carbohydrate removal accessory (cra) manual for the carbohydrate removal accessory (cra) ... 11 2.6....

for

Carbohydrate Removal Accessory(CRA)

Kit # 064418

Anuta

New Stamp

Product Manual for the Carbohydrate Removal Accessory (CRA) using an ICS-3000 and DP Module Page 1 of 17

Document No. 065778-01 ©2008 Dionex Corporation November 2008

PRODUCT MANUAL

for the

Carbohydrate Removal Accessory (CRA)Installation on an ICS-3000 System

Configured with Pump 1 plus Pump 2 of the DP ModuleAnd with Injector 1 and Injector 2 of the DC Module

(Kit P/N 064418)

©Dionex Corporation 2008

Document No. 065778

Revision 01November 10, 2008



TABLE OF CONTENTS

SECTION 1 – INTRODUCTION ...........................................................................................................31.1. Detailed Description of CRA Components ...............................................................................................4

SECTION 2 – INSTALLATION.............................................................................................................52.1. Before the Installation.................................................................................................................................52.2. Installing the Transfer Pump.....................................................................................................................52.3. Preparing the AAA System ........................................................................................................................62.4.1. Configuring Injector Valve 2 of ICS-3000 DC as a Transfer Valve .............................................................................72.4.2. Configuring Pump 2 of ICS-3000 DP as a Transfer Pump............................................................................................82.5. Liquid Connections .....................................................................................................................................92.5.1. Wet Test to Assign the Valve Positions to Chromeleon Commands ..........................................................................102.5.2. Pressure Test to Detect Leaks and Obstructions..........................................................................................................112.6. Testing Recovery of Amino Acids and Efficiency of Carbohydrate Removal ....................................12

APPENDIX A - PROGRAM FOR DIRECT INJECTION ................................................................14

APPENDIX B - PROGRAM FOR CARBOHYDRATE ELIMINATION AND SUBSEQUENT SEPARATION OF AMINO ACIDS ..............................................15



SECTION 1 – INTRODUCTION

The following instructions are for the installation and use of the Carbohydrate Removal Accessory (CRA), an Accessory Kitfor use with Dionex ICS-3000 AAA Direct systems. The CRA is used to remove carbohydrate interferences to amino acidanalysis.

This fully automated on-line technique is based on retaining amino acids on a Carbohydrate Removal Column (CRC)containing cation exchange resin prior to their separation by anion exchange. The carbohydrates are not retained on the CRCand are rinsed out before the transfer of amino acids onto the AminoPac PA10.*

The ICS-3000 AAA Direct system has to be modified to include the CRA for on-line carbohydrate removal. The CRA consistsof a short cation exchange column (the CRC), and a set of PEEK tubing of specified length and internal diameter. The ICS-3000 requires a DC (P/N 061793) with two six-port Rheodyne injection valves in the microbore configuration and a DP withone quaternary gradient pump and one isocratic pump (P/N 061710) with degasser.The replacement CRC column is available as a 2 x 15mm column (P/N 068598).

Assistance is available for any problem during the shipment or operation of Dionex instrumentation and columnsthrough the Dionex North America Technical Call Center at 1-800-DIONEX-0 (1-800-346-6390) or through any of theDionex Offices listed in "Dionex Worldwide Offices" on the Dionex Reference Library CD-ROM.

* See P. Jandik, J. Cheng, D. Jensen, S. Manz and N. Avdalovic, J. Chromatogr. B., 758 (2001) 189 - 196 for additional details.



1.1. Detailed Description of CRA Components

Check this list carefully. All parts must be available before attempting a CRA installation.

• Dionex Reference Library CD-ROM including CRA Product Manual (P/N 053891) • 1 Carbohydrate Removal Column Filled with Cation Exchange Material (P/N 068598) • 5 Feet of 0.005 inch (152 cm of 0.127 mm ID) Red PEEK Tubing (P/N 052310) (to make T1, T2, and T3 tubing

segments)• 5 Feet of 0.030 inch (152 cm of 0.762 mm ID) Green PEEK Tubing (P/N 044777) • 5 Feet of 0.010 inch (152 cm of 0.254 mm ID) Black PEEK Tubing (P/N 052306) • 5 10-32 Couplers • 15 Fittings - 10-32 • 15 Ferules - Double Cone • Clean Glass Container (V =0.2 to 1 L) for Transfer Fluid (water or 0.02% formic acid) • IonPac MFC-1 3x27 mm P/N 037017

Preparation of segments of PEEK tubing: Only T1 and T2 need to be cut exactly as specified.

