HPLC Method Development - Basics

Dr. Gesa J. Schad

Shimadzu Europa GmbH
Outline

• Factors that affect resolution

• Separation modes

• Basic principles for method development

• Retention, Selectivity, Efficiency

LC separation

Flow
A
Chromatographic resolution

Resolution Equation

 1 0.5    1  k 
R S
 
 4 N 
  
  
 1 k 
Efficiency Selectivity Retention
Chromatographic resolution

12
Selectivity
10

8 Retention
Efficiency
6
Rs

0
2 4 6 8 10 12 k
N
1000 2000 3000 4000 5000 6000

0 1.5 2 2.5 3 3.5 4 4.5 α

Resolution visualized

Increase in Efficiency (N)

Increase in Selectivity (α)

Increase in Retention (k)

Separation modes

Chromatographic Mode Relative Percentage

of users (%)

Reversed Phase 50.4

Normal Phase 24.1
Ion Exchange 14.0
Size Exclusion 6.7
Chiral 2.8
Hydrophobic Interaction 1.1
Other 1.1
[LC / GC International]
Normal phase LC

• Use of polar stationary phase and low polarity mobile

phases
• Useful for the separation of low to moderate polarity
compounds (i.e. samples not soluble in aqueous
solutions)
• Good for separating geometric and positional
isomers
• Not suitable for separating compounds with minor
hydrophobic differences
Normal phase LC

• There are two types of normal phase packings

→ Unbonded and bonded

• Silica and alumina are the most poplar

• Useful for preparative LC
• Unbonded silica is hygroscopic therefore Rt
reproducibility can be a problem if water content is
not controlled
• Bonded phases such as amino, diol and cyano are
less affected by water in the mobile phase
Normal phase LC

Polar Stationary Phase Non Polar Mobile Phase

(eg. SIL, diol, NH2) (eg. hexane, ethyl acetate)

OH
CH3 NO2
OH

OH

Least Most
OH
polar polar

Injection
Reversed phase LC

• First choice for most chromatographers

• Approx. 75 % of all HPLC separations are performed
using RP columns
• Ideal where differences in hydrophobicity are expected
• Best to run acids & bases in their ion-suppressed mode
• Wide variety of phases available
→ C18, C8, Ph, etc.
Reversed phase LC

Non Polar Stationary Phase Polar Mobile Phase

(eg. C18, C8, C4) (eg. MeCN, MeOH)

C18 CH3

C18

C18
Most Least
C18 polar polar

Injection
Ion exchange LC

• Ideal for the separation of inorganic ions, peptides,

proteins & amino acids
• Retention is controlled by the pH and ionic strength
of the mobile phase, rather than its organic solvent
content
• They consist of ionic groups bound to a support
• The concentration of the ionic groups is called the
exchange capacity of the packing.
• Supports are typically – silica (pH range 2.5-8.5)
and polymer (pH range 1-13)
 Cation exchange
Stationary Phase Mobile Phase (aqueous buffer)
eg. SCX = SO3- eg. Phosphate, Na+ counter cation
WCX = COO-
S O3- Na+ Y2+ Y2+
Y+
S O3- Na+

Y+
S O3- Na+
Y2+
S O3-Na+ Y+

Y+ Y2+

(R-SO3-)sNa+ + Y+m (R-SO3-)sY+ + Na+m

Injection
Anion exchange
Stationary Phase Mobile Phase (aqueous buffer)
eg. SAX = N(R)3+ eg. diethanolamine, Cl- counter anion
WAX = NH3+
N(CH3)3 + Cl- X2- X2-
X-
N(CH3)3+ Cl-
X-
N(CH3)3+ Cl- X2-
N(CH3)3 Cl-
+ X-

X- X2-

(R-NR3+)sCl- + X-m (R-NR3+)sX- + Cl-m

Injection
Size exclusion LC

• Is used for samples which contain high molecular

weight compounds and for samples where
components are significantly different in molecular
size
• It can be used to determine the molecular weight
distribution of polymers
• Retention is determined primarily by the size of the
pores in the packing rather than the eluent
Size exclusion (SEC)

