the length of the piston (it is not necessary to remove the piston from the housing to clean the piston). Rotate the pad frequently to assure the entire surface is scrubbed.

Do not exert pressure perpendicular to the length of the piston, as this may cause the piston to break.

After scouring, use a lint-free cloth, dampened with alcohol, to wipe the piston clean.

7.4.3 REPLACING THE SEAL

Materials Needed:

• Seal Insertion/Removal Tool (supplied with Seal Kit)

• Isopropanol/Water (50/50) or Methanol/Water (50/50)

To replace the seal:

1. Place a high-pressure replacement seal on the rod-shaped end of the seal insertion/removal tool so thatthe spring is visible when the seal is fully seated on thetool.

2. Insert the tool into the pump head so that the open sideof the seal enters first, facing the high-pressure cavityof the pump head. Be careful to line up the seal withthe cavity while inserting.

3. Withdraw the tool, leaving the seal in the pump head.When you look into the pump head cavity, only thepolymer portion of the seal should be visible.

4. Attach the pump head in the reverse order of removal.

5. Condition the seal by running the pump with a 50:50solution of isopropanol (or methanol) and water for 30minutes at a flow rate of about 50mL/min using theColumn Flushing feature under Tools and ManualControl (Section 3.3.6.2 ).

Use water and organic solvents to break in new seals. Buffer solutions and salt solutions should never be used to break in new seals. If buffers or salt solutions are employed, the seals will not set properly and have a short useful life.

7.4.4 CHECK VALVE CLEANING AND REPLACEMENT

Materials Needed:

• Liquid Laboratory Detergent

• Isopropanol/Water (50/50) or Methanol/Water (50/50)

• Open-end wrench, ½”, 9/16" x 5/16"

• Torque wrench ½”

Check valve problems are frequently the result of small particles that are present in the mobile phase (e.g. dust and non-dissolved buffers and salts). Most check valve problems can be solved by pumping a strong solution of liquid laboratory grade detergent through the check valves at a rate of 20mL/min for one hour. If the pump is used for delivering solvents that are not miscible with the laboratory detergent, pump an intermediate solvent such as isopropanol for a few minutes before introducing the detergent solution. After washing with detergent, pump distilled water through the pump for 15 min. Either the Column Flushing (Section 3.3.6.2 ) or Solvent Line Flushing (Section 3.3.6.6 ) can be used to pump solvents through the pump. If cleaning the check valve does not solve the problem, try sonicating the check valve for 10 min in the appropriate solvent. (Normal Phase Solution: HPLC-grade Ethyl Acetate or a strong solvent, Reversed



Phase Solution: HPLC-grade 50:50 methanol: water solution). If cleaning or sonicating the check valve does not fix the problem, it should be replaced.

To replace the check valve:

1. Disconnect the mobile phase tubing from the inlet(bottom) of the pump head using the 9/16” open-endwrench.

2. Disconnect the mobile phase outlet tubing from the topof the pump head using the 5/16” open end wrench.

3. Remove both check valve housings from the pump headusing the ½” open-end wrench. The check valvecapsule will then be accessible as shown in Figure 7-5 .

Figure 7-5: Check Valve Capsule and Check Valve Housing

4. Replace the old check valves with the new checkvalves. Ensure that the replacement check valvecapsule is installed in the same orientation as the oldcheck valve.

5. Install the check valve housings back into the pumphead. Tighten the check valve housing to 75 inch-pounds with the ½” torque wrench.

7.5 UV DETECTOR

7.5.1 CLEANING THE FLOW CELL

A noisy UV signal may be due to the accumulation of extraneous materials in the flow cell. Cleaning the flow cell can be done by bypassing the column and pumping a strong solvent such as methanol at a flow rate of 50mL/min for a few minutes. The Column Flushing feature under Tools and Manual Control (Section 3.3.6.5 ) can be used for this process.

7.5.2 REPLACING THE LAMP

The lamp is accessed via the left side panel of the instrument. Make certain that the Reveleris ® PREP purification system is powered down, and disconnect the power cord from the power supply before the left side panel is removed. Only trained technician may access the left side panel.

To replace the Lamp for the UV detector:

1. Remove the hand tight screw on the shield coveringthe lamp (Figure 7-6 ).

Figure 7-6: Lamp Shield Mounting Screws

2. Remove the shield.

3. Unplug the lamp from the UV board by pulling the blackplug.

4. Unscrew the Allen setscrew from the lamp mount.

5. Pull the old lamp out from the mount.

6. Install the new lamp.

7. Tighten the Allen setscrew on lamp mount.

8. Plug in the new lamp to the UV board.

9. Attach the shield and install the screw that affixes it tothe system.

After replacing the lamp, the UV assembly may need to be recalibrated. Please contact your BUCHI Service Representative.

7.6 ELSD DETECTOR

7.6.1 CLEANING THE NEBULIZER

The nebulizer is accessed via the front door of the instrument. Make certain that the Reveleris ® PREP purification system is powered down, and disconnect the power cord from the power supply before the front door is removed. Only trained technician may access the front door.

