Practical High Performance Liquid Chromatography

Course Number H5930A

Student Manual

Gas Chromatography

Liquid Chromatography

Mass Spectrometry

Capillary Electrophoresiss

Data Systems

Manual Part Number H5930A-90000

Printed in January, 2001

Practical High Performance Liquid Chromatography

H5930A

Student Manual

ii

Notice

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2000 by Agilent Technologies, Inc.

All rights reserved

Printed in the United States of America

iii

Table Of Contents

INTRODUCTION TO HIGH PERFORMANCE LIQUID CHROMATOGRAPHY..............1

IN THIS SECTION YOU WILL LEARN: ............................................................................................2 HISTORICAL ASPECTS ...................................................................................................................3 SEPARATION PROCESS...................................................................................................................4 TYPICAL COLUMN PACKING SUPPORT ..........................................................................................5 TYPICAL COLUMN SUPPORTS – STYRENE DVB ............................................................................6 MODES OF LIQUID CHROMATOGRAPHY ........................................................................................7 DIFFERENCES IN GAS AND LIQUID CHROMATOGRAPHY ................................................................8 INSTRUMENTATION .......................................................................................................................9 CHROMATOGRAPHIC PARAMETERS.............................................................................................10 SEPARATION PARAMETERS .........................................................................................................11 REVIEW QUESTIONS ....................................................................................................................12

THE SEPARATION PROCESS..................................................................................................15

IN THIS SECTION YOU WILL LEARN: ..........................................................................................16 THE GOAL OF SEPARATION .........................................................................................................17 TOOLS FOR ACHIEVING A SEPARATION .......................................................................................18 RESOLUTION VALUES .................................................................................................................19 CALCULATE A RESOLUTION VALUE ............................................................................................20 FACTORS INFLUENCING RESOLUTION .........................................................................................21 RESOLUTION FACTORS................................................................................................................22 SEPARATION SELECTIVITY ..........................................................................................................23 PARAMETERS WHICH WILL AFFECT SELECTIVITY......................................................................24 COLUMN SELECTION ...................................................................................................................25 EFFECT OF TEMPERATURE ON SEPARATION ................................................................................26 CAPACITY FACTOR K’ .................................................................................................................27 CAPACITY FACTOR......................................................................................................................28 USEFUL SOLVENTS FOR REVERSED-PHASE CHROMATOGRAPHY.................................................29 SOLVENT STRENGTH: NORMAL PHASE ......................................................................................30 NORMAL PHASE: SOLVENT STRENGTH ......................................................................................31 EFFICIENCY .................................................................................................................................32 CALCULATING EFFICIENCY .........................................................................................................33 DISPERSION: EDDY DIFFUSION ..................................................................................................34 DISPERSION: LONGITUDINAL DIFFUSION ...................................................................................35 DISPERSION: MASS TRANSFER...................................................................................................36 DISPERSION.................................................................................................................................37 TYPICAL FLOW RATES ................................................................................................................38 SMALL BORE AND MICROBORE COLUMNS..................................................................................39 FAST OR HIGH SPEED COLUMNS .................................................................................................40 EXTRA-COLUMN BAND BROADENING ........................................................................................41 INCREASING RESOLUTION ...........................................................................................................42 PEAK SYMMETRY........................................................................................................................43 WORKSHEETS..............................................................................................................................44 MOBILE PHASE COMPOSITION – THE GENERAL ELUTION PROBLEM...........................................46 MOBILE PHASE COMPOSITION – GRADIENT ELUTION .................................................................47 GRADIENT DEVELOPMENT ..........................................................................................................48 WORKSHEETS..............................................................................................................................49

iv

PRACTICAL CONSIDERATIONS FOR GRADIENT ELUTION .............................................................52

PRACTICAL ASPECTS OF PERFORMING ANALYSES.....................................................53

IN THIS SECTION YOU WILL LEARN: ..........................................................................................54 MOBILE PHASE PREPARATION FILTRATION.................................................................................55 MOBILE PHASE DEGASSING ........................................................................................................56 VACUUM DEGASSING..................................................................................................................57 SOLVENT MISCIBILITY ................................................................................................................58 MOBILE PHASE UV CUT-OFF .....................................................................................................59 COLUMN CARE............................................................................................................................60 PRE-COLUMNS AND GUARD COLUMNS.......................................................................................61 SYRINGE WASH: HP 1090..........................................................................................................62 PRIMING......................................................................................................................................63 COLUMN EQUILIBRATION ...........................................................................................................64 PREPARING SAMPLES: FILTERING ..............................................................................................65 PREPARING SAMPLES ..................................................................................................................66 WORKSHEET ...............................................................................................................................67

HPLC INSTRUMENTATION.....................................................................................................69

IN THIS SECTION, YOU WILL LEARN: .........................................................................................70 HPLC TUBING ............................................................................................................................71 FITTINGS .....................................................................................................................................72 FILTERS............................................................................................................................... ........74 FUNCTIONS OF THE SDS..............................................................................................................75 MULTICHANNEL GRADIENT VALVE ............................................................................................76 DUAL PISTON PARALLEL PUMP...................................................................................................77 DUAL PISTON SERIES PUMP ........................................................................................................78 BALL VALVES .............................................................................................................................79 METERING PUMP SEALS AND PISTONS ........................................................................................80 DIAPHRAGM PUMP ......................................................................................................................81 SIEVES AND FILTERS ...................................................................................................................82 DAMPING UNIT ...........................................................................................................................83 1090 SDS....................................................................................................................................84 QUATERNARY PUMP ...................................................................................................................85 MANUAL INJECTION....................................................................................................................86 AUTO-INJECTION SYSTEM ...........................................................................................................87 ROTOR SEALS .............................................................................................................................88 NECESSITY FOR MORE THAN ONE DETECTOR.............................................................................89 UV-VIS DETECTORS...................................................................................................................92 FLUORESCENCE DETECTION........................................................................................................98 REFRACTIVE INDEX DETECTION................................................................................................101 LIGHT SCATTERING DETECTION................................................................................................102 ELECTROCHEMICAL DETECTION ...............................................................................................103 CONDUCTIVITY DETECTION ......................................................................................................104 HPLC-MS.................................................................................................................................105 RADIOMETRIC DETECTORS .......................................................................................................107 WORKSHEET .............................................................................................................................108

HPLC TROUBLESHOOTING .................................................................................................111

IN THIS SECTION YOU WILL LEARN: ........................................................................................112 RECORD KEEPING .....................................................................................................................113 PROPER CARE OF THE HPLC.....................................................................................................114 PEAK RETENTION TIME AND PRECISION....................................................................................115 COMMON PUMP PROBLEMS.......................................................................................................116 PRESSURE PROBLEMS................................................................................................................117 BASELINE FLUCTUATIONS.........................................................................................................118

v

NOISY BASELINE.......................................................................................................................119 MIXING PROBLEMS ...................................................................................................................120 MANUAL INJECTION VALVE......................................................................................................121 AUTO-INJECTORS ......................................................................................................................122 GOOD COLUMN PRACTICES.......................................................................................................123 COLUMN FRIT REPLACEMENT...................................................................................................124 COLUMN REGENERATION..........................................................................................................125 DETECTOR PERFORMANCE........................................................................................................126 DETECTOR TIME CONSTANT .....................................................................................................127 DETECTOR HEAT EXCHANGERS ................................................................................................128 NOISY BASELINES .....................................................................................................................129 DRIFTING BASELINES................................................................................................................130 GHOST PEAKS ...........................................................................................................................131 EXTRA-COLUMN DISPERSION ...................................................................................................132 PEAK SHAPE..............................................................................................................................133 WORKSHEET .............................................................................................................................138

vi

Introduction to High Performance Liquid Chromatography

Introduction to High Performance Liquid Chromatography In This Section You Will Learn:

2

In This Section You Will Learn:

2

In This Section, You Will Learn

• The Historic Progression of Liquid Chromatography• About the Separation Process• Modes of HPLC• The Basics of the HPLC Instrumentation• About the Chromatogram

In this section you will learn how liquid chromatography has progressed throughout this century. You will also learn the basic separation mechanism and the main modes of high performance liquid chromatography. The layout of a modern liquid chromatograph will be presented and the qualitative and quantitative aspects of the chromatogram discussed.

Introduction to High Performance Liquid Chromatography Historical Aspects

3

Historical Aspects

3

Historical Aspects

• 1906 - Mikhail Semenovich Tswett (1872-1919)Calcium Carbonate, Petroleum Ether

• 1940’s - Partition and Paper Chromatography

• 1950’s - Gas, Thin-Layer, Gel Filtration and Gradient Elution Chromatography

• 1960’s - Introduction of Commercial HPLC

M.S. Tswett. Ber. Dtsch. Bot. Ges. 24: 384-393 (1906)

0

NN

NN

Mg

CHCH2 3

CH3

CH3

H C3

0

CH3

CH3

CH3

0CH

30

0

• Modern separation science began at the turn of the century with M. Tswett’s separation of plant chlorophylls on a calcium carbonate stationary phase with petroleum ether as the mobile phase. An apparatus similar to the one above was used in the separation.

• In the 1940’s, Martin and Synge introduced the concept of partition chromatography.

• During the 1950’s, gradient elution was introduced by Tiselius and the theory of separation was described by Van Deemter.

• Finally, in the 1960’s, the first commercial liquid chromatographs were introduced.

Introduction to High Performance Liquid Chromatography Separation Process

4

Separation Process

4

Separation Process

Stationary Phase

Mobile Phase

Chromatography is a separation process in which the components to be separated are distributed between two phases, a stationary phase and a mobile phase. Components of the sample mixture separate when they have differential migration in the column. Differential migration depends on the equilibrium distribution of the sample components between the stationary and mobile phase. Compounds whose molecules are found to reside most of the time in the mobile phase will elute first. Compounds whose molecules spend most of their time in the stationary phase will move through the column more slowly and elute at later retention times.

Introduction to High Performance Liquid Chromatography Typical Column Packing Support

5

Typical Column Packing Support

5

Typical Column Packing Support

Si

Si

Si

Si

Si

Chemically Modified

Silica Gel

Pores

Silica Gel

Surface

3

CH

CH

3

Si - O - Si - (CH ) CH2 17 3

OH

Si

O

Si

OH

O

O

Si

OH

O

Si

O

O

SiOH

Silica gel is commonly used as a stationary phase in adsorption chromatography (normal-phase) and is the support for numerous chemically bonded stationary phases. The surface of silica gel is covered with silanol groups which can interact with molecules or serve as a reaction site for chemical bonding. Common bonded phases include octadecysilyl (C-18), cyano, amino, C-8, C-4 or C-2.

Introduction to High Performance Liquid Chromatography Typical Column Supports – Styrene DVB

6

Typical Column Supports – Styrene DVB

6

Typical Column Supports - Styrene DVB

• Support for:• Reversed and Normal Phases• Ion Exchangers• Size-Exclusion Chromatography

CH = CH 2

+

- CH - CH - CH - CH - CH - CH - CH -222 2

Styrene Divinyl Benzene- CH - CH - CH - CH - CH - CH - CH -222 2

CH = CH 2

CH = CH 2

Cross-linked polystyrene is made from the copolymerization of styrene and divinylbenzene. Polymer stationary phases such as styrene divinylbenzene are stable in the pH range from 1-13 and can often withstand higher temperatures than silica gel. They are most often used as supports for ion exchange columns or size exclusion columns.

Introduction to High Performance Liquid Chromatography Modes of Liquid Chromatography

7

Modes of Liquid Chromatography

7

Modes of Liquid Chromatography

Gel Filtration-Aqueous

Gel Permeation-Organic

Polystyrene SilicaSize ExclusionHigh MW CompoundsPolymers

Aqueous/BufferCounter Ion

Anion or CationExchange Resin

Ion ExchangeIonicsInorganic Ions

OrganicsSilica, Amino, Cyano Diol

Normal PhaseCompounds insoluble in water, Organic isomers

Water/OrganicIon Pair Reagent

C-18, C-8Ion PairIonics, Bases, Acids

Water/Organic Modifiers

C-18, C-8, C-4, C-2Reversed PhaseNeutralsWeak AcidsWeak Bases

Mobile PhaseStationary PhaseModeTypes of Compounds Separated

The five major HPLC separation techniques are shown in the chart above. The most widely used mode is reversed-phase. This technique has a wide application range including neutrals, weak acids, weak bases, and ionics when used in conjunction with an ion-pairing reagent. Normal- phase liquid chromatography traditionally involved the use of bare silica or alumina columns and was known as adsorption chromatography. Today bonded polar stationary phases are also available. Ion-exchange is exclusively used for the separation of ions in solution. Size exclusion separates molecules of high molecular weight based upon their size.