Stationary Phase Mobile Phase

A
A

B B A B C
Pores B

C C

C
Injection
Method Development

Resolution Equation

 1 0.5    1  k 
R S
 
 4 N 
  
  
 1 k 
Efficiency Selectivity Retention
Method Development

12
Selectivity
10

8 Retention
Efficiency
6
Rs

0
2 4 6 8 10 12 k
N
1000 2000 3000 4000 5000 6000

0 1.5 2 2.5 3 3.5 4 4.5 α

Method Development
Retention [1 < k < 10] :
• characteristics of the analytes
• bonded phase chemistry
• choice of mobile phases
• mobile phase composition
Selectivity [α ≥ 1.2] :
• stationary phase
• mobile phase
• pH
• temperature
Efficieny [N] :
• flow rate
• column length
• particle size
• quality of column packing
Basic Principles

• Hydrophilic or hydrophobic (unspecific) interaction of the analyte with

the polar / non-polar surface of the stationary phase (-C18, -C8, -C4,
Phenyl, Silica, Diol, Amino .. etc.)

• Separation based on differences in affinity to the bonded phase

• Mobile phase determines state of ionisation

• Steric hindrance - steric selectivity can be an important factor in

the separation of structural isomers
Choice of Stationary Phase

 NOT ALL C18 ARE THE SAME!!!

Choice of Stationary Phase

Type Mechanisms of Strength of Interaction

Separation
C18 Hydrophobic binding Strong
Shape selectivity Weak
Target Analytes Polar, moderately polar and nonpolar analytes
Uncharged acids and bases
Ionized acids or bases using ion-pairing
Recommended Analytes differing in hydrophobicity
Applications Homologous compounds differing by –CH2
Ideal starting point for method development
Choice of Stationary Phase

Type Mechanisms of Strength of Interaction

Separation
Aromatic bonded π-π interactions Strong
phase
Dipole-dipole Moderate
interactions
Hydrophobic binding Moderate
Shape selectivity Moderate
Target Analytes Analytes with π bonding and conjugated
systems
Analytes with electron delocalization and
electron withdrawing groups, such as halogens,
nitro groups,
ketones, esters and acids
Analytes with different dipole moments
Analytes differing in hydrophobicity
Polar and moderately polar analytes
Recommended Stereoisomers
Choice of Stationary Phase

Type Mechanisms of Strength of Interaction

Separation
Pentafluorophenyl π-π interactions Strong
Dipole-dipole Strong
interactions
Hydrophobic binding Moderate
Shape selectivity Strong
Target Analytes Analytes with π bonding
Analytes with electron donating groups, such
as phenols, aromatic ethers and amines
Analytes with proton donor groups
Analytes with different dipole moments
Analytes differing in hydrophobicity
Polar and moderately polar analytes
Recommended Structural isomers, Steroids, Taxanes,
Applications Substituted aromatics
Suitable for use in up to 100% aqueous mobile
Method Development

Example: ionizable compounds

Aspartam Phenylalaninmethylester
[logP: 1.1] non-polar
O Saccharin
O O [logP: 0.9] O
NH CH3
OH NH2 O
Acesulfam O
pka 3.19 pka 7.87 NH
[logP: 0.31]
S
NH
O O
S O
H3C O pka ~ 2
O
Choice of Mobile Phase

Silica Me
support of
O Si
stationary
phase Me
pka ~ 3.5
OH
Me
O Si
Me
OH
Choice of Mobile Phase

Silica Me
support of
O Si O
stationary HO
phase Me H + pka ~ 7.87
H N
pka ~ 3.5 H O

O-
HN

O
Me O
H3C

O Si
Me
OH  Supress ionisation of
ionizable groups by
mobile phase pH !!
Choice of Mobile Phase

Choice of mobile phase in RP-LC:

• solvent: less polar = stronger solvent
• water = weakest solvent
• Ion pairing reagent ?
• Ionsuppression necessary? pH !!
• Buffer needed? Watch the buffer range!
Choice of Mobile Phase

Highest buffer capacity at pH = pka:

buffer pka buffer range

H3PO4/H2PO4- 2.12 1.1 – 3.1
H2PO4-/HPO42- 7.21 6.2 – 8.2
HPO42-/PO43- 12.67 11.6 – 13.6
CH3COOH/CH3COO- 4.75 3.8 – 5.8
HCOOH/HCOO- 3.74 2.7 – 4.7
Log k vs. % organic solvent

→ linear relationship
→ Estimation: Increase % organic solvent by 10 reduce log k by 3
log k

% organic solvent
Log k vs. % organic solvent

→ paralell graphs = same separation mechanism

log k

% organic solvent
Log k vs. % organic solvent

→ Different slope = differences in separation mechanism

→ better separation at lower % organic solvent
log k

% organic solvent
Log k vs. % organic solvent

→ Choice of organic solvent defines elution order

→ larger peaks should elute after smaller peaks
log k

% organic solvent
Isocratic or gradient elution ?

