1

Part III – Introduction to Method Part III – Introduction to Method Development of HPLCDevelopment of HPLC

Procedure for method developmentOptimization of separation in reversed phase

HPLCCalibration methods

2

Method Development of HPLCMethod Development of HPLC

What mode ? Reverse phase, normal phase or ….

What column?Types of columns, temperature …

What mobile phase? Isocratic or gradient Buffer and pH

What detector?What calibration method?

3

Procedure for HPLC Procedure for HPLC method developmentmethod development

1. Information on sample, define separation goals

2. Need for special HPLC procedure, sample pretreatment, etc.

3. Choose detector and detector settings

4. Selecting a HPLC method; preliminary run to estimate best separation conditions

5. Optimize separation conditions

6. Check for problems

7. Validate method for release to routine laboratory

4

Selecting an HPLC modeSelecting an HPLC mode

Method development for most samples is often begun with reversed phase mode.

Ion pair reversed phase and normal phase are secondary choice.

Samples exhibiting any of the following characteristics will often require special consideration. high molecular weight samples, such as synthetic polymers, carbohydra

te or proteins mixtures of optical isomers (enantiomers) mixtures of other isomers samples composed of inorganic salts

5

Reversed phase conditions Reversed phase conditions that affect HPLC separationthat affect HPLC separation

Separation Variable Preferred Initial ChoiceCOLUMN Dimensions (length, I.d.) 15 , 25 cmL x 2 - 6 mmID, Particle size 3-5 um Stationary phase C-8 or C-18MOBILE PHASE Solvents A/B Water/ACN %-B Variable Buffer (compound, pH, concentration) 10-100 mM phosphate, pH 2-7 Additives (eg., ion-pair reagents, amines) 1-20 mM Heptanesulfonic acid sodium

salt for cations or TBA for anions Flow rate 0.1-2 ml/minTEMPERATURE 10-60 oCSAMPLE SIZE Volume 100 ul Mass 100 ug

Reversed phase modeUses water / organic mobile phaseColumn: C-18(ODS),C-8, phenyl, trimethylsilyl(TMS), cyano

Ion pair revered phase modeUses water / organic mobile phase, a buffer to control pH, and an ion pair reagentColumn: C-18(ODS),C-8, cyano

Normal phase modeUses mixtures of organic solvent as mobile phaseColumn: silica, amino, cyano, diol

First choice for neutral or non ionizedcompounds that dissolve in water /organic mixtures

Good choice for ionic or ionizable compounds, especially bases or cations

Good second choice when reversed phase or ion pair reversed phase mode are ineffective: Lipophilic smples that do not dissolve well in water / organic mixtures

For mixtures of isomers and for preparative scale HPLC(in this case, silica is best)

Characteristics of primary HPLC methodsCharacteristics of primary HPLC methods

Ion exchange modeUses aqueous mobile phase plus buffer for pH control

Column: anion or cation exchange

Size exclusion chromatographyUses either aqueous (gel filtration) or organic (gel permeation) mobile phase

Column: diol for gel filtration; polystyrene or silica for gel permeation

Good choice for separation of protein and nucleic acid sample and related compoundsSeparation of inorganic ions (ion chromatography) is also used.

Good choice for separating high molecular weight samples such as proteins and synthetic polymersGPC is used for molecular weight distribution measurements.

Characteristics of secondary HPLC methodsCharacteristics of secondary HPLC methods

8

Improving the separationImproving the separation

Resolution value for complete separation of two neighboring peaks should be value of Rs>1.5.

Value of Rs>2 should be the goal during method development of simple mixtures.

In case of multi component sample, value of Rs>1 may be the separation goal because of difficulty.

9

Separation goals Separation goals in HPLC method developmentin HPLC method development

Goal CommentResolution Precise and rugged quantitative analysis

requires Rs > 1.5Separation time < 5-10 min is desirable for routine

proceduresQuantitation RSD<1% for assays; <5% for trace

analysesPressure <150 kgf/cm2 is desirable.

<200 kgf/cm2 is usually essentialPeak shape Narrow peaks for large signal to noise

ratio are desirableSolvent consumption Minimum mobile phase usage per run is

desirable

10

Completing the methodCompleting the method

The method should be robust in routine operation and usable by all the laboratories.

In this regard, HPLC method must meet stringent standards of precision accuracy ruggedness transferability

Method validation is important to complete method development.

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Optimization of Separation in

Reversed Phase HPLC

12

ResulotionResulotion

Resolution (R or Rs) value for complete separation of two neighboring peaks should be >1.5.