The nebulizer can become blocked over time due to deposition of sample and particulate matter from the mobile phase. A dirty or blocked nebulizer can cause increased baseline noise, poor peak shape, and decreased sensitivity.

To clean the nebulizer:

1. Power off the unit and disconnect the power cord fromthe power supply.

Hand tight shield Screws



2. Remove the front door by using the appropriate tool.3. Disconnect the finger tight gas inlet fitting (with gas

tubing attached) from the nebulizer.

4. Disconnect the inlet line (Nebulizer tubing) from thenebulizer (Figure 7-7 ).

Figure 7-7: ELSD Detector

5. Remove the nebulizer from the drift tube bypushing it in and then turning it firmly a quarter turncounterclockwise.

6. Remove the orange wear band from the nebulizer andset aside.

7. Place the nebulizer in a beaker f i l led with amethanol: water (50:50) solution.

8. Sonicate the nebulizer in an ultrasonic bath for10 minutes.

Do NOT sonicate the wear band

9. If the nebulizer is still completely blocked, connect a high-pressure airline to the nebulizer inlet to help removethe blockage.

10. If the nebulizer is permanently blocked or cannotbe cleaned, it should be replaced.

11. Replace the nebulizer wear band and nebulizer gas inlet.

12. Put the nebulizer back into the unit by aligning thegrooves and turning a quarter turn clockwise untilthe nebulizer locks firmly into place.

13. Connect the liquid and gas inlet lines to the nebulizer.

14. Replace the front door.

7.6.2 REPLACING THE NEBULIZER TUBING

The nebulizer tubing is accessed via the front door of the instrument. Make certain that the Reveleris® PREP purification system is powered down, and disconnect the power cord from the power supply before the front door is removed. Only trained technician may access the front panel.

The tubing between the ELSD Sampling Valve and the nebulizer (nebulizer tubing) (Figure 7-8) can become blocked over time due to particulate matter from the sample. A dirty or blocked nebulizer tubing can cause poor ELSD peak shape or a decrease in ELSD sensitivity. It is best to replace the

nebulizer tubing every time the nebulizer is partially or completely blocked. Occasionally, the blocked nebulizer tubing can be cleaned by sonicating it in an IPA: water (50:50) solution for 10min.

To replace the nebulizer tubing:

1. Power off the unit and disconnect the power cord.2. Remove the front door by using the appropriate

tool and set it aside.

Figure 7-8: Nebulizer Tubing

3. Gently unscrew the tubing from the nebulizer andthe ELSD Sampling Valve and replace it with a newnebulizer tubing.

The tubing going into the nebulizer should be inserted all the way until it bottoms out. If there is a gap, it can cause poor ELSD peak shape. This tubing usually extends past the fitting by approximately 11 mm.

4. Replace the front panel.

7.6.3 CLEANING THE DRIFT TUBE

The drift tube can become dirty over time from sample and particulate matter from the solvents. A dirty drift tube can cause an increase in baseline noise and a decrease in sensitivity. If the drift tube needs cleaning, please contact your BUCHI Sales Representative.



7.7 REPLACING THE FUSE

Disconnect the power cord from the pow er supply before changing the fuse(s). Make certain that you replace the fuse with one of the same rating (e.g. replace a 5A fuse with a 5A fuse).

To replace the fuse:

1. Disconnect the instrument from the main power.

2. Remove the cover plate for the fuse compartment(above the power inlet socket) (Figure 7-9 ) by gentlysqueeze the side tabs and pulling it straight out.

3. Remove the two fuses and replace them with two newfuses of the same rating.

Figure 7-9: Fuse Compartment

7.8 CLEANING THE DISK SPACE

The Reveleris® PREP software can crash or perform poorly when the disk space is full or almost full. It is recommended that you remove files from your system regularly to maximize its performance.

7.8.1 DELETING OLDER VERSION OF REVELERIS® PREP PURIFICATION SYSTEM SOFTWARE

Only the latest version of the Reveleris® PREP purification software should be stored in the instrument. To remove the older version software: 1. Close the software.2. Right click the mouse on the start menu and then OpenWindows® Explorer. 3. Then go to Computer, Reveleris Prep (C :) and Releases.4. If there are multiple software versions in the Releasesfolder, delete all except the latest version.

7.8.2 DELETING THE LOG FILES FROM THE CURRENT SOFTWARE VERSION

The Reveleris® PREP software stores log files in the system and should be cleared periodically. Please delete the files regularly to increase disk space. To delete the log files:

1. Close the software.2. Right click the mouse on the start menu and then OpenWindows® Explorer. 3. Go to Reveleris Prep (C :), and Releases.4. Select the current software version, then Coriolis folder,and then logs. 5. Delete all Server.exe files. DO NOT delete the SWInstallfile.

7.8.3 TRANSFERRING AND DELETING THE RUN AND METHOD FILES

It is also recommended that you delete or transfer the run and method files regularly to increase disk space.

