Phase Separations Phase Separations Customer Education Pro gram Customer Education Pro gram
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Advanced HPLC Column Selection Using the Hydrophobicity Chart
Time (min) 50
neutralbase
neutral
base0
Copyright 1998 Phase Separations
Copyright 1998 Phase Separations
Course Librar yCourse Librar y* Strategies and Techniques for Faster HPLC
Methods Development
* Methods Development Optimization Software --HIPAC
* Advanced HPLC Column Selection Using The Hydrophobicity Chart
* Solid Phase Extraction Technology
* Selecting Optimum HPLC Column Dimensions and Stationary Phase Particle Size
* Troubleshooting Common HPLC Problems
* Purchasing High Performance Chromatographic Supplies
* Understanding RP-HPLC Separations -- Effect of Silica Type/Activity
* Reversed-Phase HPLC Methods Development
Copyright 1998 Phase Separations
Course List - Order Number & DurationCourse List - Order Number & Duration* Strategies and Techniques for Faster HPLC PSL908200 2.5 hours
Methods Development
*Methods Development Optization Software -- HIPAC PSL908201 45min
* Advanced HPLC Column Characterization & Selection PSL908203 1.5 hoursUsing The Hydrophobicity Chart
* Solid Phase Extraction Technology PSL908204 4 hours * Selecting Optimum HPLC Column Dimensions and PSL908205 1 hour
Stationary Phase Particle Size
* Troubleshooting Common HPLC Problems PSL908206 2 hours
* Purchasing High Performance Chromatographic PSL908207 7 hours Supplies
* Understanding RP-HPLC Separations PSL908208 1.5 hours -- Effect of Silica Type/Activity
*Reversed-Phase HPLC Methods Development PSL908202 7 hours
...because Chromatography is still a ScienceSM
Strategies & Techniques forFaster HPLC Methods Development
A Certificate of Achievement will be sent to allattendees, which can be used for your files asdocumentation of this training program.
Order Course # PSL908200
This course is designed for those individualsdeveloping new reversed-phase HPLC methodsor those planning to improve existing methods inorder to increase the methods' sample through-put, robustness and cost effectiveness. A logicaland efficient methods development process isdescribed which covers column selection basedon analyte type and sensitivity requirementsutilizing different selectivities of chromatographicpackings to solve difficult application problems.We will explore solvent system optimizationstrategies, as well as review the role thatMethod Development Optimization Software canplay in this process. Key examples are shown,and each attendee will receive a complete set ofthe slides in hard- copy form to keep for futurereference.
Duration: 2.5 hours
Course Abstract:
© 1998 Phase Separations
Strate gies & Techniques for Faster
HPLC Methods Development
presents:
Copyright 1997 Phase Seprations
Copyright 1997 Phase Seprations
Packings forPackings forReversed-Phase ChromatographyReversed-Phase Chromatography
silica-based vs. polymericchain length/typesilica qualityendcappingpackings with embedded polar function
use a well-bonded, endcapped C8 based on a high-purity silica from a reputable manufacturer!
Copyright 1997 Phase Seprations
Reduce particle size Reduce particle size
Use shorter columnsUse shorter columns
Maintain constant ratio of column length to Maintain constant ratio of column length to particle sizeparticle size
How to achieve shorter analysis How to achieve shorter analysis times -- times -- without sacrificing resolution without sacrificing resolution and efficiency and efficiency
a. 5 µm 3.9 x 150 mm; Symmetry® C
Chlordiazepoxide (x) Degradation Products
Faster Anal ysis for Stabilit y Studies
Columns: 18
b., c. 3.5 µm 4.6 x 100 mmMobile Phase: acetonitrile/methanol/
TETA-MeCOOH pH 7.0Detection: UV at 240 nmSample: 100 µL of 40 µg/mL
10.00 20.00 30.00
10.00 20.00 30.00
Minutes
a. 5 µm 0.7 mL/min
b. 3.5 µm 1.0 mL/min
10.00 20.00
c. 3.5 µm 1.4 mL/min
2
2
2
Peak 2Analysis USP Times Plates
a. 40 min 11,240b. 30 min 13,400c. 20 min 12,500
x
x
x
D. J. PhillipsCopyright 1997 Phase Seprations
water/methanol/glacial
USP Plates USP Tailing Peak 4
a) 4.6 mm ID 5780 1.1 b) 3.9 mm ID 5710 1.1 c) 3.0 mm ID 4350 1.2 d) 2.1 mm ID 4140 1.2
Conditions:Column: Symmetry® C 18 5 µmMobile Phase:
acetic acid 79:20:1Flow Rates: a) 1.4 mL/min
b) 1.0 mL/minc) 0.6 mL/mind) 0.29 mL/min
Sample: mixture of 6 sulfa drugs,10 to 39 µg/mL
Injection vol.: a) 14 µL, b) 10 µL, c) 6 µL and d) 3 µL
4
0.00 10.00Minutes
0.00 10.00
0.00 10.00
a)
4
0.00 10.00
4
4
c)
d)
b)
Performance of Different DiameterColumns at Equal Linear Velocities
4.6 mm x 150 mm
3.9 mm x 150 mm
3.0 mm x 150 mm
2.1 mm x 150 mm
D. J. PhillipsCopyright 1997 Phase Seprations
Method Development Strate gy
Gradient at low pH Starting Solvent
Gradient at high pH Starting Solvent
Gradient withSecond Slope
Isocratic Run inStarting Solvent
Isocratic Run inSecond Solvent
Isocratic Run inThird Solvent
Mixture Optimization
Calculate Isocratic Mobile Phase
Calculate Isoeluotropic Mobile Phases
Copyright 1997 Phase Seprations
Limit of Quantitation
Method Validation Criteria
Precision
Accuracy
Limit of Detection
Specificity
Linearity and Range
Ruggedness
Robustness
MethodValidation
Copyright 1997 Phase Seprations
Copyright 1997 Phase Seprations
Method RuggednessMethod Ruggedness
Anal yst to Anal ystAnal yst to Anal ystInstrument to InstrumentInstrument to InstrumentLab to LabLab to LabColumn to ColumnColumn to ColumnBatch to BatchBatch to Batch
Copyright 1997 Phase Seprations
Method RobustnessMethod Robustness
Content of Or ganic SolventContent of Or ganic SolventIonic Stren gthIonic Stren gthpHpHTemperatureTemperatureOther AdditivesOther Additives
...because Chromatography is still a ScienceSM
Methods Development Optimization Software – HIPAC™
Detailed use of the software is shown, as wellas examples of the thorough, tutorial informationincluded in the software. This tutorial information,and the use of the built- in chromatographicoptimization examples, can also be effectivelyused in the training of lab personnel new to HPLCtechnology.