13

Resolution for complete separation Resolution for complete separation

Rs = 1.0 Rs = 1.50Peak shape is not triangle Peak shape is not triangle but Gaussian distribution.but Gaussian distribution.

14

Ratio of overlapping areaRatio of overlapping area

15

Resolution is a function of …Resolution is a function of …

N : average of N1 and N2

k' : average of k'1 and k'2

R Nk

k

1

41

1

'

'

Capacity factor (k’) Selectivity () Column efficiency (N)

16

Capacity Factor, k’Capacity Factor, k’

t1 = retention time of a solute peakt0 = column void time solvent peak (non retained peak)

t0

t1

k’ = t0

t1 - t0

17

Selectivity,

Selectivity is an indication of the degree

of separation between two peaks.

t0

t1 t2

= k’1

k’2

t1 - t0

t2 - t0=

18

The Number of The Number of Theoretical Plate, NTheoretical Plate, N

Equation : N = 16 x ( Rt / W )2

Modified equation for actual measurement :N = 5.54 x ( Rt / W 1/2 )2

Modified equation for integrator :N = 6.28 x (Rt x H / Area)2

W

W1/2

H1/2

H

Rt

Area

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ResolutionResolutionContribution of CapacityContribution of Capacity

1

14

1k

kNR

k' (k'/k'+1) contribution1 1/2 0.5002 2/3 0.6673 3/4 0.7504 4/5 0.80010 10/11 0.90920 20/21 0.95250 50/51 0.980

100 100/101 0.990

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ResolutionResolutionContribution of SelectivityContribution of Selectivity

(-1/) contribution1 0/1 0.0002 1/2 0.5003 2/3 0.667

10 10/11 0.90913 12/13 0.92317 16/17 0.94120 19/200 0.950

100 99/100 0.990

R Nk

k

1

41

1

'

'

21

ResolutionResolutionContribution of EfficiencyContribution of Efficiency

(N)1/2 contribution8000 89 ---10000 100 0.1215000 122 0.3720000 141 0.5830000 173 0.94

R Nk

k

1

41

1

'

'

22

Factors Affecting ResolutionFactors Affecting Resolution

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How to increase N ?How to increase N ?

H = Length of Column / N

Van Deemter Equation

H

Linear velocity

Optimum flow rateOptimum flow rate Each column has a Each column has a optimum flow rate.optimum flow rate.

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Optimum Flow RateOptimum Flow Rate

To get a good column efficiency,

4.0 mmID 0.6 mL/min4.0 mmID 0.6 mL/min4.6 mmID 0.8 mL/min4.6 mmID 0.8 mL/min6.0 mmID 1.0 mL/min6.0 mmID 1.0 mL/min

These setting are preferable.

25

4.0 mmID 0.6 mL/min4.6 mmID 0.8 mL/min6.0 mmID 1.0 mL/min

N

Flow rate

3 m

10 m

5 m

Using smaller particle size!

Smaller Particle Size Smaller Particle Size

26

By decreasing % of organic solvent

However, k’= 2 - 10 is preferable.

k < 2- Insufficient separation

k > 10- Long running time- Broad band, cause (1) low sensitivity (2) poor accuracy

in quantitation 0 2 4 6 8 10 12 14 16 18 20

0 2

4

6

8 1

0

k’

Rs

1- 20

2- 10

How to increase k’ ?How to increase k’ ?

27

Solvent OptimizationSolvent Optimization

MeOH / water mixtures for a mobile phasesample component: nitorbenzene, benzene, 2,6-dinitrotoluene, 2-nitrotoluene, 4-nitrotoluene, 3-nitrotoluene, toluene, 2-nitro-1,3-xylene4-nitro-1,3-xylene, m-xylene

100% MeOH 0.1<K’<0.3

80% MeOH0.6<K’<1.7

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Relationship between k’ and Relationship between k’ and percent organic solvent (B%)percent organic solvent (B%)

log(k’) = (B%) + (, coefficient)

For small molecules,a 10% increase in percent organic solvent (B%) decreases the k’ of every band by a factor of about 3.