• T1: Red PEEK Tubing 1 length: 60 cm (23.622 inches) (Injector Valve port 5 to Transfer Valve (TV) port 6) • T2: Red PEEK Tubing 2 length: 18 cm (7.087 inches) (TV port 1 to outlet of CRC) • T3: Red PEEK Tubing 3 length: 18 cm (7.087 inches) (Inlet of CRC holder to TV port 4) • T4: Red PEEK Tubing 4 (TV port 2 to AminoPac PA10 column inlet) • T5: Black PEEK Tubing 5 (Gradient mixer to TV port 3) • T6: Yellow PEEK Tubing 6 (from IonPac MFC at the end of tubing 7 to injector port 6) • T7: Black PEEK Tubing 7 (Pump 2 of DP to IonPac MFC at tubing 6) • T8: PTFE Tubing 8 (Transfer Fluid container to Pump 2 of ICS-3000 DP) • T9: Green PEEK Tubing 9 (Injector Valve port 3 to waste) • T10: Green PEEK Tubing (TV port 5 to waste)



SECTION 2 – INSTALLATION:

2.1. Before the Installation

We are assuming that your ICS-3000 AAA-Direct system is configured and functioning according to the Dionex AAA-DirectManual. Before installing the CRA accessory, generate one chromatogram of an 8 µM amino acid standard and a secondchromatogram of the 8 µM amino acid standard containing 100 µM glucose. Use the program in Appendix A if you haveChromeleon 6.8.

2.2. Installing the Transfer Pump

The second isocratic pump of the ICS-3000 DP is used as a transfer pump. Please refer to the ICS-3000 operator's manualregarding the installation and set-up of the DP pump. The following steps describe how to connect Pump 2 of the DP to theInjector Valve:

A. The black PEEK tubing T7 is installed between Pump 2 and IonPac MFC-1 inlet.

B. The yellow tubing T6 is installed between the outlet of IonPac MFC-1 and port 6 of Injector Valve (Use ca. 1 meter ofyellow tubing to achieve a backpressure >500 psi when pumping transfer fluid at 0.080 mL/min). We recommend rinsingthe CRC cartridge with at least 7 mL of 0.02% formic acid prior to its initial use. This initial rinse can be performed withthe cartridge already installed at the Transfer Valve and using the connections just described.

Figure 1Liquid connections from Pump 1 and Pump2 of ICS-3000 DP

T7

Ion Pac MFC-1

Pump 2

Pump 1

T6

Pump 1 to Injector



2.3. Preparing the AAA System

A. Switch off the cell voltage from the electrochemical detector and stop the pump. B. Remove the entire length of black PEEK tubing between the gradient mixer and injector valve from the system. C. Remove the entire length of the red PEEK tubing connecting the injection valve to the AminoPac PA10 Guard column. D. Remove the Guard column and the red tubing connecting it to the inlet of the AminoPac PA 10 analytical column. E. Seal the Guard column by connecting the loose end of the red tubing into the Guard column outlet. Do not leave the

AminoPac PA 10 column inlet or outlet open for longer than ca. 5-10 minutes. Use blind plugs in case you have to interruptthe installation at this point.

Figure 2 Liquid Connections between Injector Valve and Transfer Valve

T1 CRC T6

T4Transfer ValveInjector Valve



2.4.1. Configuring Injector Valve 2 of ICS-3000 DC as a Transfer Valve

Go to the Chromeleon configuration and ensure that the Injector Valve 2 (InjectValve_2) is activated by checking the checkbox. In the column "Controlled by" specify "DC." Note: "Controlled by AS" is a default setting.



2.4.2. Configuring Pump 2 of ICS-3000 DP as a Transfer Pump

Go to Chromeleon configuration and ensure that both pumps are assigned to the same time base to which both Injector Valvesof ICS-3000 have also been assigned.