To transfer and delete run files:

1. Close the software and insert a flash drive (64MB memorydrive or greater) into one of the USB ports located on the side of the LCD display screen. 2. Right click the mouse on the start menu and then OpenWindows® Explorer. 3. Then, go to Libraries, Documents, Roadrunner, and runs.You may delete the custom folders or just the run files. 4. Right click in properties to determine the size of the data inthe folder to be copied. 5. Verify that the USB drive is large enough and then copythe custom run folders/ run files onto it. (NOT the main Run folder itself) 6. After the run files have been transferred to a USB drive,delete all of the runs in this folder from the Reveleris® Prep unit (DO NOT delete the main Run folder itself)



To transfer and delete method files: 1. Close the software.2. Right click the mouse on the start menu and then OpenWindows Explorer. 3. Go to Libraries, Documents, Roadrunner, and methods.4. Transfer the individual custom method folder/methods tothe USB drive or delete the custom folders/methods from the main Method folder (DO NOT copy or delete the main Method folder itself).

7.8.4 DELETING THE FILES FROM THE RECYCLE BIN

After deleting all or some of the files described above, please delete the deleted files from the Recycle bin. 1. Right click the mouse on the start menu and then OpenWindows Explorer. 2. Go to Computer, Local Disk (C :), $Recycle. Bin andRecycle Bin. 3. Delete all of the files in the Recycle Bin.



8. APPENDICES

8.1 SPECIFICATIONS

REVELERIS® PREP PURIFICATION SYSTEMS SPECIFICATION

Mode of Operation Flash Chromatography or Preparative Liquid Chromatography

Fluidics

Solvent Delivery System 4 Independent channels (4L)

Gradient Formation Binary Gradient with High Pressure Mixing

Flow Rate 1-200mL/min

Flow Rate Accuracy +/- 3% (4-200mL/min)

Maximum Pressure 200psig (Flash mode); 1700psig (Prep LC mode)

Sampling

Sample Modes Liquid or Solid Sample(Flash) Modes

Sample Loader Sample loaders for 3g, 25g, and 60g can be employed

Sample Loader Plunger Three sizes (15mL, 75mL, and 150mL). Includes sleeve with locking mechanism to provide for safe use at pressures up to 200psig.

Absorbance Detector

Wavelength Range 200-500nm (optional visible 200 – 850 nm)

Number of Wavelengths monitored

3 wavelengths can be monitored and used to trigger fraction collection

ELSD Detector

Light Source Laser (1.0mW)

Detector Element Silicon Photodiode

Product Class Class 1 laser product

Fraction Collection

Triggers UV signal, ELSD signal

Collection Racks Multi position racks for 12, 13, 16, 18, 25mm tubes, 480mL bottles

Rack Size 5" x 12", 2 racks for tubes, 1 rack for bottles

Communications:

Operating Parameter Selection & Display:

Windows®-Based Graphical LCD with f u l l QWERTY keyboard

Power Requirements: 120/240V, 50/60Hz, 10 A

Dimensions: 16.75" H x 21.50" W x

20.50" D (42.54cm H x 54.61 cm W x 52.07 cm D)

Weight: 92 lbs (41.73kg)

Environmental Operating Temperature:

15 to 30°C

Relative Humidity: 10 – 90%, Non-Condensing

Maximum Altitude: 2000m

8.2 SPARE PARTS AND OPTIONAL ACCESSORIES

SPARE PARTS /OPTIONAL ACCESSORIE S

Part No. Description

148633212 Reveleris® X2/Prep ELSD Sampling Valve Kit (includes a stator and rotor)

142120263 Reveleris® X2/Prep Sample Injection Valve Kit (includes a stator and rotor)

142129852 Stator for Reveleris® PREP Mode Switching or Prep Injection Valve

142129853 Rotor for Reveleris® PREP Mode Switching or Prep Injection Valve

142120814

Reveleris® , Reveleris® X2, and Reveleris® Prep Main Pump Check Valve Capsule (4/pack; needs 2 check valves per pump head)

142120912 Reveleris® X2 Main Pump Piston Seals (2/pack)

142129854 Reveleris® Prep Main Pump Piston Seals (2/pack; needs 1 per pump head)

142119162 Reveleris® X2 and Reveleris® Prep Main Pump back Pressure Regulator Cartridge

148633134 Reveleris® X2/Prep Air Check Valve

142129804 Solvent Back Flow Preventer Valve

142129807 30psi Back Pressure Regulator

142129811 Main Solvent Pressure Sensor P1 (3000psi)

148633171 Reveleris X2/Reveleris Prep Solvent Pressure Sensor P2 (300psi)

142129805 Pressure Sensor Snubber

145125333 1/8” Flange free PEEK fitting (internal and SIV) (5/pack)

143112418 1/8” Flange free ferrule (10/pack)

145125340 1/16” Flange free PEEK fitting (shuttle to nebulizer) (5/pack)

145125376 1/16” Flange free ferrule (10/pack)

