1

Part III Introduction to Method

Development of HPLC
Procedure

for method development

Optimization

of separation in reversed phase

HPLC
Calibration

methods

Method Development of HPLC

What

mode ?

Reverse

What

column?

Types

What

phase, normal phase or .

of columns, temperature

mobile phase?

Isocratic

or gradient
Buffer and pH
What

detector?
What calibration method?

Procedure for HPLC

method 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

Selecting 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, carbohydrate

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

Reversed phase conditions

that affect HPLC separation
Separation Variable
COLUMN
Dimensions (length, I.d.)
Particle size
Stationary phase
MOBILE PHASE
Solvents A/B
%-B
Buffer (compound, pH, concentration)
Additives (eg., ion-pair reagents, amines)
Flow rate
TEMPERATURE
SAMPLE SIZE
Volume
Mass

Preferred Initial Choice

15 , 25 cmL x 2 - 6 mmID,
3-5 um
C-8 or C-18
Water/ACN
Variable
10-100 mM phosphate, pH 2-7
1-20 mM Heptanesulfonic acid sodium
salt for cations or TBA for anions
0.1-2 ml/min
10-60 oC
100 ul
100 ug

Characteristics of primary HPLC methods

Reversed phase mode
Uses water / organic mobile phase
Column: C-18(ODS),C-8, phenyl,
trimethylsilyl(TMS), cyano

First choice for neutral or non ionized

compounds that dissolve in water /organic
mixtures

Ion pair revered phase mode Good choice for ionic or ionizable

Uses water / organic mobile phase, a compounds, especially bases or cations

buffer to control pH, and an ion pair
reagent
Column: C-18(ODS),C-8, cyano

Normal phase mode

Uses mixtures of organic solvent as
mobile phase
Column: silica, amino, cyano, diol

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 secondary HPLC methods

Ion exchange mode
Uses aqueous mobile phase plus buffer
for pH control
Column: anion or cation exchange

Size exclusion chromatography

Uses 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
compounds
Separation of inorganic ions (ion
chromatography) is also used.

Good choice for separating high

molecular weight samples such as
proteins and synthetic polymers
GPC is used for molecular weight
distribution measurements.

Improving 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.

Separation goals
in HPLC method development
Goal
Resolution
Separation time
Quantitation
Pressure
Peak shape
Solvent consumption

Comment
Precise and rugged quantitative analysis
requires Rs > 1.5
< 5-10 min is desirable for routine
procedures
RSD<1% for assays; <5% for trace
analyses
<150 kgf/cm2 is desirable.
<200 kgf/cm2 is usually essential
Narrow peaks for large signal to noise
ratio are desirable
Minimum mobile phase usage per run is
desirable

10

Completing 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

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

13

Resolution for complete separation

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

14

Ratio of overlapping area

15

Resolution is a function of

Capacity factor (k)

Selectivity ()
Column efficiency (N)

1
1

R 4

N : average of N1 and N2
k' : average of k'1 and k'2

k'

N
k'1

16

Capacity Factor, k

k =

t1 - t 0
t0
t1

t0
t1 = retention time of a solute peak
t0 = column void time solvent peak
(non retained peak)

17

Selectivity,

k2
k1

t2 - t0
t1 - t 0
t1

t2

t0
Selectivity is an indication of the degree
of separation between two peaks.

The Number of
Theoretical Plate, N

18

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

Rt
Area
H
W1/2
W

H1/2

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

19

Resolution
Contribution of Capacity
1
1
R
4
k'
1
2
3
4
10
20
50
100

(k'/k'+1)
1/2
2/3
3/4
4/5
10/11
20/21
50/51
100/101

k 1

contribution
0.500
0.667
0.750
0.800
0.909
0.952
0.980
0.990

20

Resolution
Contribution of Selectivity
1
1

R 4

1
2
3
10
13
17
20
100

(-1/)
0/1
1/2
2/3
10/11
12/13
16/17
19/200
99/100

k'

N
k'1

contribution
0.000
0.500
0.667
0.909
0.923
0.941
0.950
0.990

21

Resolution
Contribution of Efficiency
1
1

R 4

8000
10000
15000
20000
30000

(N)1/2
89
100
122
141
173

k'

N
k'1

contribution
--0.12
0.37
0.58
0.94

22

Factors Affecting Resolution

23

How to increase N ?
H

= Length of Column / N

Van Deemter Equation

Optimum flow rate

Linear velocity

Each column has a

optimum flow rate.