2.5. Liquid Connections 1st and 2nd pump of DP and Adjusting/Confirming Transfer Flow Rate

Confirm that the tubing connections in the injection valve are as follows (Figure 3): 25 µL sample loop between ports 1 and 4,with the injection port and waste tubing at ports 2 and 3 respectively. Ports 5 and 6 should be open after the removal of red andblack tubing as described in Section 2.4.

Figure 3 Liquid Connections

IMPORTANT T1, T2 and T3 should have the exact length that is specified in the Introduction.

Connect the tubing T6 from the IonPac MFC-1 outlet (at Transfer Pump) to port 6. Complete all tubing connections shown inFigure 3 but do not install the CRC yet.

Figure 4 shows the two positions of the Transfer Valve. In the Delay position, sample is loaded onto the CRC. In the In-Lineportion the sample is being transferred to the analytical column.

1 1

2

43

56

2

34

5

6Tubing 1

Tubing 2

Tubing 3

Tubing 4

Tubing 6

Tubing 5Tubing 10Tubing 9

CRC

AminoPac PA10

1 1

2

43

56

2

34

5

6Tubing 1

Tubing 2

Tubing 3

Tubing 4

Tubing 6


1 1

2

43

56

2

34

5

6Tubing 1

Tubing 2

Tubing 3

Tubing 4

Tubing 6


CRC

AminoPac PA10pmuP

refsnarTdiulF

reniatnoC 7gnibuT

caPnoI1-CFM

Injection Valve Transfer Valve

)EFTP(8gnibuT



Figure 4 Two Positions of Transfer Valve

The CRC is connected to the Transfer Pump when the Transfer Valve is in the "Delay" position. When thetransfer valve is in the "In-Line" position, the transfer fluid flows directly to port 6 and from there to waste(T10 green). At this time, the eluent from the ICS-3000 pump flows through the CRC. The injection valveremains in the inject position most of the time during system operation. The injection valve is rotated into theload position only when the autosampler is loading the sample loop installed between ports 1 and 4.

2.5.1. Wet Test to Assign the Valve Positions to Chromeleon Commands

The Rheodyne valve can be in one of its two possible positions. We now have to carry out a wet test to verify that the valve-position-to-command assignment is correct (see Figure 5).

A. Start a constant flow (80 µL/min) from the Transfer Pump.

IMPORTANT Do not use the priming flow rate.

B. Carry out several changes of InjectValve_2 (Transfer Valve) between Load Position and Inject Position using Chromeleoncommands and verify that at Load Position, the Transfer Fluid is exiting from tubing T2, and at Inject Position, the samefluid is exiting from tubing T10 (see Figure 5).

C. Correct the command assignment if necessary.

1 1

2

43

56

2

34

5

6

Delay Inject

1 1

2

43

56

2

34

5

6

Delay Inject

1 1

2

43

56

2

34

5

6

In-Line Inject

1 1

2

43

56

2

34

5

6

In-Line Inject



Figure 5 Assigning the Valve Positions to Chromeleon Commands

2.5.2. Pressure Test to Detect Leaks and Obstructions

By conducting this test, users can detect leaks and obstructions in the flow path.

A. Install the CRC column and make sure that all other connections that are shown in Figure 3 are in place. The column inletshould be oriented toward port 4 of the Transfer Valve. This ensures that the system pressure (In-Line) is applied onto thecolumn in the correct direction.

B. Start the ICS-3000 pump, and the constant flow from the Transfer Pump (80 µL/min). Turn on the electrochemical cellvoltage.

C. Note the pressure reading on the ICS-3000 pump.

D. Verify the set flow rate of the Transfer Fluid by collecting and weighing fractions of liquid captured at tubing T10 (see figure 5).

E. Rotate the Transfer Valve using a Chromeleon command. Verify that the pressure change is <200 psi and remains the sameduring several valve rotations. Verify that there are no upsets in the electrochemical detector reading.

F. Check for leaks.

At the low flow rates of the Transfer Fluid, the leaks are slow to develop. Allow sufficient time for spotting theleaks.