Key examples are shown, and each attendee willreceive a complete set of the slides inhard- copy form to keep for future reference.
A Certificate of Achievement will be sent to allattendees, which can be used for your files asdocumentation of this training program.
Order Course # PSL908201
One of the most significant trends today is theneed for rapid HPLC Methods Developmentefforts. Recently, a tool to aid the analyticalchemist in reducing the time necessarydeveloping new applications has becomeavailable. This tool utilizes the chromatographicexperience of analytical chemists combined withdesktop computers and software, based onchromatographic behavior models, to rapidlyoptimize application conditions. In addition, thistool can be used to assess the robustness ofpotential applications, without the need for manymultiple trial runs.
A review of the HIPAC™ brand softwaredesigned for the optimization of binary, isocraticreversed- phase applications, and systemoptimization is given.
Duration: 45 minutes
Course Abstract:
© 1998 Phase Separations
HIPACHIPACTMTM BB andand SS
BBinar y-Isocratic/inar y-Isocratic/ SSystem ystem OptimizationOptimization
HPLC Methods Development HPLC Methods Development Optimization SoftwareOptimization Software
Copyright 1997 Phase Seprations
Copyright 1997 Phase Seprations
Software Package Designed To Combine A Software Package Designed To Combine A Chromatographers' ExperienceChromatographers' Experience , , Experimental DataExperimental Data And And HPLC TheoryHPLC Theory To Speed Methods Development Process To Speed Methods Development Process
Chromatographers'Chromatographers'ExperienceExperience
ExperimentalExperimental Fast HPLC Fast HPLC DataData Theory Processing Theory Processing
(Software)(Software)
HIPACHIPACTMTM Chromato graph y Optimization SoftwareChromato graph y Optimization Software
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Copyright 1997 Phase Seprations
Complete Chromato graph y Methods Complete Chromato graph y Methods Development Software Packa ge Available Development Software Packa ge Available In In Individual ModulesIndividual Modules Targeted For The Targeted For The Type Of Applications Type Of Applications In Your LabIn Your Lab
* HIPAC B* HIPAC B Isocratic Reversed-Phase And Isocratic Reversed-Phase And Binary Normal-PhaseBinary Normal-Phase
* HIPAC S* HIPAC S Optimize System And Column Optimize System And Column ParametersParameters
* HIPAC G* HIPAC G Reversed-Phase Binary GradientsReversed-Phase Binary Gradients
* HIPAC TQ* HIPAC TQ Ternary And Quarternary IsocraticTernary And Quarternary Isocratic
HIPACTM Chromato graph y Optimization Software
Copyright 1997 Phase Seprations
Quickly Determines The Quickly Determines The Optimum Conditions Optimum Conditions For Your Application Based On Your Inputs For Your Application Based On Your Inputs
HIPACTM Chromato graph y Optimization Software
HIPACTM Chromato graph y Optimization Software
Copyright 1997 Phase Seprations
Copyright 1997 Phase Seprations
Isoeluotropic Mobile Phase SelectionIsoeluotropic Mobile Phase Selection
HIPACTM Chromato graph y Optimization Software
HIPACTM Chromato graph y Optimization Software
Copyright 1997 Phase Seprations
HIPACTM Chromato graph y Optimization Software
Copyright 1997 Phase Seprations
...because Chromatography is still a ScienceSM
Advanced HPLC Column Characterization & SelectionUsing the Hydrophobicity Chart
This course provides the chemist with a completeunderstanding of chromatographic particletechnology and performance. A wide range ofHPLC columns from different vendors wereevaluated. The development of the performancetest, and how the chart is constructed isexplained in detail. Examples of using the chart tosolve application problems are given.
A complete booklet of the course presentationmaterials will be furnished for future reference.
A Certificate of Achievement will be given to allattendees, which documents your participation inthis training program.
Order Course # PSL908203
Today, analytical chemists are frequently facedwith the very challenging assignment ofdeveloping new HPLC methods, which aresimple, rugged and robust, all in a short period oftime. Traditional methods development techniques,coupled with the improper selection of HPLCcolumn, could result in the spending of muchunnecessary time to achieve the goals of thedevelopment effort, thus delaying the introductionof new products, which can be very costly.
Phase Separations has developed a new,reversed- phase HPLC Column Selection chart,which scientifically differentiates many brands ofHPLC columns. The resultingHYDROPHOBICITY Chart provides the analyticalchemist with a performance ranking for eachcolumn. This allows the chemist to quickly selectthe best column for his/her application. Withproper use, this selection can result in muchfaster methods development success, due to use
Duration: 1.5 hours
Course Abstract:
© 1998 Phase Separations
...because Chromatography is still a ScienceSM
Reversed-Phase HPLC Methods Development
Examples are given, and a complete set ofhandouts is provided for future reference.
A Certificate of Achievement will be given to allattendees, which documents your participation inthis training program.
Order Course # PSL908202
This is a full day course is designed to providecritical information important to those developingnew methods, as well as those redevelopingexisting methods to improve performance in theareas of sensitivity, robustness and samplethroughput.
Key topics include:
• Impact of Particle Technology onChromatographic Performance
• Resolution• Suggested Methods Development Strategy• Benefits of Optimizing
Column Dimensions and Particle Size• Optimization Software• Troubleshooting Tips
Duration: 7 hours
Course Abstract:
© 1998 Phase Separations
Advanced HPLC Column Advanced HPLC Column Characterization & Selection Characterization & Selection
Usin g the Hydrophobicity ChartUsin g the Hydrophobicity Chart
. . . .. . . . . . . .
