Method Development in High-

Performance Liquid

Chromatography

The Chromatographic Process

• Diffusion in liquids is 100 times slower than

diffusion in gases. Therefore, in liquid
chromatography it is not feasible to use
capillary columns – HPLC uses packed
columns

• Small particles give high efficiency but

require high pressure. Typical particle sizes
in HPLC are 3-10 μm

Stronger solvent than

in (b)

Plate Height as a Function of Flow

Rate

Number of Theoretical Plates in

HPLC

Under optimum conditions (near H

min

), the number of

theoretical plates in a column of length L is

• Small particles reduce eddy diffusion (A term)
• Small particles reduce the distance solute must diffuse in

the mobile phase (C term)

( )

( )

μm

cm

3500

p

d

L

N

≈

Smaller Particle Size Leads to

• Higher plate number
• Higher pressure
• Shorter run time (higher sample

throughput)

• Lower detection limit

Required Column Pressure

The pressure required to drive the solvent through
a column is

f – factor depending on particle shape and packing
η – viscosity of the solvent
r – column radius

2

p

2

d

r

L

u

f

P

x

π

η

=

The Stationary Phase in HPLC

• The most common support – spherical

microporous silica particles permeable to
solvent. Silica dissolves above pH 8 and
should not be used above this pH (special
grades are stable up to pH 9 or 10)

• For chromatography of basic compounds at

pH 8-12, polymeric supports (polystyrene)
can be used

50% porosity; S = 150 m

2

/g

70% porosity; S = 300 m

2

/g

Microporous Silica Particles

Nominal pore size is 10 nm

Schematic Structure of Silica Gel

Up to 8 μmol/m

2

Si-OH

Protonated at pH 2-3

Uses of Silica in HPLC

• Bare silica is used as the stationary phase in

adsorption chromatography

• In liquid-liquid partition chromatography,

the stationary phase is chemically bonded to
the silica surface

Bidentate C

18

stationary phase stable in the pH range 2-11.5

Baseline separation of enantiomers of the drug Ritalin by HPLC

with a chiral stationary phase

Bulky isobutyl groups protect siloxane bonds from hydrolysis at low pH

Superficially Porous (Pellicular)

Particles

• A stationary phase (e.g. C

18

) is bonded to

the thin, porous outer layer

• Mass transfer of solute is 10 times faster

than into fully porous particles of the same

diameter

• Especially suitable for separation of

macromolecules (proteins), which diffuse

more slowly than small molecules

Proteins separated on C

18

-silica. 1 – angiotensin II; 2 – neurotensin;

3 – ribonuclease; 4- insulin; 5 – lysozyme; 6 – myoglobin; 7 – carbonic
anhydrase; 8 - ovalbumin

The Elution Process

• In adsorption chromatography, solvent

molecules compete with solute molecules
for sites on the stationary phase

• Elution can be described as a displacement

of solute from the stationary phase by
solvent

Eluotropic Series

• An eluotropic series ranks solvents by their

relative abilities to displace solute from a given
adsorbent

• The eluent strength (ε

°) is a measure of the

solvent adsorption energy, with the value for
pentane defined as 0 on bare silica

• The more polar the solvent, the greater is its eluent

strength and the more rapidly will solutes be
eluted from the column

Classification of HPLC Modes

• Normal-phase chromatography

– Polar stationary phase
– More polar solvent has higher eluent strength

• Reversed-phase chromatography

– Nonpolar stationary phase
– Less polar solvent has higher eluent strength

Elution Modes in HPLC

• Isocratic elution – performed with a single

solvent or constant solvent mixture

• Gradient elution – continuous change of

solvent composition to increase eluent
strength (analogous to temperature
programming in GC)