24

Optimum Flow Rate

To get

a good column efficiency,

4.0 mmID
4.6 mmID
6.0 mmID

0.6 mL/min
0.8 mL/min
1.0 mL/min

These setting are preferable.

25

Smaller Particle Size

3 m
5 m

10 m
Flow rate
4.0 mmID
4.6 mmID
6.0 mmID

0.6 mL/min
0.8 mL/min
1.0 mL/min

Using smaller
particle size!

26

How to increase k ?

By decreasing % of organic solvent

k<2

- Insufficient separation

k > 10

- Long running time

- Broad band, cause
(1) low sensitivity
(2) poor accuracy
in quantitation

Rs

0 2 4 6 8 10

However, k= 2 - 10 is preferable.

2- 10
1- 20

0 2 4 6 8 10 12 14 16 18 20

27

Solvent Optimization
100% MeOH
0.1<K<0.3

MeOH / water mixtures for a mobile phase

80% MeOH
0.6<K<1.7

sample component: nitorbenzene, benzene, 2,6-dinitrotoluene,

2-nitrotoluene, 4-nitrotoluene, 3-nitrotoluene, toluene, 2-nitro-1,3-xylene
4-nitro-1,3-xylene, m-xylene

28

Relationship between k and

percent organic solvent (B%)
log(k)

= (B%) +

(, coefficient)

1.0

log(k)

Peak 1
0.5

Peak 3

0.0

Peak 2
-0.5
0.40

0.45

0.50 0.55 0.60 0.65

(B%)

0.70

0.75

For small molecules,

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

29

How to improve
By

changing

mobile

phase and composition

pH
concentration

of buffer
column temperature
packing material (to C8, CN and Phenyl)

30

Change of Mobile Phase

a
b

a
b

cd

[ MeOH/H2O ]
critical pair
: c,d
d

[ THF/H2O ]
critical pair
: a,b

[ MeOH/THF/H2O ]
a
c
d
b

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.

31

Effect of mobile phase pH

on separation of ionic compounds
1:benzoic acid
2:sorbic acid
3:methylparaben
[analytical conditions]
1mL/min
ODS column
10mM phosphate
buffer 75%
Acetonitrile 25%
40oC
UV-240 nm

32

pH of mobile phase

For samples that contain acidic or basic compounds, retention

time vary with pH.

R-COO- and R-NH3+

Adjusting pH of mobile phase is very important to get good

repeatability.
Buffer solution must be selected correctly.

33

Relationship between k and pH

k of

organic acid and amines varies with pH of

mobile phase.
R-COOH
associated type

RCOO- + H+
(pKa=4.5)

R-NH3+

R-NH2 + H+
(pKa=6.0)

pK
a

dissociated type

pH (mobile phase)

In the range of pKa +/1.5, a linear relationship

between k and pH exists.

34

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.
associated type

dissociated
type
pH (mobile phase)

35

Relationship between pH and pKa

of buffer solution
AH

A- + H+

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

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

36

pKa of organic acid for buffer

phosphate buffer

acetate buffer

conc. pKa1 pKa2

100 2.18 6.45
90
2.19 6.77
80
2.20 6.78
70
2.23 6.80
60
2.25 6.82
50
2.29 6.84
40
2.32 6.87
30
2.38 6.91
20
2.46 6.95
10
2.64 7.01

conc. pKa
100 4.64
90
4.64
80
4.64
70
4.64
60
4.64
50
4.64
40
4.65
30
4.65
20
4.66
10
4.68

citrate buffer
conc. pKa1 pKa2
100 2.98 4.35
90
2.98 4.35
80
2.99 4.37
70
2.99 4.38
60
3.01 4.40
50
3.02 4.42
40
3.03 4.45
30
3.05 4.48
20
3.08 4.51
10
3.15 4.57

pH region of buffer action

pKa +/- 1.0

pKa3
5.61
5.63
5.65
5.68
5.71
5.75
5.79
5.85
5.91
6.02

(conc. unit : mM )