148633152 Reveleris® X2/ Reveleris® Prep ELSD Nebulizer

142113968 Nebulizer O-Ring

145167551 Reveleris® X2/Prep Nebulizer Tubing

142119201 UV Fiber light Replacement Lamp



142118905 ELSD laser (1mW)

142119171 Reveleris® X2 UV Flow Cell (0.1mm path length)

142123015 Reveleris® UV Flow Cell (0.3mm path length)



143136006 1 mL Sample Loop Assembly




143112510 0.030” ID (Green ) PEEK tubing, 10ft

143136003 1/16 SS Fitting Kit for Reveleris® Prep, 10-32 port (contains fittings and ferrules, 5/pkg)

143136001 1/8” SS Fitting Kit for Reveleris® Prep Injection Valve,1/4-28 port (contains fitting and ferrules, 5/pkg)

143136002 1/8 SS Fitting Kit for Reveleris® Prep Bulkhead, 5/16-24 port (contains fittings and ferrules, 5/pkg)

142118927 Fitting Tool

142129686 Reveleris® Prep Column Stand

142129684 BUCHI Fitting Tool for 1/4-28 Fittings

142129671 Injection Port for 22 Gauge Needles 142108751 22 Gauge Needle with Kel-F Luer Hub

142129683 Female SS Luer Adapter to 1/4"-28 Port

8.3 BUCHI REVELERIS® FLASH CARTRIDGES

REVELERIS® FLASH CARTRDIGES


145146130 Reveleris® Silica 4g, 20/pk








145170200 Reveleris® HP Silica 4g, 20/pk








145152990 Reveleris® C18 4g, Ea





145152991 Reveleris® C18 120g, Ea

145171538 Reveleris® C18 220g, Ea

145164430 Reveleris® C18 330g, Ea

145169482 Reveleris® C18-WP 4g, Ea



145157330 Reveleris® Amino 4g, Ea





145157335 Reveleris® Diol 4g, Ea







8.4 BUCHI PRE-PACKED PREPARATIVE COLUMNS



8.5 COMMON SOLVENTS FOR FLASH CHROMATOGRAPHY

Common Solvents for Flash Chromatography

Solvent (by increasing polarity) Properties

Pentane

Polarity: 0.00 Viscosity (cp 20): 0.23 Boiling Point (ºC): 36 UV Cutoff (nm): 190 Immiscible With: acetonitrile, dimethylformamide, dimethyl sulfoxide, methanol, water water solubility (%w/w): 0.004

Petroleum Ether

Polarity: 0.01 Viscosity (cp 20): 0.30 Boiling Point (ºC): 30-60 UV Cutoff (nm): 210 Immiscible With: acetonitrile, dimethylformamide, dimethyl sulfoxide, methanol, water water solubility (%w/w): data not available

Hexane


Cyclohexane


Iso-octane

Polarity: 0.10 Viscosity (cp 20): 0.5 Boiling Point (ºC): 99 UV Cutoff (nm): 215 Immiscible With: acetonitrile, dimethylformamide, dimethyl sulfoxide, methanol, water water solubility (%w/w): data not available

Cyclopentane

Polarity: 0.1 Viscosity (cp 20): 0.44 Boiling Point (ºC): 49 UV Cutoff (nm): 200 Immiscible With: acetonitrile, dimethylformamide, dimethyl sulfoxide, methanol, water water solubility (%w/w): data not available

n-Heptane






Trichlorethylene

Polarity: 1.00 Viscosity (cp 20): 0.57 Boiling Point (ºC): 87 UV Cutoff (nm): 273 Immiscible With: water water solubility (%w/w): data not available

Carbon tetrachloride

Polarity: 1.60 Viscosity (cp 20): 0.97 Boiling Point (ºC): 77 UV Cutoff (nm): 265 Immiscible With: water water solubility (%w/w): 0.08

i-Propyl ether

Polarity: 2.40 Viscosity (cp 20): 0.37 Boiling Point (ºC): 68 UV Cutoff (nm): 220 Immiscible With: dimethylformamide, water water solubility (%w/w): 1.71

Toluene


Chlorobenzene

Polarity: 2.70 Viscosity (cp 20): 0.80 Boiling Point (ºC): 132 UV Cutoff (nm): 287 Immiscible With: data not available water solubility (%w/w): data not available

o-Dichlorobenzene


Ethyl ether

Polarity: 2.80 Viscosity (cp 20): 0.24 Boiling Point (ºC): 35 UV Cutoff (nm): 215 Immiscible With: dimethyl sulfoxide, water water solubility (%w/w): 6.89

Benzene






Isobutyl alcohol


Methylene chloride (Dichloromethane)


Ethylene dichloride


n-Butanol

Polarity: 3.90 Viscosity (cp 20): 2.98 Boiling Point (ºC): 117.5 UV Cutoff (nm): 215 Immiscible With: water water solubility (%w/w): 0.43

n-Butyl acetate


n-Propanol

Polarity: 4.00 Viscosity (cp 20): 2.27 Boiling Point (ºC): 98 UV Cutoff (nm): 210 Immiscible With: none in this table (miscible with all solvents listed in this table) water solubility (%w/w): 100