1

2

34

5

6

Tubing T2

Delay = Load PositionLiquid exiting T2

1

2

34

5

6

Tubing T2

In-Line = Inject PositionLiquid exiting T10

Tubing T10Tubing T10

Tubing T1Tubing T1



2.6. Testing Recovery of Amino Acids and Efficiency of Carbohydrate Removal

In the original version of this carbohydrate removal method, the CRC was regenerated by injecting 25 µL of 1 M HCl before each injection of a sample (see the literature reference provided in the Introduction). Purewater was used as a Transfer Fluid and each sample run required two independent chromatographic runs in asequence (First run HCl, second run: sample).

We are now recommending the use of 0.02% formic acid as the Transfer Fluid. The injections of 1 M HCl areno longer necessary and the programming of sample runs is greatly simplified. It is also possible to use higherTransfer Fluid flow rates and longer "Delay" times (see Figure 4) to accommodate the removal of higherconcentrations of interfering carbohydrates.

A. Make sure that the sample vials (amino acids with and without an added excess of glucose) (Section 1) and an additionalvial with water are placed in the autosampler tray. Add another vial containing 100 µM glucose solution. At this point, theevaluation sequence of your browser should already contain the chromatograms of samples 1 and 2. (Figure 6: Lines 1 and2).

B. Program a sequence of additional injections (samples 3-6 in Figure 6) for injecting AA standard with and without an addedlevel of glucose and also a pure glucose solution. Inject a water blank first (Figure 6, sample 3) to check thechromatographic background of the system configured for carbohydrate removal.

Should the injection of glucose solution (sample 6) show too high residual level of that compound, run a series ofinjections with gradually increasing Delay times (Samples 7-12).

If necessary, rerun the amino acid standard at an optimized Delay time. Calculate the recoveries of all amino acids for the optimized Delay time.

Figure 6Evaluation Sequence



Figure 7 Samples 1 and 5 from Figure 6

Comparison of Chromatograms with and without CRC Installed

0.3 4.0 8.0 12.0 16.0 20.0 24.0 30.0

Time (Minutes)

Response(nC)



APPENDIX A - PROGRAM FOR DIRECT INJECTION (76_24Method without CRA.pgm)The direct injection program "76_24Method without CRA.pgm" can be created from the 1p8 CRA 76_24.pgm (see AppendixB) by placing a semicolon in front of the transfer valve switching command:

The green font color indicates that the command has been turned off. Remember to stop the flow of the transfer fluid frompump 2 by modifying the corresponding command

The AminoPac 10 column and Pump1 connections have to be moved from transfer valve Ports 2 and 3 toinjector valve ports 5 and 6 to enable direct injections of samples onto AminoPac PA10 column.

1.800 InjectionValve_2.InjectPosition Duration=45.00

Pump_2.Flow = 0.00



APPENDIX B - PROGRAM FOR CARBOHYDRATE ELIMINATION AND SUBSEQUENT SEPARATION OF AMINO ACIDS (1p8 CRA 76_24.pgm)

Sampler.AcquireExclusiveAccess Sampler_DiverterValve.Position_1 Compartment_TC.AcquireExclusiveAccess Pump_1.Pressure.LowerLimit = 0 [psi] Pump_1.Pressure.UpperLimit = 3000 [psi] Pump_2.Pressure.LowerLimit = 0 [psi] Pump_2.Pressure.UpperLimit = 3000 [psi] Pump_1.%A.Equate = "DI H20' Pump_1.%B.Equate = "%B 250mM NaOH" Pump_1.%C.Equate = "%C 1MAcBuffer" Pump_1.%D.Equate = "%D 3.5 M HAc" Pump_2.%A.Equate = "0.002% formic acid" Flush Volume = 100 Wait FlushState NeedleHeight = 1 [mm] CutSegmentVolume = 5 [µl] SyringeSpeed = 3 TrayTemperature = 10 CycleTime = 0 [min] WaitForTemperature = False Pump_1_Pressure.Step = Auto Pump_1_Pressure.Average = On Pump_2_Pressure.Step = Auto Pump_2_Pressure.Average = On EDet1.Mode = IntAmp EDet1.CellControl = On Data_Collection_Rate = 2.0 [Hz] Electrode = pH pH.UpperLimit = 13.0 pH.LowerLimit = 10.0 ;WaveformName = "Amino Acids (pH,Ag,AgCl Reference)" Waveform Time = 0.000, Potential = 0.130, GainRegion = Off, Ramp = On, Integration = Off Waveform Time = 0.040, Potential = 0.130, GainRegion = Off, Ramp = On, Integration = Off Waveform Time = 0.050, Potential = 0.330, GainRegion = Off, Ramp = On, Integration = Off Waveform Time = 0.210, Potential = 0.330, GainRegion = On, Ramp = On, Integration = On Waveform Time = 0.220, Potential = 0.550, GainRegion = On, Ramp = On, Integration = On Waveform Time = 0.460, Potential = 0.550, GainRegion = On, Ramp = On, Integration = On Waveform Time = 0.470, Potential = 0.330, GainRegion = On, Ramp = On, Integration = On Waveform Time = 0.560, Potential = 0.330, GainRegion = Off, Ramp = On, Integration = Off Waveform Time = 0.570, Potential = -1.670, GainRegion = Off, Ramp = On, Integration = OffWaveform Time = 0.580, Potential = -1.670, GainRegion = Off, Ramp = On, Integration = OffWaveform Time = 0.590, Potential = 0.930, GainRegion = Off, Ramp = On, Integration = Off Waveform Time = 0.60, Potential=0.13, LastStep=On, Ramp=On, GainRegion=Off, Integration=Off