... .. .. . . . . ... .
.. . .. . . .
Time (min)
50
neutral
base
neutral
base
0
Copyright 1997 Phase Seprations
Copyright 1997 Phase Seprations
Silica Pore StructureSilica Pore Structure* Porous Silica
> 99% of Surface Area in the Pores
* Pore Size Surface Area
SA ~ 3
SA - Surface Area, m 2/gSV - Specific Pore Volume, mL/gPD - Pore Diameter, or nm
Typical Surface Area 100 -- 300 m 2/g
SVPD
10010nmÅ
Å
Copyright 1997 Phase Seprations
Silanol TypesSilanol Types
H Si
O O
Si Si Si
O O O O O
Vicinal Vicinal
GeminalGeminal
LoneLone
OH OH
Si
O O
H H
O O
Si Si
O O O
Copyright 1997 Phase Seprations
Bonded Phase: Trifunctional S ynthesis
OH + SiCl
Cl
Cl
+ HCl
(Hydrolysis)
Si cc
c cc
c cc
Sio
o
Sio
o
o
Si
o
OH
Si
Si
SiSi
cc
c c c c cc
cc
c c c c cc
cc
c c c c cc
Copyright 1997 Phase Seprations
CC1818 Bonded Pore Bonded Pore
Si - OH = Silanol GroupSi-CCCCCCCCCCCCCCCCCC = C1825
Si - OH
Si - OH
Si - OH
Si -
OH S
i - O
H
50
CH
3
Si -
O -
Si -
CH
3
C
H3
CH
3
Si -
O -
Si -
CH
3
C
H3
CH3
Si - O - Si -CH3
CH3
CH3Si - O - Si -CH3 CH3 CH3
Si - O - Si -CH3 CH3
Endcapped Silanol
Si-CCCCCCCCCCCCCCCCCCSi-CCCCCCCCCCCCCCCCCC
Si-CCCCCCCCCCCCCCCCCC
Si-CCCCCCCCCCCCCCCCCC
Si-C
CC
CC
CC
CC
CC
CC
CC
CC
C
Si-C
CC
CC
CC
CC
CC
CC
CC
CC
C
Si-C
CC
CC
CC
CC
CC
CC
CC
CC
C
Si-C
CC
CC
CC
CC
CC
CC
CC
CC
C
Å
Å
Copyright 1997 Phase Seprations
Ligand Density (Surface Covera ge)Ligand Density (Surface Covera ge)
χ = %C
100 SA %C100 ]1 - [ MW - 1
nC 12
= µmoles/m 2
* Better measure of material's characteristics
SA - Specific Surface Area %C - % Carbon LoadMW - Molecular Weight of Ligand nC - # of Carbon Atoms in Ligand
Ligand Density Primary Ligand Density Silanols
Ligand Density Hydrolytic Stability
Surface Area Ligand Density
Copyright 1997 Phase Seprations
ION EXCHANGEION EXCHANGE
pH 14
pH 1
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% Ionized
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pH 12.6 Neutral
2.7 Neutral
8.6 ~100%
6.7 ~100%pH 7
Cation
Anion
Copyright 1997 Phase Seprations
Terminolo gyTerminolo gySurface Silanol GroupsSurface Silanol Groups
* Changes surface charge as pH 2 7* Changes surface charge as pH 2 7
OHOH OO++ HH++
SiSi Si Si
Behaves as a Cation ExchangerBehaves as a Cation Exchanger(pH 2)(pH 2) (pH 7)(pH 7)
Copyright 1997 Phase Seprations
Tailin g vs. Buffer pH
Buffer pH
Tailing Factor
1
2
3
4
2 3 4 5 6 7 8
Conventional C 18
Modern C 18
Ideal
B. A. Alden, T. H. Walter
Copyright 1997 Phase Seprations
Effect of Mobile Phase pH onSelectivit y
Buffer pH
Ret
. Tim
e
0
4
8
12
3 4 5 6 7 8 9
base
neutralacid
2 4 8
AcidNeutral Neutral
AcidBaseBase
3 4 8
pH 3 pH 8
Copyright 1997 Phase Seprations
Reversed-Phase Selectivity Chart
YMC-Basic
1
10Platinum™ C18
Inertsil® C8
Kromasil® C8Zorbax® XDB C8
Symmetry® C8
Hypersil® BDS C18
Inertsil® ODS-2Prodigy™ C18
Symmetry® C18
Kromasil® C18
YMC J'Sphere H80
Zorbax® Rx C18
Nova-Pak® C18
Zorbax® SB C18
Hypersil® ODS
Alltima™ C18YMC J'Sphere M 80
Waters Spherisorb ® ODS 2µBondapak® C18
Zorbax® SB C8
YMC J'Sphere L 80Lichrosorb ® Select B
Waters Spherisorb ® C8
Nova-Pak® C8
Hypersil® BDS C8
Prodigy™ C8
SymmetryShield ™ RP8
Zorbax® Rx C8
Alltima™ C8
Lichrospher® Select B
Purospher® RP18
Nucleosil® C18
Inertsil® ODS-3
1
Hydrophobicity
Sila
nol A
ctiv
ity
Waters Spherisorb ® ODS 1Resolve® C18
Zorbax® XDB C18
Hypersil Hypurity Elite®
2 3 4 5 6 7 8 9 (Log Scale)
2
3
4
5
6
7
8
9
20
(Log
Sca
le)
Copyright 1997 Phase Seprations
...because Chromatography is still a ScienceSM
Solid Phase Extraction Technology
1. Introduction and theory2. Strategies for use – cleanup, fractionation
and trace concentration3. Chemistries and device configurations4. Experimental techniques and demonstrations5. Method development and application
assistance resources6. Method troubleshooting tips7. Applications using the latest in SPE
technology
A complete booklet of the course presentationmaterials will be furnished for future reference.
A Certificate of Achievement will be issued to allattendees.
Order Course # PSL908204
Solid Phase Extraction (SPE) is a powerful tool forsample preparation and has been growing inpopularity because of the significant benefits inreduced sample preparation cost and increasedsample throughput it can bring to your laboratory.In addition, utilizing a chromatographic bed in asample preparation protocol can solve thedifficult problems of increasing method sensitivity,accuracy and precision for your analyticalapplications.