Tetrahydrofuran


Ethanol






Ethyl acetate


i-Propanol


Chloroform


Dioxane


Acetone


Acetonitrile

Polarity: 5.8 Viscosity (cp 20): 0.38 Boiling Point (ºC): 82 UV Cutoff (nm): 190 Immiscible With: cyclohexane, heptane, hexane, pentane, iso-octane water solubility (%w/w): 100

Dimethylformamide

Polarity: 6.40 Viscosity (cp 20): 0.92 Boiling Point (ºC): 153 UV Cutoff (nm): 268 Immiscible With: cyclohexane, heptane, hexane, pentane, i-propyl ether water solubility (%w/w): 100





Methanol

Polarity: 5.1 Viscosity (cp 20): 0.55 Boiling Point (ºC): 65 UV Cutoff (nm): 205 Immiscible With: cyclohexane, heptane, hexane, pentane, iso-octane water solubility (%w/w): 100

Ethylene glycol


Dimethyl sulfoxide

Polarity: 7.20 Viscosity (cp 20): 2.24 Boiling Point (ºC): 189 UV Cutoff (nm): 268 Immiscible With: cyclohexane, ethyl ether, heptane, hexane, pentane, iso-octane water solubility (%w/w): 100

Water

Polarity: 10.20 Viscosity (cp 20): 1.00 Boiling Point (ºC): 100 UV Cutoff (nm): 190 Immiscible With: benzene, n-butanol, n-butyl acetate, carbon tetrachloride, chloroform, cyclohexane, ethylene dichloride, methylene chloride, ethyl acetate, ethyl ether, heptane, hexane, pentane, i-propyl ether, toluene, iso-octane water solubility (%w/w): 100



8.6 VOLATILE MOBILE PHASE MODIFIERS

VOLATILE BUFFERS AND MOBILE PHASE MODIFIE RS

pKa pKb pH Range BP MP

Acids Trifluoroacetic Acid Formic Acid Acetic Acid Carbonic Acid

0.3 3.75 4.75 6.37

13.70 10.25 9.25 7.63

72.4°C 100.7°C 116.0°C

-

Bases Ammonia Methylamine Ethylamine Triethylamine

9.25 10.81 10.66 11.01

4.75 3.19 3.34 2.99

-33.35°C 16.6°C -6.3°C 89.3°C

Ion-Pair Reagents Pentafluoropropionic Acid Heptafluorobutyric Acid Nonafluoropentanoic Acid

~0.6 ~0.6 ~0.6

96-97°C 120°C 140°C

8.7 POLARITY ORDER OF COMMON ORGANIC SOLVENT SOLVENTS



8.8 SOLID/DRY SAMPLE PREPARATION

Users must be aware of the hazards associated with the chemicals in use and wear appropriate personal protective equipment.

For liquid loading, the dissolution solvent should be as non-polar as possible — ideally the weaker solvent of the elution mixture. The ideal volume should be between 10–15% of the column volume of the cartridge.

If a solvent other than that used for the elution is used, it is best to check the effect on the separation system first by running a TLC plate of the sample in the dissolution solvent. If the Rf of the desired component is ≤0.1 the dissolution solvent will have little effect on the separation. A good example of such a solvent is dichloromethane.

If a different dissolution solvent is used, it is often a good idea to “sandwich” the sample between two layers of the dissolution solvent to ensure no sample precipitates out, which may affect the separation or block the cartridge. Usually ¼ of a column volume (CV) before sample load followed by ¼ CV after the sample load is ideal.

If a polar dissolution solvent (strong solvent) has to be used, it is recommended that a sample dry load technique is used. However, if this is not practical, use the minimum volume of polar solvent to dissolve the sample. This will reduce the effect of the polar solvent on the separation. Alternatively, a weaker solvent can be used to dilute the polar dissolution solvent but care must be taken to ensure precipitation does not occur within the sample solution.

Drying dissolved or liquid sample onto dry silica is a common technique in which the sample is dissolved in a polar (strong) solvent. Dry silica is added and the dissolution solvent is then evaporated. This is usually carried out using a rotary evaporator and bulk Davisil® silica.

Ideally the sample to silica ratio should be between 1:1 and 1:3, but the resulting dried mix should be a free flowing powder. The actual ratio will depend on the mobility of the sample and more liquid/oily samples will usually require the 1:3 ratio.

Step 1: Add enough dissolution solvent to completely dissolve the entire sample. Weigh out the required amount of silica, based on the ratio’s mentioned in the note above, and add it to the dissolved sample in the round bottomed flask.

Step 2: Evaporate the solvent from the sample/silica mix. Care must be taken as this step can cause “bumping” which may lead to loss of sample.