Introduction to High Performance Liquid Chromatography Differences in Gas and Liquid Chromatography

8

Differences in Gas and Liquid Chromatography

8

Differences in Gas and Liquid Chromatography

Only about 20% of known organic compounds can be analyzed by GC.

10 10 10 10 100 2 4 6 8

Polarity

Solute Molecular Weight

LCGC

LCLC

GC

Gas Chromatography Liquid Chromatography

Introduction to High Performance Liquid Chromatography Instrumentation

9

Instrumentation

9

Instrumentation

Chromatogram

Injector

Detector

Column

Solvents

Pumps

Mixer

The components of a high performance liquid chromatograph include: solvent reservoirs, a pumping system to provide accurate compositions, flows and the pressure necessary to push the mobile phase through the tightly packed column, a sample delivery mechanism which will not interrupt the flow of mobile phase, a column where the separation takes place, and the detector to sense the presence of individual sample components.

Introduction to High Performance Liquid Chromatography Chromatographic Parameters

10

Chromatographic Parameters

10

Chromatographic Parameters

Detector R

esponse

Time

W WA B

tR(B)

A B

Inject

t0

R(A)t

t = retention time

t = elution time of an unretained component

R

W = peak width at base

0

Sample components typically produce gaussian shaped peaks. Components which are not retained by the stationary phase are said to elute at t0. Those sample components that have some attraction for the stationary phase elute at later retention times. Retention times provide the qualitative aspect of the chromatogram. The chromatographic peak height or peak area may be related to the quantity of analyte in the mixture when compared to standards of known concentration.

Introduction to High Performance Liquid Chromatography Separation Parameters

11

Separation Parameters

11

Separation Parameters

• Column Stationary Phase Selection• Column Length and Diameter• Mobile Phase Composition• Temperature• Flow Rate• Sample Size

The resolution of chromatographic peaks can be controlled by the selection of proper separation parameters. Based upon the sample components’ molecular structure, a column stationary phase is chosen. The length chosen may depend on the difficulty of the separation. A mobile phase composition compatible with both the samples and the stationary phase is selected and optimized to produce the best separation possible. Mobile phase composition is the primary parameter used to optimize an HPLC separation. Mobile phase selection may also depend upon the detector parameters. The column temperature and flow rate are used as secondary adjustments, fine tuning the chromatogram. The sample size is also an important parameter as large injected masses or volumes can lead to loss of resolution and degradation of peak shape.

Introduction to High Performance Liquid Chromatography Review Questions

12

Review Questions

12

Review Questions 1A

1. Name three differences in gas and liquid chromatography.

2. What is the most widely used mode of HPLC?

3. What mode may be used for separation of ions in solution?

Introduction to High Performance Liquid Chromatography Review Questions

13

13

Review Questions 1B

1. Name the parts of an HPLC instrument.

2. What is the symbol for an unretained component’s elution time?

3. What is the difference between normal and reversed phase?

Introduction to High Performance Liquid Chromatography Review Questions

14

The Separation Process

The Separation Process In This Section You Will Learn:

16

In This Section You Will Learn:

2

In This Section, You Will Learn

• What factors influence the resolution between sample components.

• How the capacity factor, selectivity and efficiency influence resolution.

• How liquid chromatographic operating parameters affect each resolution factor.

• When gradient elution can improve the separation.

In this section, the factors influencing the resolution of sample components will be discussed. Included in this discussion are the capacity factor, selectivity and efficiency. The relationship between the operating parameters such as mobile phase composition, temperature and flow rate to resolution will also be discussed. Finally, gradient elution will be explored.

The Separation Process The Goal of Separation

17

The Goal of Separation

3

The Goal of Separation: Resolution Between Sample Components

R - resolutiont - retention time of component Bt - retention time of component Aw - width at base of peakw - width at half-height

A B

tRA

tRB

0

R=2t - t RB RA

W + W A B

R=1.176t - t RB RA

W + W 1/2A 1/2B

The most important goal of the chromatographer is to achieve adequate resolution between all peaks in the chromatogram in a reasonable amount of time. A quantitative measure of resolution between two adjacent chromatographic peaks has been developed and appears above. The first equation describes the resolution based upon the width at the base of each peak. The second equation describes the resolution based upon the width at half-height.

The Separation Process Tools for Achieving a Separation

18

Tools for Achieving a Separation

4

Tools for Achieving a Separation

• Column Selection – stationary phase, particle size, etc...

• Column Length• Mobile phase composition• Column Temperature• Flow Rate

The experimental variables available to the liquid chromatographer to achieve resolution between sample components include column selection, column length, mobile phase composition, column temperature and flow rate. Column selection includes choice of appropriate stationary phase, particle diameter, particle shape, column diameter and column length. Mobile phase composition has the most profound effect upon the spacing of chromatographic peaks and this is where most development effort is usually focused. Column temperature and flow rate are of secondary importance and are utilized to fine tune the separation.

The Separation Process Resolution Values

19

Resolution Values

5

Resolution Values

For equal peak areas, R of 1.5 gives baseline separation

0.4 0.50.6 0.7

0.8 1.00 1.25

The graphic above illustrates the resolution values for overlapping chromatographic peaks. When two chromatographic peaks are baseline resolved, the resolution value is 1.5. Quantification will not be precise when two adjacent chromatographic peaks with resolution values of 1.25 or less are involved. During method development, the analyst may wish to achieve a minimum resolution value of 2 in order to insure robust method performance as the column degrades or in case of minor alterations in experimental conditions.

The Separation Process Calculate a Resolution Value

20

Calculate a Resolution Value

6

Calculate a Resolution Value

Calculate the resolution between the first two chromatographic peaks.

.048

.053

Width at half-height

t0

1 2

3

4t t

t

t

Answer

0.913

1.072

Ret Time

t

t

1

2

The Separation Process Factors Influencing Resolution

21

Factors Influencing Resolution

7

Factors Influencing Resolution

N: Total number of theoretical plates available; column efficiency.k: Capacity factor, the peak retention function.a: the relative separation of the peaks; the selectivity function.

CapacitySelectivityEfficiency

R = 1/4 N x x k’1 + k’

- 1

The degree of resolution between two chromatographic peaks is dependent upon three factors. The first term, efficiency can be varied with flow rate and column length. This term reflects how much dispersion takes place within a chromatographic peak. The second term, selectivity, illustrates how well the chromatographic system chosen can distinguish between sample components. Selectivity is dependent upon stationary phase selection, mobile phase selection and column temperature, among others. The final term is related to the capacity factor and is primarily influenced by mobile phase composition. The discussion will elaborate on each of these factors.

The Separation Process Resolution Factors

22

Resolution Factors

8

Resolution Factors

0

Capacity Selectivity

Efficiency

Capacity, selectivity and efficiency are illustrated graphically in the chromatogram presented above.

The Separation Process Separation Selectivity

23

Separation Selectivity

9

Separation Selectivity

=k’k’

B

A

A B

1 2

A B

1 2

= 1.22= 1.04

Change Mobile Phase Composition.

Change to Different Mobile Phase.

Change Mobile Phase pH.

Change Column Temperature.

Use Special Chemical Effects.

Change Stationary Phase.

To change selectivity:CapacitySelectivityEfficiency

R = 1/4 N x x k’1 + k’

- 1

The separation selectivity is quantitatively given by which is simply a measure of the spacing between the apex of two chromatographic peaks. A selectivity value of 1 indicates that no separation took place and the k’ values are identical. Increasing selectivity is a very useful way to increase the resolution, as it does not necessarily involve a concomitant increase in analysis time. Increasing the selectivity, however, can be more difficult as it usually involves a change in the actual mobile phases used, column selected or the addition of modifiers to the mobile phase.

The Separation Process Parameters Which Will Affect Selectivity

24

Parameters Which Will Affect Selectivity

10

Parameters Which Will Affect Selectivity

DECREASING INCREASING

Weaker Solvents

Stronger Solvents

IPA/Water MeOH/WaterACN/Water

High Organic Stationary Phase

C-2

C-18

Low Organic Stationary Phase

Easily

Overloaded

Not EasilyOverloaded

Change mobile phasecomposition

Change stationary phase

Notice that changing the actual mobile phase constituents can be a powerful way to change the chromatographic selectivity. Notice that different mobile phase compositions will actually cause different spacing between the apex of the chromatographic peaks. Probably, the most significant changes in selectivity can be realized with a change in stationary phase. The example given illustrates possible selectivity differences between a C-8 column and a C-18 column. Although both columns are essentially a straight chain hydrocarbon bonded to the surface of the silica gel, one will usually find greater selectivity with the greater carbon content.

The Separation Process Column Selection

25

Column Selection

11

Column Selection

Types of Compounds Separated

Mode Stationary Phase

Mobile Phase

NeutralsWeak AcidsWeak Bases

Reversed-Phase C-18, C-8, C-4, C-2

Water/Organic Phase

Ionics, Bases, Acids

Ion-Pair C-18, C-8 Water/Organic Ion-Pair Reagent

Compounds Insoluble in Water, Organic Isomers

Normal -Phase Silica, Amino, Cyano Diol

Organics

IonicsInorganic Ions

Ion Exchange Anion or Cation Exchange Resin

Aqueous/Buffer Counter Ion

High MW Compounds Polymers

Size Exclusion SilicaStyrene-Divinylbenzene

Gel Filtration-Aqueous Gel Permeation-Organic

The most significant way to change the selectivity is to change the stationary phase. Listed above are the five most frequently used modes of liquid chromatography. The most widely used mode is reversed-phase. An off-shoot of reversed-phase liquid chromatography is ion-pair chromatography for the separation of strong acids and bases. Normal-phase liquid chromatography traditionally involved the use of silica columns, but now popular bonded phases such as cyano, amino, and diol are also available. Ion exchange chromatography is utilized solely for the separation of ions. Size exclusion separates compounds based upon their size such as polymers and biomolecules. Selectivity may be improved by simply changing the type of column used, but staying within the same mode of liquid chromatography. An example would be using a cyano column in reversed-phase as opposed to a C-18.

The Separation Process Effect of Temperature on Separation

26

Effect of Temperature on Separation

12

Effect of Temperature on Separation

40º C

65º C

100 5

Time in Minutes

A B

AB

Column selectivity may also be altered with a change in column temperature although this experimental variable does not produce the dynamic changes associated with mobile or stationary phase changes. Raising the column oven temperature will increase the efficiency and decrease the retention time of solutes. Occasionally, as in the example presented, peak elution order may change as the result of a change in temperature. In addition to separation improvements, the use of a column oven will produce better retention time precision.

The Separation Process Capacity Factor k’

27

Capacity Factor k’

13

Capacity Factor k’

InjectO

t - tOR

t=k’tO

tR2

tR1

Capacity factor is characteristic of a specific compoundat a given mobile phase composition, temperature, and column type.Capacity factor is equal to the number of moles in the stationary phase divided by the number of moles n the mobile phase.

CapacitySelectivityEfficiency

R = 1/4 N x x k’1 + k’

- 1

The capacity factor is related to the ratio of the total number of moles of a given component in the stationary phase versus those in the mobile phase for any given equilibration. A higher k’ value indicates that the sample is highly retained and has spent a significant amount of time interacting with the stationary phase. The capacity factor is characteristic of a specific compound at a given mobile phase composition, temperature, and column type. One may use the capacity factor instead of retention time to identify components qualitatively. The value is independent of flow rate making day-to-day fluctuations less troublesome.

The Separation Process Capacity Factor

28

Capacity Factor

14

Capacity Factor

60% Acetonitrile40% Water

Time (min.)

Weaker Solvent Composition

2 4 6 8 10

mA

U

Stronger Solvent Composition

Time (min.)

80% Acetonitrile20% Water

mA

U

2 4 6 8 10

The single most important way to change thecapacity of a chromatographic peak is to changethe mobile phase composition.

Increasing the strength of the mobile phase decreases the capacity factor of the eluents.For reversed phase, an increase of 10% organic decreased k’ for each chromatographic peak by a factor of 2 or 3.