37

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

characteristics on separation
1:benzoic acid
2:sorbic acid
3:methylparaben
[analytical conditions]
1mL/min
40oC
10mM phosphate
buffer (pH2.6) 75%
Acetonitrile 25%
UV-240 nm

ODS column

Phenyl column

39

Addition of other organic solvents

Addition of co-solvents besides methanol, acetonitrile and THF
is sometimes effective to get better separation.
ethyl ether
isoporpyl ether
dioxane
dimethoxyethane
cyclohexne oxide
Around 10% can be
added to normal
mobile phase for
revered phase mode.

separation of Estrogens

ACN/H20

10% Ethyl ether ACN/H20

40

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 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 - 1

Poor reproducibility

Inadequate column equilibration

The 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

Problems in gradient elution - 2

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.

44

Calibration methods in HPLC

External

calibration method

Internal

calibration method

Standard

additive method

45

External Standard Calibration

Preparation of Standards
Target Compounds

Dilution

Dilution

Dilution

Dilution

46

External Standard Calibration

Analysis of Vanillin

47

Concentration

External Standard Calibration

200
180
160
140
120
100
80
60
40
20
0
0

1000

2000

Peak Area

3000

4000

48

External Standard Calibration

[Concentration]

Calculation of Results
Y = aX + b
125 ppm
2500
[Peak Area]

a : SLOP
b : Y intercept
2500

49

Internal Standard Calibration

Preparation of Standards
Internal
Standard

Target Compounds

Dilution

Dilution

Dilution

Dilution

50

Internal Standard Calibration

Analysis of Vanillin

51

Internal Standard Calibration

[Terget Conc. / IS Conc.]

Analysis of Vanillin
4
3.5
3
2.5
2
1.5
1
0.5
0
0

10

[Tergat Area / IS Area]

15

52

Internal Standard Calibration

[Target Conc. / IS Conc.]

Calculation of Results
Y = aX + b
a : SLOP
b : Y intercept

1.67
5.0
[Target Area / IS Area]

Y = Target Conc. / IS Conc.

1.67 = Target Conc./ 100 ppm
Target Conc. = 167 ppm

T 2500
500
IS

53

Advantage of external standard

calibration method
Only

the target compound separation can be

focused.
Target

Target

54

Disadvantage of external standard

calibration method
Injection

error will directly influence the

quantitative result.
10 uL injection

100 ppm

11 uL injection

110 ppm

55

Advantage of internal standard

calibration method
Injection

error can be eliminated.

10 uL injection
1000
IS

11 uL injection
2000

2000 / 1000 = 2

1100
IS

2200
T

2200 / 1100 = 2

56

Advantage of internal standard

calibration method
Recovery

in the pretreatment process can be

estimated.

standard IS (100 ppm)

IS
1000

addition of IS (100 ppm)

to actual sample
T
IS
950

Recovery = (950/1000)x100 = 95%

57

Disadvantage of internal standard

calibration method
Separation
IS

is slightly difficult.

IS

IS

58

Disadvantage of internal standard

calibration 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 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

Standard additive
calibration method
Original
Sample
Target

60

Standard additive
calibration method

61

x104

100 ppm=10x104
??? ppm= 7x104

17
12

10x4 10

Peak Area

7
7x4 10

T
-70

??=70 ppm

50
100 ppm
Added amount

62

Accurate Quantitation

[ Detector Response ]

Select Workable Range

ty
i
r
a
e
n
Li

c
e
p
Ex
t
i
s
Sen

R
d
te

e
g
n
a

y
t
i
iv

[ Concentration of Solute ]