Step 3: Once the sample is dry it is often required to scrape down the sample/silica mix from the sides of the flask with a spatula. After this step, the sample/silica mix should be a free flowing powder. If it is still damp with solvent, evaporate further either on the rotary evaporator or by covering the neck with a porous paper (a filter paper secured with tape or elastic band is often used) and placing in a vacuum chamber.



Step 4: Once the sample/silica mix is a free flowing powder, it is ready to be poured into the empty dry load tube. In this case the 15g size is being used. See order block above for part number ordering information.

Step 5: Ensure a lower frit is inserted in the dry load tube. Then pour the dry sample/silica mix into the dry load tube. Gently tap the tube to settle the sample/silica bed. Insert the top frit. The easiest way to do this is to insert the frit sideways then turn it flat inside the top of the tube. Then push the frit down to meet the silica surface using the frit insertion rod.

Step 5a

Step 5b

Step 5c

Step 5d

Step 6: Install the solid loader tube into the assembly.



9. INTRODUCTION TO THE REVELERIS®

DRY AIR SUPPLY

9.1 ABOUT THE REVELERIS ® DRY AIR SUPPLY

The Reveleris® Dry Air Supply is designed to be used with Reveleris® X2 Flash Chromatography and Reveleris® Prep Purification systems. It simplifies the operation of the Reveleris® systems by producing particle free dry air on demand without the need for operator attention. Increase productivity, reduce operating costs, and improve lab safety by eliminating the need to replace expensive and dangerous air or nitrogen cylinders. Air is used in the Reveleris® systems to operate the evaporative light scattering detector (ELSD), which is part of the Reveleris® X2 and Reveleris® Prep detection systems, to carry samples to the ELSD, and to purge solvents out of spent cartridges and sample loaders.

The Reveleris® Dry Air Supply has a small, compact footprint that fits easily on the lab bench next to your Reveleris® system. It produces an on demand supply of particle free dry air at 85 psi and at flow rates up to 15 L/min. at atmospheric pressure.

9.1.1 PRINCIPLE OF OPERATION

The Reveleris® Dry Air Supply consists of a compressor pump, a particle filter, a moisture removal device, and associated connectors and tubing to connect to the Reveleris® X2 and Reveleris® Prep systems.

Figure 9-1 shows the flow schematic of the Dry Air Supply. The air pump generates compressed air which is then passed through a coalescing filter to remove any liquid and particulate matter. The 0.01um filter is equipped with a float drain which automatically opens to empty any liquid (water) that may accumulate inside the filter housing. The particle and liquid free air is then passed through the moisture membrane air dryer to remove moisture before exiting the Reveleris® Dry Air Supply.

Figure 9-1: Schematic Diagram of the Reveleris ® Dry Air Supply

9.1.2 SYSTEM CONTROL

The operation of the Reveleris® Dry Air Supply is controlled by the Reveleris® X2 and Reveleris® Prep system software.

The DC power required for its operation is supplied from the Reveleris® systems.

9.1.3 WHERE TO GO FOR FURTHER INFORMATION

Additional information about Flash Chromatography, the Reveleris® X2 Flash Chromatography system, and the Reveleris® Prep Purification system can be obtained from your BUCHI Sales Representative.

9.2 INSTALLATION AND OPERATION

9.2.1 LOCATION/SYSTEM REQUIREMENTS

The Reveleris® Dry Air Supply may be installed on the lab bench next to your Reveleris® X2 flash chromatography or Reveleris® Prep purification system. The identical environmental condition suitable for the Reveleris® system is suitable for the Air Supply:

• The temperature range of the laboratory should bebetween 18°C and 30°C (64°F - 86°F). The Reveleris ®Dry Air Supply should be installed in an area in whichthe temperature range is fairly constant (the suggestedtemperature variation is no more than +/- 2°C/hr (+ /-5°F/hr)). The relative humidity of the area should meetthe ASHRE standard for human comfort.

• Corrosive vapors, solvents and dust should not beallowed in the laboratory as these can affect systemperformance.

The dimensions of the Reveleris® Dry Air Supply are 7" (17.8cm) W x 7" (17.8cm) D x 7" (17.8cm) H. A minimum of 6" (15cm) should be provided at the rear of the unit to allow for proper ventilation.

The Reveleris® Dry Air Supply requires a DC power to operate. This is supplied through the connection to the back of the Reveleris® X2 flash chromatography or Reveleris® Prep purification system.

9.2.2 UNPACKING

The Reveleris® Dry Air Supply and its accessories are shipped in one container. Unpack components carefully, making sure all items in the list below have been included and are in good condition.

The shipping container for the Reveleris® Dry Air Supply contains:

• The Dry Air Supply

• A power cable (6 ft)

• 1/8” Flange free PEEK fitting (2 pieces)

• 1/8” Flange free ferrule (2 pieces)

• 1/8” PTFE tubing (10ft)

• ¼” ID x 3/8” OD Drain tubing (6ft)

The Reveleris® Dry Air Supply has been carefully packed and shipped to ensure that it is received in proper condition. If you find any evidence of damage to the unit or the box, contact the shipping company that delivered the unit in order


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local distributor orto file a claim. You may also contact your BUCHI Service Representative.

