Phase Separations Customer Education Program · ...because Chromatography is still a Science SM Strategies & Techniques for Faster HPLC Methods Development A Certificate of Achievement

Phase Separations Phase Separations Customer Education Pro gram Customer Education Pro gram

��

Advanced HPLC Column Selection Using the Hydrophobicity Chart

Time (min) 50

neutralbase

neutral

base0

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


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


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!


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


Limit of Quantitation

Method Validation Criteria

Precision

Accuracy

Limit of Detection

Specificity

Linearity and Range

Ruggedness

Robustness

MethodValidation



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


Method RobustnessMethod Robustness

Content of Or ganic SolventContent of Or ganic SolventIonic Stren gthIonic Stren gthpHpHTemperatureTemperatureOther AdditivesOther Additives


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.


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:


HIPACHIPACTMTM BB andand SS

BBinar y-Isocratic/inar y-Isocratic/ SSystem ystem OptimizationOptimization

HPLC Methods Development HPLC Methods Development Optimization SoftwareOptimization Software



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

��

��


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


Quickly Determines The Quickly Determines The Optimum Conditions Optimum Conditions For Your Application Based On Your Inputs For Your Application Based On Your Inputs





Isoeluotropic Mobile Phase SelectionIsoeluotropic Mobile Phase Selection







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.


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:



Reversed-Phase HPLC Methods Development

Examples are given, and a complete set ofhandouts is provided for future reference.



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:


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



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


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


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


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


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


ION EXCHANGEION EXCHANGE

pH 14

pH 1

��

��

��(-)

��

��

�� (+)

�� (+)��pKa 10.6 ��

% Ionized

��(-) pKa 4.7��

pH 12.6 Neutral

2.7 Neutral

8.6 ~100%

6.7 ~100%pH 7

Cation

Anion


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)


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


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


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)



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 Certificate of Achievement will be issued to allattendees.


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:


Solid Phase Extraction Solid Phase Extraction TechnologyTechnology

A Powerful Tool in Sam ple PreparationA Powerful Tool in Sam ple Preparation



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



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



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)



How Does Chromato graph y Work?

Stationar y Phase

Analyte A

Analyte B

Mobile Phase



Chromatographic Bed (Sorbent)

Housing/Body

Filters/Frits

Luer Tip



Vacuum ManifoldsVacuum Manifolds



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



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)



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



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



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



Fraction 1 (Clear)WaterPolar Analytes (Clear)

Fraction 2 8% IPAPure Red

Fraction 335% IPAPure Blue

Fraction 470% IPANon-PolarAnalytes (Clear)Fractionation



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


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



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


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


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


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.



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:


Selectin g Optimum Selectin g Optimum HPLC Column Dimensions HPLC Column Dimensions and and Stationar y Phase Particle SizeStationar y Phase Particle Size



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


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


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


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


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



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


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%


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%


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


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


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


( ) =( )( ) 100 (µL) 1min 1 mL 1000µL 20 cm min. mL

2cm PW( )


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.



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:


Troubleshootin g Common Troubleshootin g Common HPLC ProblemsHPLC Problems

SM



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


Problem

CHEMISTRY HARDWARECOLUMN/GUARDSOLVENTSAMPLE

PUMPINJECTORDETECTORINTEGRATOR

Always do the easiest thing first:1. Stop flow

2. Remove the column

TroubleshootingTroubleshooting


0.090 Parker Style

0.130 Waters

other Waters Columns

Waters Spherisorb

Installation and EquilibrationInstallation and Equilibration


Extra-Column Band SpreadingExtra-Column Band Spreading

Column Connection


.009"

.020".040"

note the differences of the inner diameter of this tubing



Effect of Connecting Tubing on System Bandspreading

.009"

.020"

.040"


sample band dispersion inside tubing


The Observed Bandwidth (TOT) * Sum of the Bandspreading Contributions

- Column (COL) - Extra-Column (EC) Instrument

components

σ2= σ2

+ σ2


TOT COL EC


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


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 )


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


voids - high back pressure, distorted and/or double peaks

Column CollapseColumn Collapse


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


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


Retention Time ReproducibilityRetention Time Reproducibility

Solvent Composition- Hydrophobic CollapseTemperaturepHIon Pairin gIonic Stren gthExtraneous PeaksGradient Control


pH - ControlpH - Control


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.




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:


Purchasing High Performance Purchasing High Performance Chromatographic SuppliesChromatographic Supplies

A Course For A Course For Purchasing ProfessionalsPurchasing Professionals



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


Chromato graph yChromato graph y Technolo gy Technolo gy Back grounderBack grounder


HPLC Column

Pump Waste

Data

Detector

Injector

Solvent


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




* How Does A Chromatographic Column Work? * How Does A Chromatographic Column Work?


Sample Band

Analyte Bands



* How Does A Chromatographic Column Work? * How Does A Chromatographic Column Work?


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



* How Do You Get Peaks? * How Do You Get Peaks?


Detector

Detector Cell

Sample BandSample Peak



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???


Time

I Know From The Peaks' Retention Time Determined By Pure Standards



How Do You Know How Do You Know How Much Is PresentHow Much Is Present In The In The Sample???Sample???


Time

I Know From The Peaks' Area And Height




Look What Happens When It RainsRight After I WAX My Car! [ Non-Polar Surface ]

Water Beads Up Polar Water

DISLIKES Non-Polar

Surface




* 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




Mol. Wt.

Retention Time

Calibration Curve

Molecular Weight Distribution"Finger Print"

Big Ones


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


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


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



Manufacturer/Supplier ConsiderationsManufacturer/Supplier Considerations


Primary

Packer/Repacker

Distributor

End User

Distributor

End User

Packer/Technical Distributor

Secondary Manufacturer/Packer


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



Understanding Reversed-Phase HPLC Separations –Effect of Silica Type/Activity

Examples are given, and a complete set ofhandouts is provided for future reference.



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:


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



Review of Chromatographic TerminologyReview of Chromatographic Terminology

RETENTION, k' = (V1-V0)/V0

SELECTIVITY, α = k'2/k'1PLATE COUNT, N = 16(V/W)2


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)


k', N, k', N, α −α − How They Control How They Control ResolutionResolution

Documents

Phase Separations Customer Education Program · ...because Chromatography is still a Science SM Strategies & Techniques for Faster HPLC Methods Development A Certificate of Achievement