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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.
11
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
19
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
20
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
23
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.
24
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
28
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
29
How to improve How to improve
By changing mobile phase and composition pHconcentration of buffercolumn temperaturepacking material (to C8, CN and Phenyl)
30
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 ]
31
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
32
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)
34
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 ]
36
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 ]
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