Isocratic separation:
k3 significantly higher than k2
Long retention → broad peak
2
1 Gradient separation recommended

0 5 10 15

t0 = 1.3 min
t R  t 0  k1 = 2.8
tR1 = 4.9 min k k2 = 4.6
tR2 = 7.3 min t0 k3 = 13
tR3 = 18.2 min
Isocratic or gradient elution ?

∆%B > 25 % → Gradient separation recommended

%B
t1 – (2.5 x t0) = t*
100
8.83 – (3.75) = 5.08
77 %
t0 = 1.5 min } 19 %
t1 = 8.83 min 58 %

33 %

0 5 10 15

t0 t*
Effect of Temperature

→ Plotting log k versus 1/T (in Kelvin) produces a

straight line
→ Different slope indicates differing selectivities

log k

1/T (K)
→ Temperature will affect the degree of ionization
of the analytes & silanol groups on the column
Effect of Temperature

1 4
14 °C 3
6
5 7
2

1 4
20 °C 3
6
5 7
2

25 °C 1 4
3

5, 6
7
2

0 1 2 3 4 5 6 7
Effect of pH

Remember the charge on both the analyte

and the stationary phase may change
as a function of mobile phase pH
Effect of pH

pKa = 3.5
p - Aminobenzoic acid
Folic acid
pKa = 2.7
pKa = 4.8

pKa = 4.8

pKa = 7.8
Effect of pH
1
pH 3.5 3
6
4

2
5 7

pH 2.7
1 6
3
2
5 4 7

pH 2.2 6
1 1. Vitamin B6 (pKa > 5.5)
3 2. Vitamin B5 (pKa = 4.4) 4, 5
2 3. p-Aminobenzoic acid
4. Folic acid 7
5. d-Biotin (pKa = 4.65)
6. Vitamin B12 (pKa < 2 + > 6)
1 2 3 4 5 6 7 8 9 10
7. Riboflavin (pKa > 7)
Factors affecting resolution
Retention [1 < k < 10] :
• characteristics of the analytes
• bonded phase chemistry
• choice of mobile phases
• mobile phase composition
Selectivity [α ≥ 1.2] :
• stationary phase
• mobile phase
• pH
• temperature
Efficieny [N] :
• flow rate
• column length
• particle size
• quality of column packing
Factors affecting resolution

Remember the Resolution Equation ?

 1 0.5    1  k 
R S
 
 4
N 
  
  
 1 k 
Efficiency Selectivity Retention

Increase N without affecting α or k !

Changes in column dimension
→ adapt flow rate
→ adjust gradient time
Method Transfer to UHPLC

→ transferring of HPLC to UHPLC while maintaining selectivity when

changing column dimensions and flow rate.
→ In a gradient separation the relative retention factor (k*) is
influenced by gradient time tg, flow rate (F), change in organic
solvent (Δ%B), and column dead volume (V0) according to:

 tg F
k 
% BVm S

→ k* must be kept constant !

→ gradient time has to be adjusted when column dimensions and /
or flow rate are changed !
Method Transfer to UHPLC

Adjust flow rate:

2
d c 2 d p1
F2 F1  2 
d c1 d p 2
F2 = new flow rate
F1 = original flow rate
dc2 = inner diameter of the original column
dc1 = inner diameter of the new column
dp2 = particle size of the original column
dp1 = particle size of the new column
Method Transfer to UHPLC

Adjust run-time:

F1 V02
t 2 t1  
F2 V01
t2 = new flow run-time
t1 = original run-time
F2 = new flow rate
F1 = original flow rate
V02 = dead volume of new column
V01 = dead volume of old column
Method Transfer to UHPLC
Adjust injection
volume:

2
d 2 L2
I 2  I1  2 
d1 L1

I2 = new injection volume

I1 = original injection volume
d2 = inner diameter of the original column
d1 = inner diameter of the new column
Method Transfer to UHPLC
Method Transfer Example
125
3
100

75

50 2
1 4 5 10
6
25 7 8 9 11 12
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 min

125 125 3
100 100

75 75

50 50 2 10
4 5
1 6
25 25 78 9 11 12
0 0
0.0 1.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 min

→ transfer from a 20 min HPLC method to a 1 min separation in

UHPLC conditions, where gradient time, column dimensions and
flow rate were adjusted to maintain selectivity in the faster
analysis.