The capacity factor is usually changed by modifying the mobile phase composition. The example provided is that of a reversed-phase separation on a C-18 column. In reversed-phase, a weak mobile phase will be more polar than a strong mobile phase. A weaker mobile phase composition is produced by increasing the amount of water in the mobile phase, thus increasing the retention of components. Conversely, an increase of 10% organic decreases the k’ for each component by a factor of 2 or 3.

The Separation Process Useful Solvents for Reversed-Phase Chromatography

29

Useful Solvents for Reversed-Phase Chromatography

15

Useful Solvents for Reversed-Phase Chromatography

• Water• Methanol• Acetonitrile• Isopropanol• Dioxane• Tetrahydrofuran

Elution Strength

Reversed-phase mobile phases generally consist of mixtures of water or aqueous buffer with various water -miscible organic solvents. The stronger the organic mixed with water, the faster sample components will elute from the column. One hundred percent organic will flush a reversed-phase column.

The Separation Process Solvent Strength: Normal Phase

30

Solvent Strength: Normal Phase

16

Solvent Strength - Normal Phase

Silica AluminaFluoroalkanes -0.2 -0.25

n-Pentane 0.0 0.01-Chlorobutane 0.20 0.26

Xylene 0.24 0.26Toluene 0.28 0.29Benzene 0.20 0.32

Chloroform 0.26 0.40

Methylene Chloride 0.32 0.42

Tetrahydrofuran 0.44 0.57Acetonitrile 0.50 0.65Methanol 0.7 0.95

Solvent Strength ∈º

The elutropic series for normal-phase liquid chromatography is provided above. Solvents with larger solvent strength values will cause sample components to elute more quickly from the column. In the adsorption chromatography model, strong mobile phases are strongly adsorbed to the stationary phase. Sample molecules will have little ability to knock these mobile phase molecules from the substrate and therefore sample molecules elute quickly. When a mobile phase molecule is weak enough to be displaced from the stationary phase, sample molecules are retained and a separation occurs.

The Separation Process Normal Phase: Solvent Strength

31

Normal Phase: Solvent Strength

17

Normal Phase - Solvent Strength

Mobile Phase

B = Benzene

X = Xylene

CH3H C3

T = Toluene

CH3

n-Pentane

X

T

B

Methylene Chloride

X + T + B

∈°= 0.42∈°= 0.00

To separate xylene, toluene, and benzene, a mobile phase should be chosen that is less strongly adsorbed to the stationary phase. Pentane with a relative elution strength of 0.0 is weak enough to be displaced by these sample components. Methylene chloride with an elution strength of 0.42 is more strongly retained than any of the sample components so no separation occurs.

The Separation Process Efficiency

32

Efficiency

18

EfficiencyD

etec

tor

Res

pons

eIn

ject

Time

High Efficiency

Low Efficiency

CapacitySelectivityEfficiency

R = 1/4 N x x k’1 + k’

- 1

Another one of the factors that influence resolution is the column efficiency. Column efficiency is expressed as N, or plate number. In an ideal chromatographic system, a chromatographic peak would appear as a vertical line in the chromatogram. In reality, dispersion occurs causing the peak to take on a guassion shape. The better the column efficiency (less dispersion) the easier it will be to achieve resolution between chromatographic peaks.

The Separation Process Calculating Efficiency

33

Calculating Efficiency

19

Calculating EfficiencyIn

ject

tR

W

W

Time

1/2

B

N = 16 = 5.54tRW

2 t R2

HETP = L

N

W1/2

= 2 ∏ hptr 2

AB

N: EfficiencyHETP: Height Equivalent to a Theoretical PlateL: Column Lengthhp: Peak HeightA: Peak Area

To calculate the column efficiency use one of the equations presented here. Make certain the chromatographic peak chosen has a k’ value greater than 2. A typical plate number for a new 4.6 X 100 mm column with 5 um particles is 8 or 9000 plates. The number of theoretical plates is proportional to the column length. The HETP, or height equivalent to a theoretical plate, is also a measure of the column efficiency, which describes the efficiency of a given column for unit length of column.

The Separation Process Dispersion: Eddy Diffusion

34

Dispersion: Eddy Diffusion

20

Dispersion - Eddy Diffusion

Pack Columns CarefullyUse Narrow Mesh Range

A = Eddy Diffusion - The Multi-Path Effect

A

Linear Velocity

HETP

InitialBand

FinalBandWidth

Width

Dispersion of a chromatographic peak occurs as a result of differing migration rates through the column. Differing migration rates are a result of physical processes, such as eddy diffusion. The A-term results from inhomogeneity of flow path velocities around stationary phase particles. The A-term can be considered independent of linear velocity. To diminish dispersion resulting from this term, columns should be carefully packed using a narrow mesh range. Smaller particles will also decrease this effect, as well as smaller column diameters.

The Separation Process Dispersion: Longitudinal Diffusion

35

Dispersion: Longitudinal Diffusion

21

Dispersion - Longitudinal Diffusion

B

Small Effect in LCSignificant at Low Flow Rates

Linear Velocity

HETP

7KH�%� �WHUP�RU�ORQJLWXGLQDO�GLIIXVLRQ�WHUP�GHILQHV�WKH�HIIHFW�RI�UDQGRP�molecular motion on dispersion. Although not as serious a consideration in LC, this term becomes more significant at lower linear velocities.

The Separation Process Dispersion: Mass Transfer

36

Dispersion: Mass Transfer

22

Dispersion - Mass Transfer

StagnantMobilePhase

MobilePhase

StationaryPhase

C. u

C = Mass Transfer Between Phases

Reduce Effect with Low Flow Rate

Reduce Effect with Small Particles

HETP

Linear Velocity

Dispersion due to mass transfer has both a component relating to the sample molecules interaction with the stationary phase as well as a component relating to the sample molecules interaction within the mobile phase. The stationary phase interaction requires a finite rate of equilibration between the sample molecule and the stationary phase. The mobile phase interaction relates to the diffusion of analytes. The structure of the stationary phase is porous. The mobile phase within these pores is on the whole stagnant. Once a sample molecule finds itself with a stagnant pore, the only way for it to rejoin the other sample molecules in the mobile phase is for the molecule to diffuse out of the pore. This term is adversely affected at higher flow rates.

The Separation Process Dispersion

37

Dispersion

23

Dispersion

h = A + + C.u

HE

TP

Bu

C. u

A

Linear Velocity

A = Eddy Diffusion (Multi-Path Effect)B = Random Molecular DiffusionC = Mass Transfer Between Phases

The Van Deemter Equation

Bu

The total effect of the three terms, eddy diffusion, longitudinal diffusion, and mass transfer is additive. The graphic illustrates that there is an optimal linear velocity for each chromatographic column indicated by the dip in the graph. Typically, one operates the column at a linear velocity just above the dip in the curve.

The Separation Process Typical Flow Rates

38

Typical Flow Rates

24

Typical Flow Rates

4.6 1-2

3.0 0.4-0.8

2.1 0.2-0.4

1.0 0.05-0.09

mm i.d.(5um particles) mL/min

flow rate =

i.d.

i.d.

( µ bore)

(analytical)

2

flow rate(analytical)

From the previous discussion, it is apparent that each chromatographic column will have an optimal operating flow rate. Typical flow rates presented on the basis of column diameter are shown above. Flow rate as an experimental variable produces only small changes in resolution and is used for fine tuning the chromatogram.

The Separation Process Small Bore and Microbore Columns

39

Small Bore and Microbore Columns

25

Small Bore and Microbore Columns

Advantages• Decreased solvent consumption.• Good for trace analysis if sample

amount is limited.• Easy flow rate conversion to change

method from analytical column tomircrobore column.

Disadvantages• Instrumentation must have very low

extra-column volume.• Frits must be changed more

frequently.

flow rate =( bore)i.d.i.d.

2(bore)

(analytical)flow rate

(analytical)

Conventional

Microbore

4.6 mm

4.6 mm

2.1 mm

d = 10 µmp

0 2 4 6 8 10

1.00 mL/min

0 4 6 8 10

0.01 mL/min

2

or1.0 mm 100 mm

200 mm

d = 5 µmp

d = 5 µmp

A conventional HPLC column is 4.6 mm i.d. with 5 um particles. Small bore (2.1 mm i.d.) or microbore columns (1.0 mm i.d. or <) may be utilized with the following advantages: 1) decreased solvent consumption; and, 2) decreased peak dispersion resulting in better peak signal to noise ratio. A 4.6 mm i.d. column method may be transcribed to smaller internal diameter column using the flow rate conversion provided. Some disadvantages include the need for low dead volume instrumentation and that fact that the column inlet frits clog more easily.

The Separation Process Fast or High Speed Columns

40

Fast or High Speed Columns

26

Fast or High Speed Columns

Advantages• Very short analysis time.• Easy flow rate conversion to change

analytical method to fast column method.

Disadvantages • Instrumentation must have very low

extra-column volume.• Time constant and cell volume of

detector must be low; signal acquisition and integration must be fast.

High Speed

5.00 mL/min

0 0.5 1.0

Conventional

0 2 4 6 8 10

1.00 mL/min

60 mm100 mm

200 mm

4.6 mm

4.6 mm4.6 mm

d = 10 µmp

d = 5 µmp

d = 3 µmp

(analytical) flow rate = particle diameter (analytical)

particle diameter (fast)(fast) flow rate

High speed columns have typically a 4.6 mm internal diameter, but utilize 3 um SDUWLFOHV���$�� P�SDUWLFOH�FROXPQ�PDLQWDLQV�KLJK�HIILFLHQFLHV�HYHQ�DW�KLJK�IORZ�rates. As a result, analyses may be run at flow rates from 3-5 mL/min shortening the analysis time. These columns are typically 60mm in length due to the increased back pressure. Instrumentation with low dead-volume characteristics is required because of reduced dispersion.

The Separation Process Extra-Column Band Broadening

41

Extra-Column Band Broadening

27

Extra-Column Band Broadening

• Sample Volume• Volume of Connective Tubing• Detector Volume• Detector Electronic Time Constant

Dispersion that occurs outside of the column is termed extra column band broadening. In a well designed HPLC, this dispersion is negligible as compared to internal band broadening. Chromatographers must endeavor not to add extra lengths of tubing or wide diameter tubing to the liquid chromatograph between the injector and detector. One must also be careful to match HPLC fittings and set appropriate time constants on detectors.

The Separation Process Increasing Resolution

42

Increasing Resolution

28

Increasing Resolution

Increase k’

Increase SelectivityIncrease Efficiency

In summary, the three factors which influence resolution between chromatographic peaks are capacity, efficiency, and selectivity. The most effective way to increase the capacity factor is to change the mobile phase composition. Selectivity can be increased through mobile phase composition or component changes, column stationary phase changes or temperature adjustment. Column efficiency can be increased by lengthening the column, using the appropriate flow rate, and increasing the column temperature.

The Separation Process Peak Symmetry

43

Peak Symmetry

29

Peak Symmetry

A B

S = B/A

ExcellentS = 1.0 - 1.05

AcceptableS = 1.2

UnacceptableS = 2

AwfulS = 4

10% of peak height

Ideally, chromatographic peaks should be guassion in shape. However, most of the time they have a tail. The calculation above is used to mathematically describe peak symmetry. Values greater than 3 are not acceptable.

The Separation Process Worksheets

44

Worksheets

30

Worksheet

The analysis was performed on a 100 x 4.6 mm i.d., 10 um, C-8 column. The flow rate was 2 mL/min with 70/30 IPA/water.

List ways to improve the separation.

0

The Separation Process Worksheets

45

31

Worksheet

1. What happens to resolution when k’ is increased?

2. What happens to resolution when the column efficiency is increased?

3. What parameters affect column efficiency?

4. What parameters affect k’?

The Separation Process Mobile Phase Composition – The General Elution Problem

46

Mobile Phase Composition – The General Elution Problem

32

Mobile Phase Composition - The General Elution Problem

Isocratic elution of an homologous series(wide range of k’ values)

Poor resolution of early eluting peaks.Increase in peak width and decrease in peak height for later eluting peaks.Long analysis times.

When a single mobile phase composition is used for an analysis, analytes may elute over a wide range of k’ values. Early eluting peaks may not be completely separated and late eluting peaks are broad, therefore losing peak detectability due to their decreased peak height. Long analysis times are also characteristic of such separations.

The Separation Process Mobile Phase Composition – Gradient Elution

47

Mobile Phase Composition – Gradient Elution

33

Mobile Phase Composition - Gradient Elution

• "Ideal" chromatogram of an homologous series.