Column_TC.AcquireExclusiveAccess Column_TC.Mode = On Column_TC.TemperatureSet = 30.00 [°C] Compartment_TC.Mode = On Compartment_TC.TemperatureSet = 25.00 [°C] Wait Compartment_TC.TemperatureState Wait Column_TC.TemperatureState Wait SampleReady Pump_2.Flow = 0.08

0.000 Pump_1.Flow = 0.25 Pump_1.%B = 24.0 Pump_1.%C = 0.0 Pump_1.%D = 0.0 Pump_1.Curve = 5 Load Inject ED_1.AcqOn 0.2 Sampler.ReleaseExclusiveAccess 1.800 InjectValve_2.InjectPosition Duration=45.00 2.000 Pump_1.Flow = 0.25 Pump_1.%B = 24.0 Pump_1.%C = 0.0 Pump_1.%D = 0.0 Pump_1.Curve = 5 8.000 Pump_1.Flow = 0.25 Pump_1.%B = 36.0 Pump_1.%C = 0.0 Pump_1.%D = 0.0 Pump_1.Curve = 8 11.000 Pump_1.Flow = 0.25 Pump_1.%B = 36.0 Pump_1.%C = 0.0 Pump_1.%D = 0.0 Pump_1.Curve = 8 18.000 Pump_1.Flow = 0.25 Pump_1.%B = 20.0 Pump_1.%C = 40.0 Pump_1.%D = 0.0 Pump_1.Curve = 8 21.000 Pump_1.Flow = 0.25 Pump_1.%B = 16.0 Pump_1.%C = 40.0 Pump_1.%D = 0.0 Pump_1.Curve = 5



23.000 Pump_1.Flow = 0.25 Pump_1.%B = 16.0 Pump_1.%C = 70.0 Pump_1.%D = 0.0 Pump_1.Curve = 8 42.000 Pump_1.Flow = 0.25 Pump_1.%B = 16.0 Pump_1.%C = 70.0 Pump_1.%D = 0.0 Pump_1.Curve = 8 42.100 Pump_1.Flow = 0.25 Pump_1.%B = 80.0 Pump_1.%C = 0.0 Pump_1.%D = 0.0 Pump_1.Curve = 8 44.100 Pump_1.Flow = 0.25 Pump_1.%B = 80.0 Pump_1.%C = 0.0 Pump_1.%D = 0.0 Pump_1.Curve = 5 44.200 Pump_1.Flow = 0.25 Pump_1.%B = 24.0 Pump_1.%C = 0.0 Pump_1.%D = 0.0 Pump_1.Curve = 5 74.000 ED_1.AcqOff Pump_1.Flow = 0.25 Pump_1.%B = 24.0 Pump_1.%C = 0.0 Pump_1.%D = 0.0 Pump_1.Curve = 5 Compartment_TC.ReleaseExclusiveAccess Column_TC.ReleaseExclusiveAccess End

carbohydrate removal accessory (cra) manual for the carbohydrate removal accessory (cra) ... 11 2.6....

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