This seminar has been specifically designed forthe chromatographer and will include:
Duration: 4 hours
Course Abstract:
© 1998 Phase Separations
Solid Phase Extraction Solid Phase Extraction TechnologyTechnology
A Powerful Tool in Sam ple PreparationA Powerful Tool in Sam ple Preparation
Copyright 1997 Phase Seprations
Copyright 1997 Phase Seprations
Solid Phase Extraction (SPE) TechnologySolid Phase Extraction (SPE) Technology
Goals of Sample Preparation
+ Simpler + Improve Accuracy+ More Convenient + More Reproducible+ More Cost Effective + Improve Safety
* Remove Interferences* Make Matrix Compatible with Analysis* Concentrate Sample to Increase Sensitivity* Derivatization* Protect Analytical Column
Copyright 1997 Phase Seprations
Solid Phase Extraction (SPE) TechnologySolid Phase Extraction (SPE) Technology
PrecipitationPrecipitation
Chemical TechniqueChemical Technique
Utilizing the Addition of Utilizing the Addition of a Solvent to the Samplea Solvent to the Samplewhich causes One, or which causes One, or Some of the Compounds Some of the Compounds to Fall Out of Solution to Fall Out of Solution as Particulatesas Particulates
Copyright 1997 Phase Seprations
Solid Phase Extraction (SPE) TechnologySolid Phase Extraction (SPE) Technology
Chemical TechniqueChemical Technique
Where an Immiscible Solvent Where an Immiscible Solvent is Added to the is Added to the SampleSample which which then then Separates intoSeparates into 2 Distinct 2 Distinct Liquid Phases.Liquid Phases. Some Sample Some Sample Analytes will go into the Analytes will go into the Bottom PhaseBottom Phase (Aqueous) (Aqueous) , , Some will Separate into the Some will Separate into the Top Phase,Top Phase, (Organic) (Organic)
Liquid-Liquid Extraction Liquid-Liquid Extraction (LLE)(LLE)
Copyright 1997 Phase Seprations
Solid Phase Extraction (SPE) TechnologySolid Phase Extraction (SPE) Technology
How Does Chromato graph y Work?
Stationar y Phase
Analyte A
Analyte B
Mobile Phase
Copyright 1997 Phase Seprations
Solid Phase Extraction (SPE) TechnologySolid Phase Extraction (SPE) Technology
Chromatographic Bed (Sorbent)
Housing/Body
Filters/Frits
Luer Tip
Copyright 1997 Phase Seprations
Solid Phase Extraction (SPE) TechnologySolid Phase Extraction (SPE) Technology
Vacuum ManifoldsVacuum Manifolds
Copyright 1997 Phase Seprations
Solid Phase Extraction (SPE) TechnologySolid Phase Extraction (SPE) Technology
SPE Strate gies
• Elute the product of interest, retain interferences• want k � 0 for analyte
• want k large for interferences
• Elute interferences, retain product• want k � 0 for interferences• want k large for analyte
* Concentrate product of interest
^ want large for analyte / load large sample volume^ elute concentrated analyte^ enhanced sensitivity
k
Copyright 1997 Phase Seprations
Solid Phase Extraction (SPE) TechnologySolid Phase Extraction (SPE) Technology
The The Chromatographic BedChromatographic Bed In InThe Cartridge Can Perform The Cartridge Can Perform Three Critical Functions:Three Critical Functions:
1)1) Chemical Clean-up (Pure Red) Chemical Clean-up (Pure Red)
2)2) Pre-Concentration (Dark Purple) Pre-Concentration (Dark Purple)
3)3) Fractionation Fractionation (Pure Blue & Pure Red) (Pure Blue & Pure Red)
1 2 3 Solid Phase Extraction Solid Phase Extraction (SPE)(SPE)
Copyright 1997 Phase Seprations
Solid Phase Extraction (SPE) TechnologySolid Phase Extraction (SPE) Technology
Breakthrou gh Study
* Series of Experiments -- Passing Increasing Volumes of Sample Matrix Thru SPE Device
* Follow Elution Protocol* Determine Analytical Results (% Recovery)* Plot Results vs Sample Volume* Determine Maximum Volume for Each
of the Analytes -- Find Maximum Volume for Method
Copyright 1997 Phase Seprations
Solid Phase Extraction (SPE) TechnologySolid Phase Extraction (SPE) Technology
5 10 20 30 40 50
Volume Thru SPE Device (mL)
0
20
40
60
80
100
120
% R
ecov
ery
k = 10k = 30
% Recover y as a Function of k
Copyright 1997 Phase Seprations
Solid Phase Extraction (SPE) TechnologySolid Phase Extraction (SPE) Technology
Mass Balance -- Measuring Analyte Concentration in all Fractions -- Will Show Breakthrough During Loading
Proper LoadSample AnalyteFully RetainedDuring Loading OverLoad
Sample AnalyteBreaks ThroughDuring Loading Poor Conditioning
Drying OutSample AnalyteBreaks ThroughDuring Loading
Poor Recovery
Copyright 1997 Phase Seprations
Solid Phase Extraction (SPE) TechnologySolid Phase Extraction (SPE) Technology
Fraction 1 (Clear)WaterPolar Analytes (Clear)
Fraction 2 8% IPAPure Red
Fraction 335% IPAPure Blue
Fraction 470% IPANon-PolarAnalytes (Clear)Fractionation
Copyright 1997 Phase Seprations
Solid Phase Extraction (SPE) TechnologySolid Phase Extraction (SPE) Technology
Low Concentration Analyte (Light Purple) Initially Retained and Concentrated as Large Sample Volume is Processed
Interferences Discarded
Then Concentrated Analyte isEluted by a Different ElutionSolvent
Trace Concentration
Copyright 1997 Phase Seprations
Methods Development Approach Methods Development Approach Determine Nature of Analytes, and
Sample MatrixSimilar to Existing Method in Lab?
Review SPE Bibliography, and Literature References
for Exact or Similar Applications
Any?