If replacement parts are needed, please refer to9.6.2, Spare Parts for part numbers.

9.2.3 INSTALLING THE INSTRUMENT

Make sure there is free flow of air to the front of the unit and tothe cooling fan at the back panel.

Please lift the Reveleriswhen positioning or re- locating it on the bench.drag on the bench as it can damage the feet.

The Reveleris® Dry Air Supply requires a DC power tooperate. Connect the power cable provided in the accessorykit to the “Power In” port at the rear panel (Reveleris® Dry Air Supply. Connect the other end of thepower cable to the “Dry Air Supply Power Out” port at the rearpanel (Figure 2-2 ) of the Reveleris® X2chromatography or Reveleris® Prep purification

Only use the power cable included inthe accessory kit.

Connect the PTFE tubing using the 1/8” Flange free PEEKfitting and ferrule to the “Air Out” port at the rear panel(Figure 9-2) of the Reveleris® Dry Air Supply. Connect theother end to the “Air Inlet” port at the rear panelReveleris® X2 (Figure 9-3) or Revelerissystem using the other fitting and ferrule.

Depending on the location of theReveleris ® Dry Air Supply in relation to the RevelerisX2 or Reveleris ® Prep system , you may want to cut thetubing to the appropriate length.

Figure 9-2: Rear Panel of the Dry Air Supply


yo

If replacement parts are needed, please refer to Section

THE INSTRUMENT

Make sure there is free flow of air to the front of the unit and to

Reveleris ® Dry Air Supply locating it on the bench. Do not

drag on the bench as it can damage the feet.

The Reveleris® Dry Air Supply requires a DC power to operate. Connect the power cable provided in the accessory kit to the “Power In” port at the rear panel (Figure 9-2 ) of the

ly. Connect the other end of the power cable to the “Dry Air Supply Power Out” port at the rear

) of the Reveleris® X2 flash purification system.

Only use the power cable included in

Connect the PTFE tubing using the 1/8” Flange free PEEK to the “Air Out” port at the rear panel

of the Reveleris® Dry Air Supply. Connect the Inlet” port at the rear panel of the

or Reveleris® Prep (Figure 9-4) system using the other fitting and ferrule.

Depending on the location of the Dry Air Supply in relation to the Reveleris ®

, you may want to cut the

of the Dry Air Supply

Connect the drain tubing included in the shipping containerthe “Drain” port on the rear panelReveleris® Dry Air Supply. accumulate inside the filter housingthrough this drain port. Place the other endan aqueous waste container located lower than theinstrument.

Do not place the Revelerisdrain tubing in an organic solvent wastubing is not compatible with most organic solvents.

Figure 9 -3: Rear Panel of theChromatography System

Figure 9- 4: Rear Panel of thePurification System

77

drain tubing included in the shipping container to the “Drain” port on the rear panel (Figure 9-2 ) of the

Dry Air Supply. Any liquid (water) that may te inside the filter housing will be discharged

Place the other end of the tubing in an aqueous waste container located lower than the

Do not place the Reveleris ® Dry Air Supply drain tubing in an organic solvent was te container as the

is not compatible with most organic solvents.

3: Rear Panel of the Reveleris ® X2 Flash Chromatography System

4: Rear Panel of the Reveleris ® PREP Purification System



9.2.4 OPERATING THE DRY AIR SUPPLY

The operation of the Reveleris® Dry Air Supply is controlled by the Reveleris® X2 Flash or Reveleris® Prep Purification system software. Every time the Reveleris® software is started, it performs a pressure check to determine the type of air source connected to the system. If the Dry Air Supply is used, the pressure will be low initially. The Reveleris® software will then turns on the air compressor pump inside the Reveleris® Dry Air Supply for a few seconds to check for pressure. If the pressure meets specification, this information is then stored in the software for its operation. If there is a leak in the air line or the air line tubing is not connected properly, the pressure will remain low and an error will be displayed (Figure 9-5 ). Check or correct the connections, then press the Reset button. Press the Reset button a second time to clear the error message. If the pressure specification is met, this screen will disappear and you can start using the Reveleris® X2 Flash or Reveleris® Prep Purification system. For more information on the operation of the Reveleris® X2 Flash or Reveleris® Prep Purification system, please refer to the operating manual.

Figure 9-5: Low Pressure Error screen

The Reveleris® Dry Air Supply air compressor pump is turned on only as needed during the operation of the Reveleris® X2 flash chromatography or Reveleris® Prep purification system. Air is used in the Reveleris® X2 flash chromatography / Reveleris® Prep purification system to operate the evaporative light scattering detector (ELSD), and to purge solvents out of spent cartridges and sample loaders. When the ELSD is not used during a run, or the instrument is sitting idle, the compressor pump will be turned off.

9.3 MAINTENANCE AND TROUBLESHOOTING

9.3.1 MAINTENANCE

Users m ust be aware of the hazards when performing maintenance. Use appropriate PPE, power down and unplug the system when removing any panel and follow all applicable regulations and your organization’s safety program.