• Optimum overall resolution.• Equal band widths for all peaks.• Shorter analysis times.

Other Uses: • To quickly check unknown

samples.• To clean the column.

Gradient elution is the solution to the general elution problem. When using a gradient elution program, the initial solvent strength is selected to give an elution strength capable of eluting the early chromatographic peaks with adequate resolution. The elution strength is increased in a predetermined way in order to bring other peaks off the column with optimum overall resolution, increased peak height, and shortened analysis times.

The Separation Process Gradient Development

48

Gradient Development

34

Gradient Development

10%Organic

100%

Organic

Consider:• Choice of Organic Solvent• Initial Composition of Mobile Phase• Ramp Rate• Gradient shape• Flow Rate• Column Re-equilibration• No Peaks k’ < 2• No Peaks k’ > 10

A scouting run can be the initial step during gradient development. A linear gradient is run from 5-10% organic to 100% organic over a set time period, then the composition is held for some additional time to insure all sample components have eluted. The results are examined to determine the proper initial gradient composition and gradient profile.

The Separation Process Worksheets

49

Worksheets

35

Worksheet - How can this gradient be improved?

10%Organic

100%Organic

Linear Gradient

1. What is the problem with this gradient?

2. How would you improve it?

The Separation Process Worksheets

50

36

Worksheet - How can this gradient be improved?

10%Organic

100%Organic

Linear Gradient

1. What is wrong with this chromatogram?

2. How can it be improved?

The Separation Process Worksheets

51

37

Worksheet - How can this gradient be improved?

10%Organic

100%Organic

Linear Gradient

1. What is wrong with this chromatogram?

2. How can the chromatogram be Improved?

The Separation Process Practical Considerations for Gradient Elution

52

Practical Considerations for Gradient Elution

38

Practical Considerations for Gradient Elution

Ghost Peaks

0% MeOH 100%

• Solvents must be pure or ghost peaks will occur.

• Make certain the buffer is soluble at final gradient mobile phase composition.

• Allow time for column reconditioning between runs.

• Different LC models will have different delay volumes.

Solvents utilized in gradient elution must be pure. Water quality is of particular importance. Impurities are retained on the column while the composition of the mobile phase is weak. As the elution strength is increased, the impurities appear as peaks in the chromatogram. To avoid precipitation in the instrument, test that the buffer is soluble in the final mobile phase composition. Finally, to increase retention time precision, make certain that adequate re-equilibration time is allowed between each chromatographic run.

Practical Aspects of Performing Analyses

Practical Aspects of Performing Analyses In This Section You Will Learn:

54

In This Section You Will Learn:

2

In This Section, You Will Learn

• Mobile phase preparation

• Column care

• Sample preparation

Practical Aspects of Performing Analyses Mobile Phase Preparation Filtration

55

Mobile Phase Preparation Filtration

3

Mobile Phase Preparation Filtration

Filtration Apparatus

Vacuum

• Always use HPLC grade solvent• Change HPLC grade water daily.

– Prevents particulate matter from damaging the instrument or column head.

• Use at least 0.5 mm filters.– Organics - PTFE – Water - Nylon– Inorganic Membrane Filter

All solvents used on the HPLC should be at HPLC grade. These solvents are pre-filtered and purified to have minimal absorbance in the UV. After dissolving buffers and additives, the mobile phase should be filtered with at least a 0.5 um filter to remove particulate matter, which may damage the instrument or column. The apparatus shown is one possible device for filtration. A vacuum is applied to pull solvent through the filter housed inside the screw cap. The reservoir is plastic coated to avoid implosion. Handle the filters with tweezers and make certain the filtration apparatus is clean at all times. Nylon is a good filter for aqueous mobile phases, while PTFE is an excellent filter for most organic solvents. Inorganic membranes are resistant to a wide range of HPLC solvents.

Practical Aspects of Performing Analyses Mobile Phase Degassing

56

Mobile Phase Degassing

4

Mobile Phase Degassing

• Purpose– Removes dissolved oxygen and

nitrogen from the mobile phase• Methods

– He sparging– On-line vacuum degassing– Refluxing– Vacuum filtration– Ultrasonication

Degassing the mobile phase is an important step before beginning an HPLC analysis because water and lower molecular weight alcohols dissolve relatively large amounts of air. Degassing removes these dissolved gasses from the mobile phase. The dissolved gasses can result in bubble formation in the pumps or detector. Dissolved oxygen can quench fluorescence detection. Helium can be used to sparge mobile phases because it has a low solubility and thus can " knock out" other dissolved gasses. Boiling premixed solvents is not recommended because the more volatile component is lost more rapidly changing the composition.

Practical Aspects of Performing Analyses Vacuum Degassing

57

Vacuum Degassing

5

Vacuum Degassing

The best way to remove dissolved gasses from the mobile phase is vacuum degassing. The mobile phase is pulled through gas permeable tubing coiled within a vacuum chamber on the way to the pump. Besides adequately removing dissolved gasses, the other advantages include: real-time degassing and less expense as helium is not required.

Practical Aspects of Performing Analyses Solvent Miscibility

58

Solvent Miscibility

6

Solvent Miscibility

Name

Acetic Acid

Acetone

Acetonitrile

Benzene

Butyl Alcohol

Carbon Tetrachloride

Chloroform

Cyclohexane

Cyclopentane

Dichloroethane

Dichloromethane

Dimethylformamide

Dimethyl Sulfoxide

Dioxan

Ethylacetate

Ethyl Alcohol

Di-Ethylether

Heptane

Hexane

Methyl Alcohol

Methylethyl Ketone

I-Octane

Pentane

I-Propyl Alcohol

Tetrachloroethane

Tetrahydrofuran

Toluene

Buty

l Alc

ohol

I-Pro

pyl A

lcoh

ol

Di-P

ropy

leth

er

Tric

hlor

oeth

ane

Acet

ic Ac

idAc

eton

eAc

eton

itrile

Benz

ene

Carb

on Te

tC

hlor

ofor

mCy

clop

enta

ne

Cycl

ohex

ane

Dich

loro

etha

neCH

Cl

2

2D

MF

DMSO

Dioxa

nEt

hyla

ceta

teEt

hyl A

lcoh

olDi-E

thyl

ethe

rHe

ptan

eHe

xane

Met

hyl A

lcoh

olM

EKI-O

ctan

ePe

ntan

e

2

2

C H

Cl 4

THF

Tolu

ene

Wat

er

Xyle

ne

Trichloroethane

Water

Xylene

Di-Propylether

Immiscible

Miscible

2-Propanol is anexcellent intermediatesolvent

Not all common HPLC solvents are miscible. If immiscible solvents are mixed several problems may result such as an unstable baseline, fluctuating pressures and high pressure. If you are uncertain about the last solvent system used in your HPLC, flush the flow path with isopropanol. This solvent is miscible with most common HPLC solvents. To move from a normal-phase separation to a reversed-phase separation, remove the normal phase column, add a capillary tube in its place and flush the liquid chromatograph with isopropanol. After, you may proceed with the reversed-phase analysis.

Practical Aspects of Performing Analyses Mobile Phase UV Cut-Off

59

Mobile Phase UV Cut-Off

7

Mobile Phase UV-Cutoff

Solvent UV Cutoff (nm)

Acetonitrile 190Water 190Cyclohexane 195

Hexane 200Methanol 210Ethanol 210Diethyl Ether 220

Dichloromethane 220

Chloroform 240Carbon Tetrachloride 265

Tetrahydrofuran 280 (210)

Toluene 280

UV cutoff is the wavelength at which absorbance equals 1 AU.

Listed above are the UV-cutoffs for common HPLC solvents. When utilizing a UV detector, care should be taken not to use the solvent below or near its UV-cutoff or an unacceptable noise level will result limiting your detectability. For instance, if you were monitoring a compound at 220 nm, you would select acetonitrile over methanol because acetonitrile’s UV-cutoff is lower than methanol resulting in better detection performance. Other factors must be addressed for different detectors. For instance, when using a mass spectrometer with a particle beam interface as the detector, one must consider the molecular weight of the mobile phase and additives.

Practical Aspects of Performing Analyses Column Care

60

Column Care

8

Column Care

á Filter all Solvents and Samples á Store Column in Appropriate Solvent

á Use a Guard Column á Do Pay Attention to the Safe Operating pH Range of the Column

á Flush to Remove Buffers at End of Use

á Do Not Pressure or Solvent Shock the Column

á Cap When Not in Use á Do Not Operate Silica or Bonded Phases for Extended Periods at High Temperatures

By following the manufacturers recommendations and applying the suggestions above, you may extend the lifetime of your analytical HPLC columns. To prevent clogging of the column inlet frit and damage to the column bed, Filter all solvents and samples. Guard columns, positioned between the injector and analytical column will extend the life of your analytical column by trapping particulate matter and strongly retained sample components. Make certain that your column is flushed and free of buffers and damaging additives before storage. Caps the ends of the column firmly to prevent the column from drying out. The normal operating range of silica based bonded phase columns is from pH 2 to 8. Silica is soluble in the ppm range at pH 7.5 and above. Silica columns will degrade more quickly when operated at elevated temperatures.

Practical Aspects of Performing Analyses Pre-Columns and Guard Columns

61

Pre-Columns and Guard Columns

9

Pre-columns and Guard Columns

Mobile phase

from pump

Pre-columnInjector

Guard column

Analytical

column

To detector

Pre-column acts on mobile phase.Alternative: Polymer analytical columns

Guard column acts on sample.

Pre-columns are positioned prior to the injector and serve to condition the mobile phase. The lifetime of a silica column may be extended because the pre-column will saturate the mobile phase with dissolved silica before the mobile phase ever reaches the analytical column. Extreme pH’s, high ionic strength, or high mobile phase polarity all contribute to dissolution of silica. Guard columns are placed between the injector and the analytical column. Guard columns should be the same stationary phase and internal diameter as the analytical column, but they are very short. These columns protect the analytical column from impurities and particulates. Many are sold as cartridges to facilitate frequent replacement.

Practical Aspects of Performing Analyses Syringe Wash: HP 1090

62

Syringe Wash: HP 1090

10

Syringe Wash 1090

Normal Mode Wash Mode

You should perform a syringe wash:• Daily• After changing mobile phase

composition• When you experience

reproducibility problems• When air has been found in the

solvent delivery system

For best reproducibility of peak area and height, an HP 1090 requires a syringe wash on at least a daily basis. Syringe washes should be performed any time the mobile phase composition changes, when you perform any maintenance on the auto-injector area, or as part of your start up procedure each day. The syringe wash function simply removes air bubbles from the syringe. The wash function has nothing to do with sample contamination. The sample loop is continuously flushed during normal operation. Syringe washes are not required on the HP 1050 or HP 1100 series HPLC’s.

Practical Aspects of Performing Analyses Priming

63

Priming

11

Priming

Removes air bubbles from the solvent composition.Allows easy solvent system change.

Priming the HPLC Pump

Flow

Purge Valve

to waste

When a liquid chromatograph has been idle, there is always the possibility that air has managed to permeate the flow path. Priming the liquid chromatograph involves pumping each channel at 100% composition and high flow rate until steady pressure and flow is obtained. The mobile phase will forcibly expel any trapped air. Priming on a daily basis, when the mobile phase is changed, or when maintenance work is required will lead to more reproducible peak areas and retention times. With the HP 1090, the capillary to the column should be disconnected and the end placed into a beaker before priming. For the HP 1050 or 1100, the purge valve may be opened and the flow channeled to waste.

Practical Aspects of Performing Analyses Column Equilibration

64

Column Equilibration

12

Column Equilibration

• Assures reproducible results• 5-10 column volumes for equilibration of Reversed-phase columns

Before you begin an analysis, the column must be equilibrated with the mobile phase. Reversed-phase columns using a simple mobile phase without buffers and modifiers require only 5-10 column volumes for equilibration. Some applications may take much longer. A column, which has not been equilibrated properly, will exhibit irreproducible retention times. Other symptoms of an unequilibrated column are unstable pressure and a drifting baseline.

Practical Aspects of Performing Analyses Preparing Samples: Filtering

65

Preparing Samples: Filtering

13

Preparing Samples - Filtering

• Nylon - hydrophilic nature works with aqueous and solvent based samples, autoclavable to 121ºC, pH range 3-12, no concentrated acids.