No
Try Conditions - Evaluate for Capacity/ Breakthru, RecoveryReproducibility, Robustness
and RuggednessMeets Goals?
Yes
Validate Method
Yes
No
Determine Method Goals, and Strategy
Call SPE VendorChromatography Mode
Develop Method Conditions
YesNo
Copyright 1997 Phase Seprations
Solid Phase Extraction (SPE) TechnologySolid Phase Extraction (SPE) Technology
Some Causes of Poor Recovery
* Poor/No Condtioning* Drying Out Before Loading* Poor Chromatographic Conditions
- k too Low (Breahthrough)- k too Large (Still Adsorbed)
* Basic Compounds Strongly Retained ByDeprotonated Silica Silanols of Sorbent
* Metals in Silica Based Sorbents Can Interact With Metal Chelator Analytes
Copyright 1997 Phase Seprations
SalineMethanol
Acetonitrile
Spiked Solutions
0
20
40
60
80
100
% N
omin
al C
once
ntra
tion
3 Day 5 Day
Betamethasone Valerate(20 µg/mL)
Room Temperature Stability of Room Temperature Stability of Sample in SolutionsSample in Solutions
0 5 10 15 20 25Minutes
t=0 stock
t=5 days saline
t=5 daysMeOH
t=5 daysMeCN
a
a: betamethasone valerateb: breakdown products
b b
b
b
Copyright 1997 Phase Seprations
Results: TetracyclinesResults: Tetracyclines
Column: SymmetryShield™ RP8, 5 µm, 3.0 x 150 mm
Mobile Phase: 0.1% TFA in Water:Acetonitrile: Methanol (91:7:2)
Detection: UV at 270 nmFlow Rate: 0.9 mL/min.Injection Volume: 20 µLSample Identification:
Peak 1: MinocyclinePeak 2: TetracyclinePeak 3: Demeclocycline (I.S.)
Compound Concentration % Recovery % RSD
Minocycline 2.5 µg/mL 94.8 1.4
Tetracycline 2.5 µg/mL 104 0.55
0.000
0.008
0.012
0.004
AU
0.016
0.020
10.0 30.020.0Minutes
32
1
Cheng
...because Chromatography is still a ScienceSM
Selecting Optimum HPLC Column Dimensionsand Stationary Phase Particle Size
A discussion of the impact of the ratio of columnlength to particle size is given. This critical ratiodetermines the optimum performance of thecolumn in the method, as it relates to efficiency,analysis time and back pressure. Examples aregiven, and a complete set of handouts isprovided for future reference.
A Certificate of Achievement will be given to allattendees, which documents your participation inthis training program.
Order Course # PSL908205
In order to speed the introduction of new productsto market, analytical chemists are now oftenfaced with two additional challenges; analyzingcompounds at lower concentration levels, aswell as analyzing more samples per unit time.Developing new HPLC methods with highsensitivity and high sample throughput isbecoming a significant priority.
Phase Separations has created a new course thatcovers the key elements of optimizing HPLCcolumn dimensions, and stationary phase particlesize to meet these methods development goals. Duration: 1 hour
Course Abstract:
© 1998 Phase Separations
Selectin g Optimum Selectin g Optimum HPLC Column Dimensions HPLC Column Dimensions and and Stationar y Phase Particle SizeStationar y Phase Particle Size
Copyright 1997 Phase Seprations
Copyright 1997 Phase Seprations
Benefits of Optimizin g Column Benefits of Optimizin g Column DimensionsDimensions
Reduction in analysis timeReduction in analysis time with with decreasing column lengthdecreasing column length
Increase in mass sensitivityIncrease in mass sensitivity with with decreasing column diameterdecreasing column diameter
Increase in resolutionIncrease in resolution with increasing ratio of with increasing ratio of column length to particle sizecolumn length to particle size
Increase in loadabilityIncrease in loadability with increasing with increasing column diametercolumn diameter
Copyright 1997 Phase Seprations
Reduce particle sizeReduce particle size Use shorter columnsUse shorter columns
Maintain constant ratio of column Maintain constant ratio of column length to particle sizelength to particle size
How to Achieve Shorter How to Achieve Shorter Analysis Times --Analysis Times --
Without Sacrificin g Resolution and Without Sacrificin g Resolution and Efficienc yEfficienc y
Copyright 1997 Phase Seprations
Ratio of Column Len gth to Ratio of Column Len gth to Particle SizeParticle Size
Determines 3 Key Performance Measures:
* Maximum Column Efficiency
* Shortest Analysis Time for Given Pressure
* Pressure Drop at Given Analysis Time
Copyright 1997 Phase Seprations
Selection of Proper L/dp RatioSelection of Proper L/dp Ratio
Type of Anal ysis L/dp
Difficult > 50,000
Normal ~ 30,000
Fast < 15,000
Column
300mm -- 5µm
150mm -- 5µm
50mm -- 3µm
Copyright 1997 Phase Seprations
0
20
40
60
80
100
120
1 10 100 1000
Analysis Time [min]
Res
olut
ion
10 µm x30 cm
5 µm x15 cm
3 µm x9 cm
1 µm x3 cm
Constant Ratio of Column Len gth Constant Ratio of Column Len gth to Particle Size (30,000)to Particle Size (30,000)
0
2000
4000
6000
8000
10000
12000
14000
0.1 1
Flow-Rate [mL/min]
Pla
te-C
ount
5 µm, 3.9 mm x 150 mm
3.5 µm, 4.6 mm x 100 mm
10100Analysis Time [min]
Performance Characteristics of a 5 µm and a 3.5 µm Symmetry® Column
L/dp~30,000
B. A. Alden, U. D. Neue
Copyright 1997 Phase Seprations
Copyright 1997 Phase Seprations
Enhancin g Sensitivit y
Sensitivity can be enhanced by:
reducing column diameter
decreasing capacity factor
using shorter columns
decreasing detector noise
using more sensitive detection modes
decreasing asymmetry
increasing efficiency
Copyright 1997 Phase Seprations
Solvent Consumption -- Column LengthSolvent Consumption -- Column Length
Flow rate: 1 mL/min
Column Length Elution Time Solvent % Savings
15 cm 6.5 min 6.5 mL --
10 cm 4.5 min 4.5 mL 31%
5 cm 2.5 min 2.5 mL 62%
Copyright 1997 Phase Seprations
Solvent Consumption -- Column DiameterSolvent Consumption -- Column Diameter