0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75

1.0

0.5

0.0

-0.5

log(

k’)

(B%)

Peak 3

Peak 2

Peak 1

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How to improve How to improve

By changing mobile phase and composition pHconcentration of buffercolumn temperaturepacking material (to C8, CN and Phenyl)

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Change of Mobile PhaseChange of Mobile Phase

Although one organic solvent cannot provide good resolution, 3 combination of mobile phase has a possibility to provide good resolution, if critical pair of resolution has been changed.

a

bcd

a

bc

d

[ MeOH/H2O ]critical pair : c,d

[ THF/H2O ]critical pair : a,b

a

bc

d

[ MeOH/THF/H2O ]

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Effect of mobile phase pH Effect of mobile phase pH on separation of ionic compoundson separation of ionic compounds

1:benzoic acid2:sorbic acid3:methylparaben

[analytical conditions]1mL/minODS column 10mM phosphate buffer 75%Acetonitrile 25%40oCUV-240 nm

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pH of mobile phasepH of mobile phase

For samples that contain acidic or basic compounds, retention time vary with pH.

Adjusting pH of mobile phase is very important to get good repeatability.

Buffer solution must be selected correctly.

R-COO- and R-NH3+

33

Relationship between k’ and pHRelationship between k’ and pH

k’ of organic acid and amines varies with pH of mobile phase.

pH (mobile phase)

pKa

k’

associated type

dissociated type In the range of pKa +/- 1.5, a linear relationship between k’ and pH exists.

R-COOH RCOO- + H+

(pKa=4.5)

R-NH3+ R-NH2 + H+

(pKa=6.0)

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Precaution of pH adjustment Precaution of pH adjustment

When pH of mobile phase is very close to pKa of target sample, pH adjustment much be very careful, since pH of mobile phase will influence k’.

Normally, pH which does not affect k’ should be selected.

pH (mobile phase)

k’

associated type

dissociated type

35

Relationship between pH and pKaRelationship between pH and pKaof buffer solutionof buffer solution

AH A- + H+

log(K) = log ([ A- ][ H+] / [ AH ]) = log ([ A- ] / [ AH ]) + log ([ H+])

pH = pKa + log ([ A- ] / [ AH ])

K = [ A- ] [ H+] / [ AH ]

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pKa of organic acid for bufferpKa of organic acid for buffer

conc. pKa1 pKa2 conc. pKa conc. pKa1 pKa2 pKa3100 2.18 6.45 100 4.64 100 2.98 4.35 5.6190 2.19 6.77 90 4.64 90 2.98 4.35 5.6380 2.20 6.78 80 4.64 80 2.99 4.37 5.6570 2.23 6.80 70 4.64 70 2.99 4.38 5.6860 2.25 6.82 60 4.64 60 3.01 4.40 5.7150 2.29 6.84 50 4.64 50 3.02 4.42 5.7540 2.32 6.87 40 4.65 40 3.03 4.45 5.7930 2.38 6.91 30 4.65 30 3.05 4.48 5.8520 2.46 6.95 20 4.66 20 3.08 4.51 5.9110 2.64 7.01 10 4.68 10 3.15 4.57 6.02

phosphate buffer acetate buffer citrate buffer

(conc. unit : mM )pH region of buffer action pKa +/- 1.0

37

How to prepare buffer solution?How to prepare buffer solution?

pH calculation is accurate for low-concentration buffers.

For high-concentration buffers, the pH of the buffer must be measured with a pH meter in the first preparation.

For second preparation of a buffer, pH may not necessary be measured with pH meter.

pH meter can be always used for confirmation of pH.

38

Effect of packing material Effect of packing material characteristics on separationcharacteristics on separation

ODS column Phenyl column

1:benzoic acid2:sorbic acid3:methylparaben

[analytical conditions]1mL/min40oC10mM phosphate buffer (pH2.6) 75%Acetonitrile 25%UV-240 nm

39

Addition of other organic solventsAddition of other organic solvents

Addition of co-solvents besides methanol, acetonitrile and THF is sometimes effective to get better separation.

ethyl etherisoporpyl etherdioxanedimethoxyethanecyclohexne oxide

Around 10% can be added to normal mobile phase for revered phase mode.

ACN/H20 10% Ethyl ether ACN/H20

separation of Estrogens

40

Use of gradient elution mode Use of gradient elution mode

If two neighboring peaks can not be separated by any conditions using isocratic elution mode, these peaks can not be also separated by any conditions using gradient elution mode.

It is necessary to confirm isocratic mode can provide some conditions to separate two peaks, when gradient mode is selected for even faster analysis.

Gradient elution may be helpful to reduce the analysis time without sacrificing resolution.

41

Optimization of gradient methodOptimization of gradient method

If still further optimization is required,

(1) change of longer gradient time(tG)(2) change of faster flow rate(3) use of longer column or better column(4) change of organic solvents (Methanol - Acetonitrile - THF)

42

Problems in gradient elution - 1Problems in gradient elution - 1

Poor reproducibility

Inadequate column equilibrationThe time required for column regeneration is usually

about equal to the gradient time tG.Long equilibration is required in case of only water

(or buffer) use as a initial mobile phase. So it is advisable to begin the gradient with 5% organic or greater.