• PTFE- a hydrophobic membrane which is highly resistant to solvents, acids, and alkalies. This filter is generally used for non-aqueous samples. pH range 1-14.

• Cellulose Acetate- good filter for aqueous biological samples with very low protein retention. pH range 4-8.

• PVDF- highly resistant to most solvents, exhibits low protein binding. pH 2-12.

• Ultrafilter Membranes- molecular weight cut-off filters for biological samples.

• Nitrocellulose- exhibits high protein retention. • Solid Phase Extraction.

Samples should be filtered prior to injection. Sample particulates will cause blockages in the capillary tubing, particularly at the point of injection, and in the column inlet frit. Many HPLC suppliers sell a variety of filter products, which are application and mobile phase dependent. The list above can provide you with a starting point. Do not forget solid phase extraction, which can be useful for removal of strongly retained sample components that may damage the analytical column. Solid phase extraction may also be utilized for isolation and concentration of a particular set of sample components.

Practical Aspects of Performing Analyses Preparing Samples

66

Preparing Samples

14

Preparing Samples

• Dissolve the sample in the mobile phase or in a solvent weaker than the mobile phase.

• The sample volume should be kept as small as possible.

Sample in Mobile Phase Sample in Stronger Solvent

Ideally, the sample should be dissolved in the mobile phase or in a solvent weaker than the mobile phase for best chromatographic results. If the sample is dissolved in a stronger solvent than the mobile phase, and large injection volumes are used, chromatographic peaks will become broad and begin to have a doublet appearance. Sample volumes should be kept as small as possible in order to avoid loss of resolution due to volume overloading. The injection volume limitations are related to the column internal diameter. For instance, a 2.1mm i.d. column VKRXOG�KDYH�LQMHFWLRQ�YROXPHV��� O�RU�OHVV�

Practical Aspects of Performing Analyses Worksheet

67

Worksheet

15

Worksheet

1. You are running a routine analysis when you notice a periodic perturbation in the baseline. The pressure reading is fluctuating up and down. What is the problem? How would you correct it?

2. You decide to run a reversed-phase analysis on an instrument in your lab. The previous operator does not indicate the solvents last used in the instrument. You place water in channel A and turn on the pump. The pressure increases at rapid rate and becomes variable. You cannot get a stable baseline. Suggest a possible reason for this dilemma.

Practical Aspects of Performing Analyses Worksheet

68

HPLC Instrumentation

HPLC Instrumentation In This Section, You Will Learn:

70

In This Section, You Will Learn:

2

In This Section, You Will Learn About the Following HPLC Components:

Injector

Detector

Chromatogram

Column

Solvents

Pumps

Mixer • Tubing and fittings• Solvent Delivery Systems• Injection Systems• Detectors

In this section, you will learn about the components of a high performance liquid chromatograph including fittings and tubing, solvent delivery systems, injectors, and detectors.

HPLC Instrumentation HPLC Tubing

71

HPLC Tubing

3

HPLC Tubing

• Stainless Steel– Commonly 1/16" OD with various internal diameters.

• Teflon– Good for pressures up to 1000 psi. Commonly used from the

solvent reservoirs to the pump.• PEEK

– Can be used to replace stainless steel tubing when a metal-free environment is desired. 1000-8000 psi

• Tefzel– Pressures up to 3500 psi for metal-free analysis.

HPLC tubing is most commonly stainless steel or plastic. Most stainless steel tubing is 1/16 inch o.d. with varying internal diameters. HP uses stainless steel tubing with an internal diameter down to 0.12 mm. Stainless steel handles the high pressures of HPLC well and is more robust. Teflon tubing is often used for the connections from the mobile phase reservoir to the pump. The internal diameter will be sufficiently large to deliver solvent to the pump without drawing air. PEEK (polyetheretherketone) may be used in place of stainless steel when the analyst desires to limit sample contact with metal ions. Tefzel tubing can also be used when the analyst wants to limit sample exposure to stainless steel.

HPLC Instrumentation Fittings

72

Fittings

4

Fittings

0.130 in.

0.170 in.

0.090 in.

0.090 in.

0.090 in.

Swagelok

Parker

Valco

Waters

Rheodyne

Uptight

0.080 in.

Troubleshooting LC Fittings, Part II.LC/GC Magazine 6:788 (1988)

J. W. Dolan and P. Upchurch.

There are many different fittings for stainless steel tubing which are not necessarily interchangeable. The distance from the swaged ferrule to the end of the tubing varies from manufacturer to manufacturer. When incompatible fittings are mixed, undesirable peak dispersion may result. HP HPLC instrumentation utilizes swagelock stainless steel fittings. For a leak free connection, tighten the fitting with your fingers, then using the wrench, turn the fitting one quarter turn. Over tightening the fitting may cause damage.

HPLC Instrumentation Fittings

73

Fittings

5

Other HPLC Fittings

Finger Tight Fittings• Universal, the fitting does not attach

permanently to the tubing.• Convenient, no wrenches.

Zero Dead-Volume Union• Connect two pieces of tubing.• without any dead volume between

the tubing.• Not usually interchangeable from

one manufacturer to another.

Finger tight fittings have become very popular. They are nearly universal because the ferrule is not swaged permanently to the tubing. It also very convenient not to have to get out the wrenches every time you have to change a column. Unions are used to connect two pieces of tubing together. A zero dead-volume union will connect two pieces of tubing together without adding any additional dead volume. Most manufacturers of zero dead-volume unions do not butt the tubing from one capillary directly up against the other capillary. Instead, a thin web is used between the two pieces of tubing.

HPLC Instrumentation Filters

74

Filters

6

Filters

Solvent Inlet Filter• Stainless Steel with 10 micron

porosity.• Removes particles from solvent.

Precolumn Filter• Used between the injector and

guard column.• 2 to 0.5 micron.• Removes particulates from sample

and injector wear.• Must be well designed to prevent

dispersion.

Guard column

Injector

Analytical Column

Precolumn Filter

Particulates in the mobile phase may damage the pumping system. Commonly, a 10 micron solvent inlet frit is placed in the mobile phase reservoir to trap particulates. The solvent inlet filter should be replaced or cleaned on a periodic basis or the required flow through the filter may not be possible resulting in mobile phase composition changes and air in the pump. A precolumn filter may be placed between the injector and the analytical column to catch particles in the sample and particles from injection valve wear. The filter usually consists of a 0.5 to 2 micron frit held in a cartridge. The frit can be easily replaced when the system pressure rises.

HPLC Instrumentation Functions of the SDS

75

Functions of the SDS

7

Functions of the Solvent Delivery System

The solvent delivery system has three basic functions:

1. Provide accurate and constant flow.2. Provide accurate mobile phase compositions.3. Provide the force necessary to push the mobile phase through

the tightly packed column.

A solvent delivery system must provide accurate and reproducible flow and composition. It must also provide the force necessary to push the mobile phase through the tightly packed column. The next slides will illustrate some of the ways to accomplish this task. In addition, the solvent delivery system should not produce pressure pulsations. The addition of a damping unit is usually customary.

HPLC Instrumentation Multichannel Gradient Valve

76

Multichannel Gradient Valve

8

Multi-channel Gradient Valve

• Determines mobile phase composition.• Largest solvent plug fills first.• HP 1090 PV5, HP 1050 quaternary pump, and the HP 1100 quaternary

pump.

The purpose of a multi-channel gradient valve is to produce the solvent composition. This valve provides a fixed volume packet to a pump. For example, in the case of the HP 1090 PV5 system, the flow is split into 89ul packets. Therefore, if the desired composition were to be 80% A and 20% B, the valve would remain open on the A channel for 71.2 µl then close and deliver 17.8 µl of B.

HPLC Instrumentation Dual Piston Parallel Pump

77

Dual Piston Parallel Pump

9

Dual Piston Parallel Pump

A

B

SinglePiston

Delivery

Piston ’A’ Advancing

Piston B Retracting

CombinedDelivery

P

Check

Valves

P

A B

Pumphead

Piston

RotarySwitchingValve

A dual piston parallel pump is designed to deliver a continuous flow of mobile phase to the column by operating 180 degrees out of phase. The destructive interference of alternating pump pulses dampness the total pulsation. While one piston of the pump is delivering solvent to the column, the other retracts to refill the solvent chamber. An example of a dual piston pump is the HP 1090 metering pump.

HPLC Instrumentation Dual Piston Series Pump

78

Dual Piston Series Pump

10

Dual Piston Pump in Series

• First piston chamber is twice the size of the second.

• Provides constant flow and pressure necessary to get through column.

An alternative method for delivery of a pulse free mobile phase is the dual piston series pump. The first solvent chamber is twice the volume of the second solvent chamber. While the second piston delivers mobile phase to the column, the first piston chamber refills. When the second piston chamber is empty, the first piston moves forward and not only refills the second chamber, but also continues to deliver mobile phase to the column.

HPLC Instrumentation Ball Valves

79

Ball Valves

11

Ball Valves for Reciprocating Piston Pumps

Gold Seal

Sapphire Insert

Ruby Ball

Spring

Insert

The purpose of a ballvalve or check valve is to provide unidirectional flow. The elements of a ballvalve include a sapphire seat, a ruby ball and a spring for tension. When a piston is drawing solvent from the mobile phase reservoir, the ruby ball on the inlet side is pulled upward allowing mobile phase to fill the solvent chamber. The ruby ball on the outlet side of the chamber is pulled down against the sapphire seat so that solvent which has already been displaced to the column will not be pulled back into chamber. When the piston is on the forward stroke, the mobile phase will push the outlet ruby ball away from the sapphire seat The force of the mobile phase will push the ruby ball on the inlet side into the sapphire seat preventing flow to the reservoir.

HPLC Instrumentation Metering Pump Seals and Pistons

80

Metering Pump Seals and Pistons

12

Metering Pump Seals and Pistons

2

345

1 1 = Piston2 = Support Rings3 = Seal Keepers4 = Seals5 = Wear Retainers

The pistons found in these pumps are typically made from man-made sapphire. They should be examined on a periodic basis for scratches. Piston pumps also contain piston seals which should be replaced on a periodic basis to maintain retention time and peak area reproducibility. Some styles of piston pumps also contain wear retainers so that seal wear material will be trapped and not damage other parts of the instrument.

HPLC Instrumentation Diaphragm Pump

81

Diaphragm Pump

13

Diaphragm Pump

+

PressureOverrideValve

Piston

Stainless Steel Diaphragm

Inlet Ball Valve

Oil

CheckValve

Outlet ball Valve

The reciprocating piston/diaphragm pump has the advantage of removing the piston and piston seal from the harmful mobile phase and placing them in a lubricating environment. One side of a gold coated stainless steel diaphragm contains oil while the other side comes in contact with the mobile phase. When a reciprocating piston places pressure on the diaphragm from the oil side, the diaphragm bulges downward forcing solvent from under the diaphragm out onto the column. As the reciprocating piston retracts, the diaphragm bulges upward allowing solvent to fill the space underneath the diaphragm. The HP 1090 contains such a pump to provide the high pressures necessary to force the mobile phase through the tightly packed column. It operates at 10Hz.

HPLC Instrumentation Sieves and Filters

82

Sieves and Filters

14

Sieves and Filters

Sieves and Filters are used to protect other parts of the LC from pump seal material.

)ORZ

)ORZ

Ball-Valve Housings

HP 1090

Sieve

HP 1050

Filter

Most solvent delivery systems contain an in-line solvent filter or frit before the injector. The purpose of this filter or sieve is to collect pump seal particles of the pump seals as they break off so that they don’t damage the injection valve or in the case of the HP 1090 the ball-valves. These in-line filters must be replaced on a regular basis.

HPLC Instrumentation Damping Unit

83

Damping Unit

15

Damping Unit

Damping Unit

Pressure

2% P/P

Pump Ripple

• Filled with compressible liquid separated from mobile phase by a membrane.• Pressure ripples from high pressure pump are reduced to < 2% of original

value.

The purpose of the damping unit is to reduce pressure pulsations caused by the action of the pumps to a minimum. A damping unit consists of a diaphragm separating the mobile phase from a compressible liquid. In the HP 1090, the diaphragm is placed after the diaphragm pump. In the dual piston series pump, the diaphragm is located between the first and second piston chambers.

HPLC Instrumentation 1090 SDS

84

1090 SDS

16

Solvent Delivery System - 1090

Low Pressure Compliance

High-PressureDiaphragm Pump

Dual-syringe metering pump

Switching Valve

Damping Unit

• Metering pump for each solvent reservoir - for composition and flow.