Linear Velocity: 0.118 cm/secColumn Length: 15 cmElution Time: 8 min
Column Diameter Flow Rate Solvent % Savings
4.6 mm 1.4 mL/min 11.2 mL --
3.9 mm 1.0 mL/min 8.0mL 28%
3.0 mm 0.6 mL/min 4.8 mL 57%
2.1 mm 0.3 mL/min 2.4 mL 79%
Copyright 1997 Phase Seprations
Instrument Optimization Needed Instrument Optimization Needed for Narrow Bore and Microbore for Narrow Bore and Microbore ColumnsColumnsInstrument Bandspread: 25µl
(normally > 80 µl)
Areas to optimize:
* Detector Flow Cell* Injector Sample Loop* 0.005" Tubing* Perfect Connections* Detector Time Constant < 0.2
Copyright 1997 Phase Seprations
To perform a measurement:- disconnect column from system- connect injector directly to detector
Parameter SettingFlow Rate 1.0 mL/minChart Speed 20 cm/min
Detector Sensitivity 0.5 - 1.0 AUFS
Time Constant 0.2 seconds or less
dilute test mixture 1 to 10 in mobile phase inject 2 to 5 µl of this solution
Performance Monitorin gPerformance Monitorin g
Copyright 1997 Phase Seprations
Using 5 sigma efficiency method, measure the peak width at 4.4% of peak height
Convert to microliters using the following equation:
where:1min/20cm = chart speed1 mL/min = flow rate1000 µL/mL = volume correction factor
Typical LC System should be 100µL +/- 30µL
Microbore System should be no greater than 20µL
Performance Monitorin gPerformance Monitorin g
( ) =( )( ) 100 (µL) 1min 1 mL 1000µL 20 cm min. mL
2cm PW( )
...because Chromatography is still a ScienceSM
Troubleshooting Common HPLC Problems
Key examples are shown, and each attendee willreceive a complete set of the slides in hardcopyform to keep for future reference.
A Certificate of Achievement will be given to allattendees, which documents your participation inthis training program.
Order Course # PSL908206
This course covers all commonly encounteredHPLC problems and is designed for both theusers and the developers of HPLC methods. Itwill assist the chromatographer in spottingpotential problems during the operation of theirHPLC and in eliminating instrument downtime.
Covered subjects include:
• problem prevention (column installation,equilibration, protection, storage)
• performance monitoring (instrument bandspreading performance, column efficiencyperformance)
• examples and troubleshooting of commonproblems
Duration: 2 hours
Course Abstract:
© 1998 Phase Separations
Troubleshootin g Common Troubleshootin g Common HPLC ProblemsHPLC Problems
SM
Copyright 1997 Phase Seprations
Copyright 1997 Phase Seprations
Course OutlineCourse Outline* Column Maintenance Information
* Problem Sources * Column Installation and Equilibration* Column Use* Performance Monitoring* Column Protection* Column Storage* Troubleshooting of Column Problems
- Peak Shape Problems- Retention Time Problems
^ Hydrophobic Collapse- Miscellaneous Problems
Copyright 1997 Phase Seprations
Problem
CHEMISTRY HARDWARECOLUMN/GUARDSOLVENTSAMPLE
PUMPINJECTORDETECTORINTEGRATOR
Always do the easiest thing first:1. Stop flow
2. Remove the column
TroubleshootingTroubleshooting
Copyright 1997 Phase Seprations
0.090 Parker Style
0.130 Waters
other Waters Columns
Waters Spherisorb
Installation and EquilibrationInstallation and Equilibration
Copyright 1997 Phase Seprations
Extra-Column Band SpreadingExtra-Column Band Spreading
Column Connection
Copyright 1997 Phase Seprations
.009"
.020".040"
note the differences of the inner diameter of this tubing
Extra-Column Band SpreadingExtra-Column Band Spreading
Copyright 1997 Phase Seprations
Effect of Connecting Tubing on System Bandspreading
.009"
.020"
.040"
Performance Monitorin gPerformance Monitorin g
sample band dispersion inside tubing
Copyright 1997 Phase Seprations
The Observed Bandwidth (TOT) * Sum of the Bandspreading Contributions
- Column (COL) - Extra-Column (EC) Instrument
components
σ2= σ2
+ σ2
Extra-Column Band SpreadingExtra-Column Band Spreading
TOT COL EC
Copyright 1997 Phase Seprations
To perform a Band Spread measurement:- disconnect column from system- connect injector directly to detector
Parameter SettingFlow Rate 1.0 mL/minChart Speed 20 cm/min
Detector Sensitivity 0.5 - 1.0 AUFS
Time Constant 0.2 seconds or less
dilute test mixture 1 to 10 in mobile phase inject 2 to 5 µl of this solution
Performance MonitoringPerformance Monitoring
Copyright 1997 Phase Seprations
Using 5 sigma efficiency method, measure the peak width at 4.4% of peak height
Convert to microliters using the following equation:
where:1min/20cm = chart speed1 mL/min = flow rate1000 µL/mL = volume correction factor
Typical LC System should be 100µL +/- 30µL
Microbore System should be no greater than 20µL
Performance MonitoringPerformance Monitoring
( ) =( )( ) 100 (µL) 1min 1 mL 1000µL 20 cm min. mL 2cm( PW )
Copyright 1997 Phase Seprations
Extension of column lifetime with Guard Column using a mixture of sulfa drugs as the sampleA. Initial injection on Symmetry C 8 Sentry guard columnB. After 550 injections on same Sentry guard columnC. New Sentry Guard column for injection 551 on analytical column
Column ProtectionColumn Protection
Copyright 1997 Phase Seprations
voids - high back pressure, distorted and/or double peaks
Column CollapseColumn Collapse
Copyright 1997 Phase Seprations
Isocratic LC - Time Constant Differences
left is 0.1 secs right is 10 secsnote the noisy baseline on left chromatogram
Extra Column EffectsExtra Column Effects
Copyright 1997 Phase Seprations
Hydrophobic CollapseHydrophobic CollapseChromatographers have observed complete Chromatographers have observed complete loss of loss of retentionretention when working with low organic mobile phases. when working with low organic mobile phases.