Example: Isocratic Separation of

Aromatic Compounds by RP HPLC

Solvent A – aqueous buffer
Solvent B - acetonitrile

Gradient Elution of the Same

Mixture of Aromatic Compounds

• Same column, flow rate and solvents were

used

Selecting the Separation Mode

Suppose we have a mixture of small molecules soluble in CH

2

Cl

2

“Green” Technology: Supercritical

Fluid Chromatography

Phase diagram for CO

2

Capillary SFC of aromatic compounds with CO

2

,

using density gradient elution at 140 °C

Effect of Sample Solvent

• The sample should be dissolved in a solvent

of lower eluent strength than the mobile
phase or in the mobile phase itself

n-butylaniline

Method Development for Reversed-

Phase Separations

• Adequate resolution of desired analytes
• Short run time (high sample throughput)
• Rugged (not drastically affected by small

variations in conditions)

Initial Steps in Method Development

1. Determine goal
2. Select method of sample preparation
3. Choose detector

Criteria for an Adequate Separation

• Capacity factor 0.5 ≤ k’ ≤ 20
• Resolution R

s

≥ 2

• Operating pressure P ≤ 15 MPa (150 bar)
• 0.9 ≤ asymmetry factor ≤ 1.5

Estimating Dead Time (Volume)

2

2

c

m

Ld

V

≈

F

Ld

t

c

m

2

2

≈

F – flow rate (mL/min)
d

c

2

– column diameter (cm)

d

c

= 4.6 mm

Optimization with One Organic

Solvent

•

Choice of organic solvent

1. Acetonitrile (low viscosity, low UV cutoff)
2. Methanol (higher viscosity and UV cutoff)
3. Tetrahydrofuran (less usable UV range,

slower equilibration with stationary phase)

Optimization with Two or Three

Organic Solvents

• Step 1 Optimize the separation with

CH

3

CN/buffer (chromatogram A)

• Step 2 Optimize the separation with

MeOH/buffer (chromatogram B)

• Step 3 Optimize the separation with

THF/buffer (chromatogram C)

Optimization with Two or Three

Organic Solvents (cont.)

• Step 4 Mix the solvents used in A, B, and C, one

pair at a time, in 1:1 proportion (chromatograms
D, E, and F)

• Step 5 Construct a 1:1:1 mixture of the solvents

for A, B, and C (chromatogram G)

• Step 6 If some of the results A through G are

almost good enough, select the best two solvents
and mix the solvents to obtain points between
those two

30% MeCN
70% buffer

40% MeOH
60% buffer

32% THF
68% buffer

1 – benzyl alcohol
2 – phenol
3 – 3’,4’-dimethoxyacetophenone
4 – m-dinitrobenzene
5 – p-dinitrobenzene
6 – o-dinitrobenzene
7 – benzoin

Nomograph showing volume percentage of solvents having

the same eluent strength

Temperature as a Variable

• Isocratic method development for HPLC

can use solvent composition, %B, and

temperature, T, as independent variables

• %B and T are each varied between selected

low and high values

• From the appearance of chromatograms we

can select intermediate conditions to

improve the separation

Choosing a Stationary Phase

C

18

-silica

phenyl-silica

Order of Steps to Improve Separation

of Two Closely Spaced Peaks

1. Change the solvent strength by varying the

fraction of each solvent

2. Change the temperature
3. Change the pH (in small steps)
4. Use a different solvent
5. Use a different kind of stationary phase

Gradient Elution

• Used in case of general elution problem (GEP) –

mixtures of compounds with a wide range of

polarities

• Run a broad gradient first to decide whether to use

isocratic or gradient elution

• If Δt/t

G

> 0.25, use gradient elution

• If Δt/t

G

< 0.25, use isocratic elution

• Isocratic solvent should have composition applied

to column halfway through the period Δt

Gradient Elution (cont.)

Δt – the difference in the retention time

between the first and last peak in the
chromatogram
t

G

– the gradient time: the time over which

the solvent composition is changed

Steps in Gradient Method

Development

1. Run a wide gradient (e.g., 5 to 100% B)

over 40-60 min. From this run, decide
whether gradient or isocratic elution is
best

2. If gradient elution is chosen, eliminate

portions of the gradient prior to the first
peak and following the last peak. Use the
same gradient time as in step 1

Steps in Gradient Method

Development (cont.)

3. If the separation in step 2 is acceptable, try

reducing the gradient time to reduce the
run time

4. If the separation is not acceptable, it can

be improved by going to a segmented
gradient