• Mixing in the Low Pressure Compliance.

• Diaphragm pump for high pressure• Damping unit.

The 1090 can be used to illustrate all of the components of a solvent delivery system working together. Each mobile phase reservoir has its own parallel piston pump (metering pump) for metering mobile phase flow and composition. These pumps are operating at low pressures. Mixing occurs in the low pressure compliance. After mixing, the mobile phase flows through a sieve then an inlet ball valve into the diaphragm or booster pump. Here the pressure necessary to force the mobile phase through the column is supplied. On the outlet side of this pump, there is another ball-valve and then a damping unit to reduce pressure pulsations.

HPLC Instrumentation Quaternary Pump

85

Quaternary Pump

17

Solvent Delivery SystemQuatenary Pumping System

• Multichannel gradient valve for composition.

• Dual piston series pump for flow and pressure.

• Damping unit.

A quaternary solvent delivery system consists of a multi-channel gradient valve for mobile phase composition control. The mobile phase then flows through an active inlet check-valve and then into the first chamber of the dual piston series pump. A ball-valve is placed at the outlet of the first chamber to provide unidirectional flow. The mobile phase then flows through a damping unit and into the second chamber of the dual piston series pump. A frit is located at the outlet of the dual piston’s second chamber. This pump provides the high pressure and the proper flow rate.

HPLC Instrumentation Manual Injection

86

Manual Injection

18

Manual Injection

ToWaste

Sample Loop

(Fixed Volume)From Pump

ToColumn

SampleSyringe

LoadInject

ToColumn

Manual injection valves are typically six-port valves with a sample loop across one pair of the ports. The loop is filled with sample while the mobile phase bypasses this part of the valve. The valve is then switched to the inject position and the contents of the sample loop are carried onto the column. To prevent concentration gradients, five times the sample volume is typically injected. Internal sample loops are found on injection valves with injection volumes less than 5 ml. Flushing a manual valve is extremely important to prevent blockages and sample carry-over. Do not use GC syringes in an LC. This practice will lead to a scratched rotor seal.

HPLC Instrumentation Auto-injection System

87

Auto-injection System

19

Auto-Injection Systems

Injecting a Sample

Prepare to InjectPre-Run

Load Sample Inject & Run

Auto-injectors can be operated by compressed air or electronically actuated. They can also be fixed or variable volume. For illustrative purposes, the HP 1050 auto-injector is shown. Prior to injection, the mobile phase flow is through the valve, metering device, sample loop, needle, needle seat, needle seat capillary, back to the valve and onto the column. For an injection, the valve switches so that the mobile phase flow will bypass the auto-injector. The needle arm rises and a vial is placed under the needle. The needle is forced down into the vial and the metering device is pulled back to draw sample into the needle and the loop. After the appropriate volume is drawn, the needle rises and the vial is returned. The needle moves into the seat and the valve is again switched to allow flow through the auto-injector delivering sample to the column.

HPLC Instrumentation Rotor Seals

88

Rotor Seals

20

Rotor Seals

Align notch withpin

A rotor seal is located inside injector valves. The seal is a disk with grooves to direct the mobile phase flow path. These seals need to be replaced on a periodic basis or irreproducible injection volumes will result due to cross-port leaks.

HPLC Instrumentation Necessity for More than One Detector

89

Necessity for More than One Detector

21

Necessity for More than One Detector -Sensitivity

PAH’s extracted from soil; Sup.LC-PAH 150x4.6mm;Solv.: H2O/CH3OH= 10:90

Fluorescence

UV-signal

WL

241/

394

WL

270/

388

WL

248/

411

WL

302/

420

WL

247/

504

Pyrene

Chrys

ene

Benzo

(e)py

rene

Peryle

ne

Benzo

(k)flu

oran

then

e

Benzo

(a)py

rene

Benzo

(ghi)

peryl

ene

Inden

o(123

-cd)p

yrene

There are a number of useful detectors for HPLC. While the most popular detectors are UV-VIS absorbance based, other detectors are needed. UV-VIS detectors are generally less sensitive than fluorescence or electrochemical based detectors.

HPLC Instrumentation Necessity for More than One Detector

90

Necessity for More than One Detector

22

Necessity for More than One Detector -Selectivity

Flecainide in Serum

Therapeutic concentration: 1.8mg/l, 20ul injected

UV and fluorescence signal

FL signal

UV signal

From time to time, it may be necessary to identify a trace component in a complex matrix. An instrument such as a fluorescence, electrochemical, or mass selective detector may be required to effectively quantitate the sample component when it cannot be separated from other components chromatographically. The selective detector can be programmed for a specific property of a compound or compound class.

HPLC Instrumentation Necissity for More than One Detector

91

Necissity for More than One Detector

23

Necessity for More than OneDetector - Qualitative Information

Qualitative Information

Take peak spectrum (UV)

Chlortoluron ?

44

68

58

96 132 138 158

172

215

200

Take peak spectrum (MS)

104

Mass/Charge

Atrazine ?

Wavelength (nm)60 80 100 120 140 160 180 200 220

Often, a detector is needed which will help identify unknown compounds. For gas chromatographic analysis, the mass spectrometer is such a detector. Qualitative analysis is not yet that routine in HPLC. The diode array, mass spectrometer and infrared detectors, however, are becoming increasingly useful.

HPLC Instrumentation UV-VIS Detectors

92

UV-VIS Detectors

24

Beer’s Law

Absorbance Detectors

Fixed Wavelength

Light

Flow

Source

Cell

Lenses

Slits

DetectorElements

I

Io

log _____ = A = abc

I

I o

I = Incident Radiation Intensity

I = Transmitted Radiation Intensity

A = Absorbance

a = molar absorptivity

b = path length

c = solute concentration

o

UV-Vis detectors are the most commonly used detectors in HPLC. Solutes which absorb UV or visible radiation (typically 190 - 600 nm) can be detected. The degree of absorption is a function of the molar absorptivity of the sample molecule, the path length of the detector flow cell and the solute concentration. The solute concentration is directly proportional to the absorbance allowing quantification. UV-Vis detectors can routinely achieve detection of only a few nanograms. They have a large linear dynamic range and are very robust.

HPLC Instrumentation UV-VIS Detectors

93

UV-VIS Detectors

25

Chromophores

Chromophore max(nm)Structure

Amine

Ethylene

Ketone

Ester

Aldehyde

Carboxyl

Nitro

Phenyl

Naphthyl

195

190195

205

210

200-210310202,255

220,275

-CHO

-COOH-NO2

-

-NH2

-C=C-C=O

-COOR

-

When utilizing a UV-Vis spectrophotometer, it is often advantageous to work at the absorbance maximum of a component or compound class being analyzed. It is more important, however to work at the wavelength which will provide you with the best possible signal to noise ratio. Make certain that background interferences, such as absorbance of mobile phase component, do not degrade your signal to noise. While many compounds absorb at or near 254 nm, it is advantageous to have a variable wavelength or diode array detector which can be adjusted to monitor multiple wavelengths either in sequence or simultaneously.

HPLC Instrumentation UV-VIS Detectors

94

UV-VIS Detectors

26

Variable Wavelength Detector

This type of UV-Vis spectrophotometer allows sequential monitoring of any wavelength between 190 and 600 nm. A deuterium lamp emits a continuous spectrum from 190-600 nm. The chosen wavelength of light passes through the flow cell after being mechanically determined by the grating. Most variable wavelength detectors are time programmable and you may also obtain a UV spectrum of a component of interest by stopping the flow and trapping the component in the flow cell, then rotating the grating. Some variable wavelength detectors may also have a tungsten lamp for radiation from 340-850 nm.

HPLC Instrumentation UV-VIS Detectors

95

UV-VIS Detectors

27

Diode Array Detector

Diode Array

GratingOptical

Slit

DetectorFlow Cell

HomiumFilter

AchromaticLens

UVLamp

VisLamp

The diode array detector can provide detection at a single wavelength or simultaneously at multiple wavelengths. This detector also has the ability to store spectra for peak purity analysis, library searching, and creation of extracted signals. This is the schematic for an HP 1100 diode array. The combined tungsten and deuterium lamps emit radiation from 190-850 nm. The radiation is collimated through the flow cell, then a mechanically controlled slit. The radiation is dispersed at the holographic grating into individual wavelengths of light. Each photodiode receives a different narrow wavelength band. A complete spectrum is taken approximately every 12 ms and spectra and signals are created and stored.

HPLC Instrumentation UV-VIS Detectors

96

UV-VIS Detectors

28

Diode Array Spectral Capabilities

Graphical and Numerical Results

200 200400 400

Purity Match

764

Purity Match

999

impure pure

Signal

Spectra

Wavelength (nm)Wavelength (nm)

Three dimensional data allows one to:• Perform peak purity.• Search user-created libraries.• Recreate signals from stored

spectra.

Three dimensional data can be very useful to the analyst. In peak purity analysis, spectra across the peak are compared with an average spectrum from the peak. If the data correlates well, a high purity factor is reported. If the data does not correlate well, then the peak is considered impure. With the availability of spectra, one can also compare spectra of unknown chromatographic peaks with those of known stored library spectra and identify the unknown. Finally, if enough spectra are stored during a chromatographic run, a chromatogram can be produced from any signal selection within the limits of the spectral collection. These chromatograms are called extracted signals.

HPLC Instrumentation UV-VIS Detectors

97

UV-VIS Detectors

29

Worksheet

Would UV-detection be suitable for:

a) Separation of polyvinylacetatepolymers.

b) Separation of phthalates.

c) Inorganic anions.

d) Separation of triazine pesticides.

COOR

COOR

SCH3

N

N N

NHCH

AmetrynCH

CH 3

3

2C H NH

5

C C

OCOCH3

HPLC Instrumentation Fluorescence Detection

98

Fluorescence Detection

30

Fluorescence Detection

Flow

Cell

PhotomultiplierTube

LightSource

Excitation

EmissionVariable Excitation

andEmission

Wavelengths

The fluorescence detector is a highly sensitive and specific detector for HPLC. A 1000 fold increase in sensitivity over UV detection is possible. About 20% of compounds can naturally absorb UV radiation becoming excited and subsequently emitting radiation at a lower energy and longer wavelength than the excitation energy. Many others can be made to fluoresce through derivatization. Radiation from a deuterium or xenon source is focused onto the first grating. This grating is rotated so that only the appropriate wavelength will focus upon the flow cell. The sample fluoresces and radiation is emitted in all directions. The emission radiation is only measured, however, 90 degrees to the incident radiation away from any interfering stray light. The fact that both the excitation and emission wavelengths are specific makes this detector quite suitable for trace analysis in complex matrices.

HPLC Instrumentation Fluorescence Detection

99

Fluorescence Detection

31

Derivatizing Agents

Functional Group

-NH

-NHR

-COOH

-CHO,=C=O

-CO-COOH

Reagent

o-Phthalaldehyde

9-Fluorenylmethylchloroformate

p-Bromophenylacylbromide

2-Naphthacylbromide-OH Phenylisocyanate

2,4-Dinitrophenylhydrazine

2,4-Dinitrophenylhydrazine

2

The above is a list of common derivatizing agents and what functional groups react with these agents. Derivatizing agents are useful when samples do not naturally fluoresce, but excellent detection limits are desirable. This list is not complete.

HPLC Instrumentation Fluorescence Detection

100

Fluorescence Detection

32

Pre-Column Derivatization

Pre-Column Derivatization

Reagent Sample

Mixing

Heated up to 99 C

Advantages• Reaction conditions are freely

chosen.• Derivatization reaction can occur

slowly.• Derivatization can serve as a

purification step.• Excess reagent can be removed.

Disadvantages• Artifacts and multiple peaks can

occur.• Reaction must be very reproducible.• Separation may be more difficult.

Derivatization may be applied pre or post-column. Pre-column derivatization may be carried out on-line as shown or off-line. One of the advantages of pre-column derivatization is that the reaction can occur slowly. In post-column derivatization, the reaction must occur as the mobile phase flows through tubing from the end of the column to the detector. The excess tubing in post-column derivatization can lead to band broadening. An advantage of post-column derivatization is that you do not have to separate excess reagent and other products from the sample. An example of pre-column derivatization is amino acid derivatization with OPA. An example of post-column derivatization is carbamate analysis.