When we investigated this phenomenon we found that When we investigated this phenomenon we found that retention times were retention times were stable for over 20 hrsstable for over 20 hrs (77 injections) (77 injections) using using 100% aqueous 100% aqueous mobile phasemobile phase..
However, when However, when flow was stopped,flow was stopped, then restarted, then restarted, retention retention was lostwas lost. This observation suggested that the mobile phase . This observation suggested that the mobile phase is extruded from the pores when pressure is released from is extruded from the pores when pressure is released from the column.the column.
?
40 min 40 min
Vo
Copyright 1997 Phase Seprations
Retention Time ReproducibilityRetention Time Reproducibility
Solvent Composition- Hydrophobic CollapseTemperaturepHIon Pairin gIonic Stren gthExtraneous PeaksGradient Control
Copyright 1997 Phase Seprations
pH - ControlpH - Control
...because Chromatography is still a ScienceSM
Purchasing High Performance Chromatographic Supplies
This full day course will provide you with thetechnical information you will need to intelligentlydiscuss product specifications and performance,as well as outline critical issues in thedevelopment of an improved purchasing programfor your chromatography supplies. Specificexamples will be given showing how to achievesignificant benefits by organizing yourchromatography purchasing needs in product/performance/ vendor priority.
A complete booklet of the course presentationmaterials will be furnished for future reference.
A Certificate of Achievement will be given to allattendees, which documents your participation inthis training program.
Order Course # PSL908207
This course is designed specifically forPurchasing Professionals to enhance theirunderstanding of the technology ofChromatography as it relates to improving theirability to effectively purchase these supplieswhile meeting corporate goals, such as improvedcustomer satisfaction, and reduced purchasingcosts through vendor consolidation.
Key topics include:
• Chromatography technology / terminology(with a focus on HPLC)[includes live demonstration]
• End user performance needs• Regulatory considerations• Manufacturer / Supplier considerations• Key purchasing specifications Duration: 7 hours
Course Abstract:
© 1998 Phase Separations
Purchasing High Performance Purchasing High Performance Chromatographic SuppliesChromatographic Supplies
A Course For A Course For Purchasing ProfessionalsPurchasing Professionals
Copyright 1997 Phase Seprations
Copyright 1997 Phase Seprations
TopicsTopics
* Chromatography Technology Backgrounder* Chromatography Technology Backgrounder* Terminology* Terminology* Hands-On Chromatographic Experiments* Hands-On Chromatographic Experiments* HPLC Supplies* HPLC Supplies* GC Supplies* GC Supplies* End-User Performance Needs* End-User Performance Needs* Manufacturer / Supplier Considerations* Manufacturer / Supplier Considerations* Regulatory Considerations* Regulatory Considerations* Key Purchasing Specifications* Key Purchasing Specifications
Purchasin g High PerformanceChromato graphic Supplies
Copyright 1997 Phase Seprations
Chromato graph yChromato graph y Technolo gy Technolo gy Back grounderBack grounder
Purchasin g High PerformanceChromato graphic Supplies
HPLC Column
Pump Waste
Data
Detector
Injector
Solvent
Copyright 1997 Phase Seprations
Chromato graph yChromato graph y Terminolo gy Terminolo gy
* * SampleSample - The Original Representative Material - The Original Representative Material Which Is To Be Analyzed -- Also Called Which Is To Be Analyzed -- Also Called The Sample MatrixThe Sample Matrix
* * Analyte(s) Analyte(s) - A Specific Compound(s) Contained In - A Specific Compound(s) Contained In The Sample Which Is(Are) To Be The Sample Which Is(Are) To Be
Separated And Analyzed Separated And Analyzed
** Compound Compound - - Pure Chemical Component In A Pure Chemical Component In A Sample, Also Called An Analyte Or SoluteSample, Also Called An Analyte Or Solute
Purchasin g High PerformanceChromato graphic Supplies
Copyright 1997 Phase Seprations
Chromato graph yChromato graph y Terminolo gy Terminolo gy
* How Does A Chromatographic Column Work? * How Does A Chromatographic Column Work?
Purchasin g High PerformanceChromato graphic Supplies
Sample Band
Analyte Bands
Copyright 1997 Phase Seprations
Chromato graph yChromato graph y Terminolo gy Terminolo gy
* How Does A Chromatographic Column Work? * How Does A Chromatographic Column Work?
Purchasin g High PerformanceChromato graphic Supplies
WHY Do They Separate?Yellow Likes The Mobile Phase And Goes As
Fast As It Does - And Comes Out FirstRed Likes The Stationary Phase Somewhat,
And Slows DownBlue Likes The Stationary Phase Best, And
Slows Down The Most
Copyright 1997 Phase Seprations
Chromato graph yChromato graph y Terminolo gy Terminolo gy
* How Do You Get Peaks? * How Do You Get Peaks?
Purchasin g High PerformanceChromato graphic Supplies
Detector
Detector Cell
Sample BandSample Peak
Copyright 1997 Phase Seprations
Chromato graph yChromato graph y Terminolo gy Terminolo gy
How Do You How Do You Identify Which Compounds Are WhichIdentify Which Compounds Are Which When When You Have A Series Of Peaks On You You Have A Series Of Peaks On You
Chromatogram???Chromatogram???
Purchasin g High PerformanceChromato graphic Supplies
Time
I Know From The Peaks' Retention Time Determined By Pure Standards
Copyright 1997 Phase Seprations
Chromato graph yChromato graph y Terminolo gy Terminolo gy
How Do You Know How Do You Know How Much Is PresentHow Much Is Present In The In The Sample???Sample???