The Reveleris® Dry Air Supply does not require routine maintenance. Once a year or after 1000 hours of use, the coalescing filter may need to be replaced. Please refer to Section 9.5.2 , Spare Parts for part number.

The exterior surfaces of the system should be cleaned with a cloth, using water with a mild detergent on a periodic basis. If mobile phase or sample is accidentally spilled on the system,

it should be removed immediately. If a mild detergent is not able to remove the foreign material, an organic solvent such as methanol or iso-propanol can be used.

On a daily basis, inspect the drain waste container and empty the container when it is full.

9.3.2 TROUBLESHOOTING

Perfor m all trouble shooting activities safely, in accordance with all applicable federal, state and local laws and regulations and in compliance with your organization’s safety program.

Troubleshooting refers to the steps to determine the cause of a problem. The Reveleris® Dry Air Supply consists of several components that include a compressor pump, a particle filter, a moisture removal device, and associated connectors and tubing. The most efficient method of determining the cause of the problem is to perform a systematic investigation into each potential cause.

9.3.3 SOFTWARE AND FIRMWARE ERRORS

In the event of a suspected firmware or software error:

• Log any error messages

• Reboot the instrument and then attempt to operate thesystem

• Contact your BUCHI Service Representative ifrebooting the system does not correct the problem.

When you contact BUCHI, please provide as much information as possible relating to the problem, including what the system was doing before the problem was observed, detailed error messages (the exact message would be best), any abnormal events that were noted, anything which you feel may be of assistance in troubleshooting, the system serial number and software revision. If the error message or problem can be reproduced, please indicate all actions that were done to reach the message or problem.

The operating software of the Reveleris ® X2 Flash Chromatography and the Reveleris ® Prep Purification systems includes a large number of internal procedures that automatically alert the user to any unacceptable situation and will not allow the system to start.

9.3.4 TROUBLESHOOTING STRATEGIES

The Reveleris® X2 flash chromatography and the Reveleris® Prep purification software provide simple troubleshooting steps. Press the info (i) button to obtain the troubleshooting information.

9.3.5 TROUBLESHOOTING CHECKLISTS

The following troubleshooting checklists are provided to assist the operator in determining the cause of a problem and suggest possible remedies. The problems described below are selected on the basis that they are likely occurrences in



normal operation that can be readily resolved by the user. If you encounter problems that cannot be resolved using this troubleshooting section, please contact your BUCHI Service Representative. Troubleshooting guide for the Reveleris® X2flash chromatography or Reveleris® Prep purification system is provided in the system operating manual.

9.4 GENERAL INSTRUMENTATION ISSUES/ERROR MESSAGES

Problem Possible Causes Solution The system does not power up

Power is not being supplied to the system

• Verify that the power cable is plugged into theReveleris® X2 flash chromatography or theReveleris® Prep Purification system

• Verify that the Reveleris® X2 flashchromatography or Reveleris® Prep purificationsystem is turned on

• If all of the above are OK and the instrumentdoes not power up then contact BUCHICustomer Service

Inlet gas pressure out of range – LOW (During Software Starts)

No gas or low gas flow into the Flash system

• Check the gas line connections between theReveleris® Dry Air Supply and the Reveleris®

system for leaks. Tighten fittings if needed• Press the Reset button to clear the error

message. Repeat one more time. The messagewill be cleared after the second time this buttonis pressed if the pressure value is withinspecification

Internal air leak in the Dry Air Supply or the Reveleris® system

• Contact your BUCHI Customer Service

Inlet gas pressure out of range (during operation)

No gas or gas flow is low • Check the gas lines to/in the system for leaks

The pressure gauge inside the Flash system is not set correctly


Inlet gas pressure out of range- HIGH (Before run starts)

Blockage in the gas lines • Check the gas lines in the system for blockages

The pressure gauge inside the Flash system is not set correctly












9.5 APPENDICES

9.5.1 SPECIFICATIONS

REVELERIS® DRY AIR SUPPLY SPECIFICATION

Dimension:

7" W x 7" D x 7" H

17.8cm W x 17.8cm D x 17.8cm H

Weight: 7.4 Ibs (3.4 kg)

Air delivery at atm. Pressure:

15 L/min

Max. Operating Pressure: 105 Psi (7 bar)

DC Motor: 12 V

Operating Current: 5.2 A

Operating temperature: -10°C to 52°C

Air filtration: 0.01 um

Air in temp 20 °C Air in Dew point 20 °C

Air out Dew Point -6.7°C

9.5.2 SPARE PARTS

SPARE PARTS


142127782 Coalescing filter

142127785 Moisture membrane

142127772 Power cable

143112342 Drain Tubing (4 ft)

143112416 1/8” PTFE Tubing (6 ft)

143112412 1/8” PTFE Tubing (10 ft)

143112522 1/8” Flange free PEEK fitting (10/pk)

143112418 1/8” Flange free ferrule (10/pk)

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