HPLC Instrumentation Refractive Index Detection

101

Refractive Index Detection

33

Refractive Index Detectors

Sample

Reference

Sample Cell

ReferenceCell

Photodiodes

Beam DeflectionFresnel PrismLaser Interferometer

ReferenceSample

The refractive index detector is one of the most universal LC detectors. Anything that changes the refractive index of the mobile phase can be detected. It is also one of the least sensitive LC detectors. Refractive index detectors must always be thermostatically controlled as the refractive index will change with temperature. The most common type of refractive index detector is the beam deflection device. The Fresnel prism can be used for microbore work. The laser interferometer is the most sensitive but can be the least reliable.

HPLC Instrumentation Light Scattering Detection

102

Light Scattering Detection

34

Light Scattering Detector

Glass Cell

Solvent

Laser Beam

Detector 2

Absolute weight and size of molecule may be calculated from scattered light as a function of angle

k*c = 1 + 2A CR(θ) M P(θ)w

2

R (θ): excess intensity of scattered light at angle θc: sample concentrationM : weight average molecular weightA : second virial coefficientK*: optical parameter 4∏ n (dn/dc) / λ N ,n: refractive index

dn: refractive in incrementdc

z

w

2 2 2 4A0

Detector 3Detector 1

Laser light scattering detectors allow the absolute molecular weight determination of polymers and biopolymers from MW 1000 to hundreds of millions. The polarized laser beam passes through the flow cell. The sample scatters light at all angles. Detectors placed around the flow cell receive the scattered light. Absolute molecular weight data calculations are then performed by the computer based upon the equation above.

HPLC Instrumentation Electrochemical Detection

103

Electrochemical Detection

35

Electrochemical Detectors

-1.5 -1.0 -0.5

0.5 1.0 1.5 V

Reduction

Wave

Oxidation

Wave

Currentµ Amps

Limiting

Diffusion

ResidualV

Auxillary

X Y

e-

X Y

e-

Electrode

MobilePhase

WorkingElectrode

Potentiostat

FlowCell

ReferenceElectrode

Current

Current

Current

Electrochemical detectors are sensitive devices which can detect traces of readily oxidizable or reducible compounds. The detector flow cell has three electrodes: a reference electrode, working electrode, and an auxiliary electrode. The potential between the working and auxiliary electrode may be selected based upon a voltammogram where the optimum voltage can be determined. The reference electrode provides a stable and reproducible voltage to which the potential of the working electrode can be referenced.

HPLC Instrumentation Conductivity Detection

104

Conductivity Detection

36

Conductivity Detectors

ref.capacitor

cell

variable resistances

fixed resistor

C

r

Balancecontrol A E

F

D

B

~

Schematics Applications

water

soap products

detergents

soft drinks

blood

plating baths

nuclear fuel reprocessing

streams

IonsAcidsBasesSalts

in}

Conductivity detectors are most commonly used for detection of inorganic and organic ions usually after ion exchange chromatography. This detector measures the conductance of the mobile phase. The sensitivity of the detector is largely dependent upon the initial conductance of the mobile phase.

HPLC Instrumentation HPLC-MS

105

HPLC-MS

37

HPLC-Mass Spectrometry

Selected Ion Monitoring

Total Ion

Chromatogram

Full Scan

252

200

113

126

111224

Interfaces

Particle BeamThermosprayContinuous Flow FABElectrospray

The mass spectrometer is a potentially powerful detector for liquid chromatography. The most common LC-MS interfaces include the particle beam interface, continuous flow FAB, thermospray, and electrospray. The beauty of the mass spectrometer is its ability to provide molecular weight information and sometimes, structural information.

HPLC Instrumentation HPLC-MS

106

HPLC-MS

38

Electrospray LC-MS

Electrospray combined with API and APCI is currently the most promising LC-MS technique. Eluent is injected through a stainless steel capillary which is held at 4 to 6 kV relative to a cylindrical electrode. The ions are desorbed from charged droplets and transported into the mass spectrometer. The ions typically have multiple charges. As a result, quadrupole mass spectrometers which measure the mass to charge ratio can be used for detection of high molecular weight compounds.

HPLC Instrumentation Radiometric Detectors

107

Radiometric Detectors

39

Radiometric Detectors

C-Methionine14

Scintillator

Fluorescence Event

PPOPOPOPNaphthaleneDioxane

Disintegration

-

C-Methionine12

CoincidenceElectronics

Multi-ChannelAnalyzer Computer

End-on PMT

End-on PMT

A radiometric detector monitors the amount of radioactivity in the mobile phase. Prior to analysis, the analytes are labeled with radioactive isotopes such as 14C. The radioactive isotopes undergo a disintegration to 12C along with the production of a beta particle. The beta particle interacts with the scintillator and the scintillator releases energy in the form of fluorescence. The fluorescence is followed with end-on photomultiplier tubes.

HPLC Instrumentation Worksheet

108

Worksheet

40

Worksheet

1. What is the difference between a dual-piston parallel and a dual piston series pump?

2. How does a ball valve work?

3. Describe the types of tubing used in HPLC instrumentation.

HPLC Instrumentation Worksheet

109

Worksheet

41

Worksheet

1. Name some of the maintenance considerations for an auto-injector.

2. Overall, which LC detectors are known for their sensitivity?

3. Which LC detectors are known for their universal nature?

HPLC Instrumentation Worksheet

110

HPLC Troubleshooting

HPLC Troubleshooting In This Section You Will Learn:

112

In This Section You Will Learn:

In This Section You Will Learn:

• Basic Maintenance and Troubleshooting of the Solvent Delivery System, Injection System and Detection System

• How to Troubleshoot Baseline Performance Problems

• Causes of Peak Shape Performance Problems

HPLC Troubleshooting Record Keeping

113

Record Keeping

Tests for HPLC ColumnsFor a newly acquired column perform and record the following

under isocratic conditions:

1. Record theoretical plates, N, accompanied by:� Length and internal diameter of column� Sample compound and k’ value� Mobile and stationary phase� Mobile phase flow rate� Sample and size� Temperature

2. Peak Symmetry.3. Include phenol and amine } for testing against acids and

bases.4. Record column pressure.

The Standard Chromatogram

Record:Analyses PerformedService Dates

The Logbook

New Column12/4/94

Pump Seals12/1/94

Check Valves11/2/94

Logbook ServiceDate

Record Keeping

A record of HPLC instrument maintenance should be chronicled in order to facilitate timely repairs and maintenance. From this record, one can reliably predict the need for such preventive maintenance as pump seal or ball-valve replacement. Chromatographers should have at their disposal a reliable test mixture to use when there is a need to distinguish between method problems and instrument failures. The inclusion of a weak acid and base can test the acidity of reversed-phase columns.

HPLC Troubleshooting Proper Care of the HPLC

114

Proper Care of the HPLC

pH Range

■ Instrument pH range 2.3 - 9.5■ Extended pH range 2.3 - 12.5

Corrosive to stainless steel

■ Hydrochloric acid■ Inorganic acids and strong acids■ Alkali halides (sodium chloride, lithium iodide)■ Carbon tetrachloride with 2-propanol or THF■ Complexing agents (EDTA, citric acid, acetic acid)

Attacks quartz and vespel

■ Alkaline solutions, pH>11

These substances are not recommended. If used, the pump and other parts of the HPLC should be thoroughly flushed when analyses are completed.

Proper Care of the HPLC

HPLC instrument manufacturers will specify the permissible mobile phase pH range of their instrument. The ranges here are for HP instrumentation. The extended pH range kit should be acquired when basic pH’s are required. Use of the listed mobile phase additives will necessitate more frequent maintenance. When additives such as these or buffers are used within the HPLC, make certain that the flow path is flushed before the instrument is shut-down.

HPLC Troubleshooting Peak Retention Time and Precision

115

Peak Retention Time and Precision

Peak retention time precision:� with oven: < 0.3%� without oven: < 0.7%Peak area precision: <1.5%

Causes of Peak retention time and area irreproducibility:

_____________________________________________

_____________________________________________

Peak Retention Time and Area Precision

A convenient way to assess instrument pump performance is to monitor peak retention time and area precision. Shown above are typical relative standard deviations for the 1090. If a known analysis begins to have larger than normal deviations, pump maintenance may be required.

HPLC Troubleshooting Common Pump Problems

116

Common Pump Problems

Common Pump Problems and Maintenance

• Air Entrapment– Degas Mobile Phase– Prime the Pump

• Check Valves• Piston Seals• Pistons• Leaks• Switching Valves• Diaphragm

Dual syringe metering pump

Switchingvalve

Mixing chamberand compliance

High pressurediaphragm pump

Damping unitPistonseals

Pistons

Checkvalves

Diaphragm

The most frequently experienced pump related problem revolves around periodic baseline disturbances which can be attributed to air in the pump itself. This sort of problem is quickly resolved by sparging the mobile phase and repriming the pump. Other pump problems include leaking metering pump seals, scratched pistons, damaged ball-valves and in-line filter blockage. A diaphragm pump may experience a leaking diaphragm noted by the presence of a milky eluent or oil around the pump.

HPLC Troubleshooting Pressure Problems

117

Pressure Problems

Troubleshooting Pressure Problems

Pressure Problem

Pressure T oo High Pressure T oo Low Pressure F luctuations

● Loosen capil lary at column inlet fr i t.

● Check the injector to needle seat capil lary.

● Keep breaking the system in half unti l the blockage is found.

● Make certain it is the correctmobi le phase.

● T ighten loose fi ttings or replacefittings.

● Change meter ing pump seals.● Change or clean clogged

solvent inlet fr i t.

● Degas and reprimethe pump.● Check for loose or improperly

seated fi ttings.● Check metering pump seals.● Check solvent inlet fr its.

When the system pressure exceeds the maximum pressure the liquid chromatograph will shut down. System high pressure is usually a result of particle build up on the column inlet frit, on a filter installed after the injector or on a guard column’s inlet frit whichever is in-line first. The second most likely blockage point is the needle seat capillary, the first restriction after injection. If the blockage is not found then systematically break the system in half until it is found. Lower pressure than normal is usually the result of a leak in the system, either at a fitting or metering pump seal. A clogged solvent inlet frit may also produce such an error as not as much solvent as usual can be pulled out of the reservoir. Fluctuating system pressure is the result of a leak, blocked solvent inlet frit or air in the pump.

HPLC Troubleshooting Baseline Fluctuations

118

Baseline Fluctuations

How would you tell?

Problem:

T he chromatogram has basel ine fluctuations.

I s problem in the detector or pump?

Baseline Fluctuations

There are many causes for baseline noise or wander. One of the first things that you should do is isolate the cause to the detector or pumping system. This task is easily accomplished. Monitor the chromatographic signal with the detector and pump on. Then, turn off the pump. Does the noise remain? If the noise goes away, the problem is in the pumping system or the problem is with your method. If the noise remains, the problem is probably in the detector. The chromatogram above is the result of air in the pump.

HPLC Troubleshooting Noisy Baseline

119

Noisy Baseline

Noisy Baseline: Pump Problem

• Have you changed your mobile phase composition?

• Have you changed your acquisition wavelength?

• What mobile phase was last used in your instrument?

• Do you have a miscibility problem?

• Are your solvents dirty?

If you are experiencing a noisy baseline, ask yourself the questions above. All of these problems are related to the method not instrument problems. If you have just changed the mobile phase then the new solvent may contain contaminants. Noisy baselines may also be the result of immiscible solvents.

HPLC Troubleshooting Mixing Problems

120

Mixing Problems

Problem:

A UV-absorbing mobile phase is dynamicallymixed with a non-UV-absorbing mobile phase.Residual pump noise is produced. A staticmixer reduces the noise level.

Drawback:

Addition of delay-volume.

+

Static Mixer

Mixing Problems

When a highly UV-absorbing mobile phase is mixed with a UV-transparent mobile phase, a noisy baseline can result. The baseline noise is a result of inadequate mixing. This same problem can occur when two mobile phases are slightly immiscible such as the TFA and water/acetonitrile mixtures. The addition of a static mixer can decrease noise by producing adequate mixing. The static mixer will add an additional delay volume which should be taken into consideration.