Purchasin g High PerformanceChromato graphic Supplies
Time
I Know From The Peaks' Area And Height
Copyright 1997 Phase Seprations
Chromato graph yChromato graph y Terminolo gy Terminolo gy
Purchasin g High PerformanceChromato graphic Supplies
Look What Happens When It RainsRight After I WAX My Car! [ Non-Polar Surface ]
Water Beads Up Polar Water
DISLIKES Non-Polar
Surface
Copyright 1997 Phase Seprations
Chromato graph yChromato graph y Terminolo gy Terminolo gy
Purchasin g High PerformanceChromato graphic Supplies
* Solid Phase Extraction * Solid Phase Extraction (SPE)(SPE)
Chemical Technique Using Chemical Technique Using Column ChromatographyColumn ChromatographyIn A Small, In A Small, Single Use Single Use Disposable Cartridge Disposable Cartridge Format. Can PerformFormat. Can PerformChemical Separation,Chemical Separation,Concentration AndConcentration AndFractionationFractionation
Used With Vacuum Stations,Used With Vacuum Stations,Syringes And AutomatedSyringes And AutomatedSample ProcessorsSample Processors
Copyright 1997 Phase Seprations
Chromato graph yChromato graph y Terminolo gy Terminolo gy
Purchasin g High PerformanceChromato graphic Supplies
Mol. Wt.
Retention Time
Calibration Curve
Molecular Weight Distribution"Finger Print"
Big Ones
Copyright 1997 Phase Seprations
End User Performance NeedsEnd User Performance Needs
Stages Of A "Method" Stages Of A "Method"
The Success Of The Method Is Critical:The Success Of The Method Is Critical:
- Reputation Of The Researcher- Reputation Of The Researcher
-- Reduce Cost And Time To Market For A New Reduce Cost And Time To Market For A New Product If The Method Is Not Reproducible Product If The Method Is Not Reproducible
(Has To Be Redeveloped And (Has To Be Redeveloped And Revalidated Revalidated -- Can Be Incredibly Costly)-- Can Be Incredibly Costly)
Purchasin g High PerformanceChromato graph y Supplies
Copyright 1997 Phase Seprations
Manufacturer/Supplier ConsiderationsManufacturer/Supplier Considerations Purchasin g High PerformanceChromato graph y Supplies
Column RePacker
Column Packer
Secondary Manufacturer/
Column Packer
Primary Manufacturer
Specialized Distributor
3rd Party Supplies
Distributor
Sourcin g HPLC Columns
YOU
Copyright 1997 Phase Seprations
Manufacturer/Supplier ConsiderationsManufacturer/Supplier Considerations
* Manufacturers * Manufacturers -- Some Names:-- Some Names:
Primary:Primary: AlltechAlltech Bio-RadBio-Rad GL Science GL Science Hamilton Hamilton HypersilHypersil Macherey-Nagel Macherey-Nagel E. Merck E. Merck Akso Nobel Akso Nobel Polymer Labs Polymer Labs Rockland HP Rockland HP Showa DenkoShowa Denko Separations Group Separations Group Toso Haas Toso Haas WatersWaters Whatman Whatman
YMC YMCSecondary:Secondary: BrownLeeBrownLee JonesJones
Keystone Keystone PhenomenexPhenomenex Supelco Supelco
Packer:Packer: BischoffBischoff Beckman Beckman CapitalCapitalChrompackChrompack HPLC TechnologyHPLC Technology HichromHichromHPHP Isolation Tech.Isolation Tech. Mac-ModMac-ModMetachemMetachem MicraMicra PEPEResolution SystemsResolution Systems + Many Others+ Many Others
Purchasin g High PerformanceChromato graph y Supplies
Copyright 1997 Phase Seprations
Manufacturer/Supplier ConsiderationsManufacturer/Supplier Considerations
Purchasin g High PerformanceChromato graph y Supplies
Primary
Packer/Repacker
Distributor
End User
Distributor
End User
Packer/Technical Distributor
Secondary Manufacturer/Packer
Copyright 1997 Phase Seprations
Key Purchasin g ConsiderationsKey Purchasin g Considerations
Brands Supplied By Different VendorsBrands Supplied By Different Vendors
BrandBrand # Vendors# Vendors # Columns# Columns
HypersilHypersil 33 190190InertsilInertsil 55 40 40KromasilKromasil 55 75 75LiChrospherLiChrospher 33 8 8NucleosilNucleosil 33 240240Waters SpherisorbWaters Spherisorb 88 80 80
Purchasin g High PerformanceChromato graph y Supplies
...because Chromatography is still a ScienceSM
Understanding Reversed-Phase HPLC Separations –Effect of Silica Type/Activity
Examples are given, and a complete set ofhandouts is provided for future reference.
A Certificate of Achievement will be given to allattendees, which documents your participation inthis training program.
Order Course # PSL908208
This course is designed for those individualsdeveloping new reversed-phase HPLC methods.
Key topics include:
• Resolution Theory
• Controlling Chromatographic Parameters
• Silica Particle Technology
• Bonding Process
• Packing Material Comparison
• Reproducibility Concerns
Duration: 1.5 hours
Course Abstract:
© 1998 Phase Separations
HPLC Method Development HPLC Method Development & Troubleshooting& Troubleshooting
Understandin g Understandin g Reversed-Phase Reversed-Phase SeparationsSeparations'The Effect of the 'The Effect of the Silica Chemistr y'Silica Chemistr y'
Phase Separations Customer Education
Programme
Copyright 1997 Phase Seprations
Copyright 1997 Phase Seprations
Review of Chromatographic TerminologyReview of Chromatographic Terminology
RETENTION, k' = (V1-V0)/V0
SELECTIVITY, α = k'2/k'1PLATE COUNT, N = 16(V/W)2
Copyright 1997 Phase Seprations
ResolutionResolutionResolution is a numerical measure of the separation of two Resolution is a numerical measure of the separation of two compounds and is a function of N, k' and compounds and is a function of N, k' and α.α.
This equation is only valid for isocratic separations. This equation is only valid for isocratic separations. Selectivity, retention and peak widths may be further Selectivity, retention and peak widths may be further
enhanced by using gradients.enhanced by using gradients.
Rs =( N ) . (α-1) . k'24 α (k'2+1)
Copyright 1997 Phase Seprations
k', N, k', N, α −α − How They Control How They Control ResolutionResolution