Use auto sampler to minimize the variation of equilibration time.

43

Ghost peak

Low grade of water This water has some impurities which cause ghost peak. Use pure water.Change to longer wavelength which may not show the

absorption of impurities.It is advisable to begin the gradient with 5% organic or

greater.

Problems in gradient elution - 2Problems in gradient elution - 2

44

Calibration methods in HPLCCalibration methods in HPLC

External calibration method

Internal calibration method

Standard additive method

45

External Standard CalibrationExternal Standard CalibrationPreparation of StandardsPreparation of Standards

Dilution Dilution Dilution Dilution

Target CompoundsTarget Compounds

46

External Standard CalibrationExternal Standard CalibrationAnalysis of VanillinAnalysis of Vanillin

47

External Standard CalibrationExternal Standard Calibration

020406080100120140160180200

0 1000 2000 3000 4000

Peak Area

Con

cen

trat

ion

48

External Standard CalibrationExternal Standard CalibrationCalculation of ResultsCalculation of Results

Y = aX + ba : SLOPb : Y intercept

2500

[Peak Area]

[Con

cen

trat

ion

]

125 ppm

2500

49

Internal Standard CalibrationInternal Standard CalibrationPreparation of StandardsPreparation of Standards

Target CompoundsTarget CompoundsInternalInternalStandardStandard

DilutionDilutionDilutionDilution

50

Internal Standard CalibrationInternal Standard CalibrationAnalysis of VanillinAnalysis of Vanillin

51

Internal Standard CalibrationInternal Standard CalibrationAnalysis of VanillinAnalysis of Vanillin

0

0.5

1

1.5

2

2.5

3

3.5

4

0 5 10 15

[Tergat Area / IS Area]

[Ter

get

Con

c. /

IS

Con

c.]

52

Internal Standard CalibrationInternal Standard CalibrationCalculation of ResultsCalculation of Results

Y = aX + ba : SLOPb : Y intercept

5.0

[Target Area / IS Area]

[Tar

get

Con

c. /

IS

Con

c.]

1.67

2500T500

ISY = Target Conc. / IS Conc.1.67 = Target Conc./ 100 ppmTarget Conc. = 167 ppm

53

Advantage of external standard Advantage of external standard calibration methodcalibration method

Only the target compound separation can be focused.

TargetTarget TargetTarget

54

Disadvantage of external standard Disadvantage of external standard calibration methodcalibration method

Injection error will directly influence the quantitative result.

10 uL injection 11 uL injection

100 ppm100 ppm 110 ppm110 ppm

55

Advantage of internal standard Advantage of internal standard calibration method calibration method

Injection error can be eliminated.

2000 / 1000 = 22000 / 1000 = 2 2200 / 1100 = 22200 / 1100 = 2

10 uL injection 11 uL injection

11001100 22002200

IS10001000 20002000

IS TT

56

Advantage of internal standard Advantage of internal standard calibration methodcalibration method

Recovery in the pretreatment process can be estimated.

standard IS (100 ppm)

10001000 950950

ISIS

TT

addition of IS (100 ppm)to actual sample

Recovery = (950/1000)x100 = 95%Recovery = (950/1000)x100 = 95%

57

Disadvantage of internal standard Disadvantage of internal standard calibration method calibration method

Separation is slightly difficult.

ISISTT

TTISIS TTISIS

58

Disadvantage of internal standard Disadvantage of internal standard calibration methodcalibration method

It is difficult to look for the IS compound.

The chemical structure of IS compound is similar with one of target compound.

IS sample is not existent in the actual sample.

59

Calibration MethodCalibration Method

External standard calibration Separation is not difficult Injection error will directly influence the quantitative

result Internal standard calibration

Injection error can be eliminated Recovery in the pretreatment procedure can be

estimated Separation is slightly difficult Difficult to look for the IS compound

60

Standard additive Standard additive calibration methodcalibration method

Original Sample

TargetTT

TT

TT

61

Standard additive Standard additive calibration methodcalibration method

TT

TT

TT??=70 ppm??=70 ppm

Pea

k

Are

a

Added amount0 50 100 ppm

17

7

12

x104

-70

100 ppm=10x104

??? ppm= 7x10410x4

10

7x41

0

62

Accurate QuantitationAccurate QuantitationSelect Workable RangeSelect Workable Range

Sensitivity

Expected Range

Linearity

[ Concentration of Solute ]

[ D

etec

tor

Res

pon

se ]