HPLC Troubleshooting Manual Injection Valve

121

Manual Injection Valve

Manual Injector Valves - Six Port Fixed Loop

ToWaste

Sample Loop(Fixed Volume)From

Pump

ToColumn

SampleSyringe

Load Inject

ToColumn

Common Problems:

■ Improper syringe size or type■ Valve Blockage■ Injection-port leakage■ Sample carryover■ Cross-port leaks

Manual Injection Valve

There are several common problems experienced by manual injection valve users. Blockages are common unless the operator carefully rinses the sample loop at the end of usage. Sample carryover can occur unless the sample loop is flushed between injections. Five times the sample loop volume should be injected into the manual valve in order to avoid concentration gradients within the sample. With time, the rotor seal within the injection valve will develop cross-port leaks. When this occurs, the injection volumes, thus area and peak height will not be consistent. At this point, the rotor seal will have to be replaced. GC syringes (sharp tip) should not be used for lc injection, as they will scratch the rotor seal.

HPLC Troubleshooting Auto-Injectors

122

Auto-Injectors

Waste

Column Pump

Six-Port

Switching

Valve

�Needle seat replaced when necessary

�Rotor or seals in auto-sampler valve replaced when necessary.

�Regular cleaning of optical sensors to ensure alignment.

�Syringe washes and was cycles observed.

�Sample related• Blockage of

needle or tubing when samples are not pre-filtered.

• Lack of peak light reproducibility when a density gradient forms in the vial.

�Needle:• Needle

blocked from septum.

• Bent Needles

Maintenance:Problems:

Auto-Injectors: Common Problems and Maintenance

Common maintenance of auto-injectors includes needle and needle seat replacement, rotor seal replacement in valves, and syringe seal replacement. Always filter samples in order to avoid needle and capillary blockages. Perform individual instrument requirements regarding syringe washes.

HPLC Troubleshooting Good Column Practices

123

Good Column Practices

Good Column Practices

• Filter solvents before use.• Pre-treat samples which contain strongly retained components of no

interest.• Flush column frequently with strong solvent.• Avoid extreme column temperatures > 60 C.• Keep the mobile phase pH between 3 and 7. If operating outside of

this pH range use a pre-column.• Use fresh buffer solutions and aqueous mobile phases or treat them

with sodium azide.• To prepare column for storage, purge column of buffers and leave

in appropriate solvent. Cap tightly.• Avoid physically mishandling columns: banging, dropping or over-

tightening fittings.

Good column practices will preserve the lifetime of your column. Always filter your solvents and samples to remove particulates. Remove strongly retained sample components with solid phase extraction prior to injection. Store columns tightly capped with appropriate mobile phases. Silica based columns should only be used between the pH range of 2 to 8 and temperatures below 80 degrees C. Try not to pressure or solvent shock your column.

HPLC Troubleshooting Column Frit Replacement

124

Column Frit Replacement

1. Carefully removethe column frit.

2. Make a slurry ofstationary phase.

3. Using a Pasteurpipette, mound thestationary phase on top ofthe column.

4. Place a new frit ontop of the mound.

5. Replace column endfitting.

Old fr it New

F r it

Column Frit Replacement

The frit on the inlet side of the column may become clogged causing higher than normal pressure readings. At that point, you may decide to replace the column or UHSODFH�WKH�FROXPQ�LQOHW�IULW��D�VWDLQOHVV�VWHHO��� P�SRUH�VL]H�GLVN�ZKLFK�KROGV�WKH�column material in place. To replace the frit, make a slurry of the same packing material that is currently in the column. Using a Pasteur pipette, mound the packing material at the top of the column. Place the new frit on top of the packing material and replace the column end fitting. Realize that the column will never be as good as it was when new, however performance should improve.

HPLC Troubleshooting Column Regeneration

125

Column Regeneration

Reversed-Phase:

75 mL water + 4 x 200 ml injections DMSO75 mL methanol75 mL chloroform75 mL methanol

Problem:

After prolonged use or insufficient precautions the column will be fouled by build-up of adsorbed materials.

Silica Gel:

75 mL THF75 mL methanol75 mL aqueous 2% acetic acid75 mL aqueous 2% pyridine75 mL THF75 mL methylene chlorideWash with next mobile phase

Column Regeneration

When strongly adsorbed components have become attached to the surface of the stationary phase you may see a degradation in peak shape and resolution. The column may be restored through the column regeneration procedures listed above. Procedure frequency is dependent on sample components.

HPLC Troubleshooting Detector Performance

126

Detector Performance

T ime

mAU

Baseline Noise Determination For Reference

Record width of baseline in mAU or RI units for later comparisons.

Detector Performance

In order to chronicle detector performance you may record the relative standard deviation of the baseline. Excessive baseline noise may be attributable to lamp decay, dirty flow cells or other correctable problems.

HPLC Troubleshooting Detector Time Constant

127

Detector Time Constant

MoreF i lter ing

LessF i lter ing

T oo High

T oo Low

T ime Constant

T ime (min.)

Informationlost

T oo muchinformation

Detector: Time Constant

The detector time constant must be set properly or one of two problems may occur. If the detector time constant is set too high, there will not be enough data points to adequately define the chromatographic peak shape so area and retention time data will suffer. If the detector time constant is set too low, then excessive noise will result because signal averaging is too frequent.

HPLC Troubleshooting Detector Heat Exchangers

128

Detector Heat Exchangers

Detectors: Heat Exchangers

Americas’ Technical Center

with heat exchanger

without heat exchanger

T ime (min.)

� Enhance detector performance by ensuring constant mobile phase temperature in flow cell.

Excessive baseline noise may result when an application utilizes a high column temperature along with a high column flow rate. The noise results when the mobile phase has not come to temperature equilibrium before entering the flow cell. Refractive index changes occur causing noise as the mobile phase cools. A heat exchanger before the detector flow cell can remedy this type of noise. The heat exchanger is simply a capillary welded into a metal block.

HPLC Troubleshooting Noisy Baselines

129

Noisy Baselines

Noisy Baselines

Possible Causes:

■ Dirty Flow Cell■ Detector Lamp Failing■ Pulses from Pump if Periodic■ Temperature Effects on Detector■ Air Bubbles passing through Detector

T ime (min.)

Common detector problems include poor sensitivity, drift, and high frequency noise. Poor sensitivity can be a result of dirty solvents or flow cells, improper detection wavelength or a failing detector lamp. Drift occurs when columns are not yet equilibrated or when the lamp has had insufficient time to warm up. High frequency noise can be a result of line voltage problems.

HPLC Troubleshooting Drifting Baselines

130

Drifting Baselines

� Gradient Elution� Temperature Unstable (Refractive Index Detector)� Contamination in Mobile Phase� Mobile Phase Not in Equilibrium with Column� Contamination Bleed in System

Drifting Baselines

Drifting baselines are common while performing a gradient analysis due to the changing composition of the mobile phase. In other situations, drifting baselines indicate that a column is still equilibrating or the detector is warming up. Contamination problems may also be a factor.

HPLC Troubleshooting Ghost Peaks

131

Ghost Peaks

Ghost Peaks

20% - 100%MeOH GradientNo Sample Injected

Ghost Peaks - Peaks which appear even when no sample is injected.

Problem - Dirty Mobile Phase

60

15

30

15

03 7 15 17

If ghost peaks (peaks which do not result from your sample) appear during gradient analyses the problem can usually be traced to unclean mobile phases, particularly water. At the beginning of a gradient run, impurities in water may stick to the column and the concentration of the impurity is enriched. During the gradient, a stronger mobile phase is introduced onto the column and the impurities begin to elute creating unwanted chromatographic peaks.

HPLC Troubleshooting Extra-Column Dispersion

132

Extra-Column Dispersion

■ Use short, small internal diameter tubing between the injector and the column and between the column and the detector.

■ Make certain all tubing connections are made with matched fittings.

■ Use a low-volume detector cell.

■ Inject small sample volumes.

Increasing Extra-Column Volume

Extra-Column Dispersion

Excessive extra-column dispersion will cause a loss of resolution. Extra-column dispersion is a result of too much tubing or internal diameters which are too large. The flow cell may also cause excessive extra-column dispersion when it has a large volume. Large injection volumes may also cause a loss in resolution. The maximum injection volume is dependent upon the internal diameter of the column.

HPLC Troubleshooting Peak Shape

133

Peak Shape

■ Void Volume in Column■ Partially Blocked Frit■ Only One-Peak a Doublet- Coeluting Components

Normal Doublet Peaks

Void Volume in Column

Peak Shape: Doublets

If all peaks in your chromatogram appear to have some form of doublet appearance then the cause is usually associated with the column or instrument. As the silica gel dissolves, the packing material may settle creating a void in the column. The column void can produce poor peak shapes including tailing or doublets. For small bore and microbore columns, the inlet frit may clog without a large change in pressure resulting in the formation of doublet peaks. When just a few or one peak in the chromatogram appears to have a doublet appearance, the cause can be attributed to a co-eluting peak.

HPLC Troubleshooting Peak Shape

134

Peak Shape

Peak Shape: Broad Peaks

• All Peaks Broadened:– Loss of Column Efficiency.– Column Void.– Large Injection Volume.– High Viscosity Mobile Phase.

• Some Peaks Broadened:– Late Elution from Previous

Sample.– High Molecular Weight.– Sample - Protein or Polymer.

As an HPLC column ages, the chromatographic peaks will broaden. When the resolution is no longer acceptable, the column will have to be discarded. Other causes of broad chromatographic peaks include high viscosity mobile phases and large injection volumes. If just one peak in the chromatogram appears broad it may be a late eluter from an earlier injection.

HPLC Troubleshooting Peak Shape

135

Peak Shape

Normal Tailing

Normal Tailing

Symmetry > 1.2

All Peaks Tail:

� Extra-Column Effects.� Build up of Contamination on Column

Inlet.� Heavy Metals.� Bad Column.

Causes

Some Peaks Tail:

� Secondary - Retention Effects.� Residual Silanol Interactions.� Small Peak Eluting on Tail of Larger Peak.

Peak Shape: Tailing Peaks

In reversed-phase liquid chromatography, interaction between weak acids and weak bases with residual silanol groups can cause tailing. The poor peak shape can be controlled with the proper pH or with the addition of a modifier such as triethylamine to prevent weak base tailing. If all peaks in the chromatogram appear tailed, the peak shape has resulted from a problem with deterioration of the column or because of extra-column effects.

HPLC Troubleshooting Peak Shape

136

Peak Shape

Causes:

� Column Overload� Small Band Eluting Before Large Band

Normal FrontingSymmetry < 0.9

2000

1500

1000

500

0

0 5 10 15 20 25

Time (min)

mA

U

Peak Shape: Fronting Peaks

Most peaks which have a fronting appearance are the result of mass overloading. In addition to the peak shape, an overloaded peak will also have a slight retention time shift to an earlier retention time (usually around 10%). Coelution of chromatographic peaks will also cause fronting when a small peak elutes just before a large peak.

HPLC Troubleshooting Peak Shape

137

Peak Shape

Causes:

■ Absorbance of sample is less than the mobile phase.■ Equilibrium disturbance when sample solvent passes through the

column.■ Normal with Refractive Index Detectors.

Normal Negative

Peak Shape: Negative Peaks

The presence of negative peaks is not usually something to be concerned about. Negative peaks will occur in UV detection when the sample absorbs less than the mobile phase. You may also see a negative peak when the sample solvent passes through the detector. Negative peaks are normal with refractive index detection.

HPLC Troubleshooting Worksheet

138

Worksheet

Time (min)

mA

U

Worksheet

1. Suggest a possible cause for the following non-ideal chromatogram:

The presence of negative peaks is not usually something to be concerned about. Negative peaks will occur in UV detection when the sample absorbs less than the mobile phase. You may also see a negative peak when the sample solvent passes through the detector. Negative peaks are normal with refractive index detection.

HPLC Troubleshooting Worksheet

139

Worksheet

TIME (min)

mA

U

Worksheet

1. Suggest a possible cause for the following non-ideal chromatogram:

HPLC Troubleshooting Worksheet

140

Worksheet

Worksheet

Suggest reasons for the following problems:

1. Flow rates are correct, check valves are working properly, but early peaks in gradient elution do not have reproducible retention times:

2. Baseline is very irregular - high general noise level:

3. Baseline has systematic periodic noise:

HPLC Troubleshooting Worksheet

141

Worksheet

Worksheet

Suggest reasons for the following problems:

1. With a UV detector, height is reproducible, but area and retention times are not:

2. Height and area are not reproducible but retention times are:

3. Reproducibility is good, test mixture looks good but some samplepeaks are broad and tailed: