High Pressure Liquid Chromatography

HPLC
High Performance Liquid Chromatography

HPLC-MS HPLC coupled to ESI Mass spectrometer

UPLC Ultra Performance Liquid Chromatography

HPLC-IR HPLC coupled to IR spectrophotometer

 High Pressure Liquid Chromatography

Separation by HPLC is based on various principles and can be

classified based on:

Surface adsorption
Partition Normal Phase-
Ion exchange Reverse Phase-
Size exclusion
Paired ion
Ion Pair Chromatography
It is widely used to selectively analyze acids and bases, particularly with reverse phase
chromatography. It is mostly used for the analysis of Proteins, peptides, nucleotide etc.
An ionic surfactant is added to a reversed-phase Chromatography system (mobile phase)
in order to affect retention and selectivity of ionic compounds.
 Paired-ion Chromatography

It is a technique related to RP-HPLC that affords an improvement in separation of

some highly polar compounds such as amino acids and organic acids that do not
adhere at all to the stationary phase if it is apolar.
Thus this polar character must be reduced to increase retention, to modify the
ionic charge, the pH of the mobile phase can be changed but if this is not sufficient
then a strongly ionic reagent, called a ion pairing reagent is added to the mobile
phase. This is usually a compound with a carbons chain (weakly polar) possessing a
functionality whose charge is opposite to the analyte to be separated.
e.g. heptane sulfonic acid is used if the ionic solute is a base.
This forms a neutralized ion-pair , a less polar specie, fairly stable and lipophilic,
which will initially stick to non polar stationary phase but can still be eluted,
apparently by a type of ion-exchange mechanism.
This principle permits the separation of inorganic cations or anions upon a RP-
phase.
 Polarity of solvents

PENTANE Non polar

HEXANE
HEPTANE
2, 2, 4 TRIMETHYLPENTANE
CYCLOHEXANE
CARBON TETRACHLORIDE
CHLOROFORM
TOLUENE
DICHLOROMETHANE
ETHYL ACETATE
ACETONE
TETRAHYDROFURAN
ACETONITRILE
ISOPROPYL ALCOHOL
METHANOL
ACETIC ACID Polar
HPLC can be:

Analytical Chromatography
Preparative Chromatography

Isocratic (same solvent or solvent mixture)

Or
Gradient (two or more solvents)
Capillary Fused silica (30m, 0.25m,0,25mm)

Column length Film thickness column I,d,

Stationary phase
Silica
Cationic sulphate
Anionic- Carboxylic
GPC Separation based on molecular weitht
Reverse phase matrix

A reversed phase chromatography

medium consists of hydrophobic
ligands chemically grafted to a
porous, insoluble beaded matrix. The
matrix must be both chemically and
mechanically stable. The base matrix
for the commercially available
reversed phase media is generally
composed of silica or a synthetic
organic polymer such as polystyrene.
Figure shows a silica surface with
hydrophobic ligands.
Ion-pair Chromatography

Ion pair chromatography (IPC) is one technique used to separate charged substances.
When a sample contains ionic components that tend to be very hydrophilic, and so reversed-
phase retention can be problematic.
It is widely used to selectively analyze acids and bases, particularly with reverse phase
chromatography.
An ionic surfactant is added to a reversed-phase Chromatography system (mobile phase) in
order to affect retention and selectivity of ionic compounds.
Selecting pair ions (ions with the opposite charge of target components) for adding to mobile
phases is very important.
Normally used when other changes in RPC conditions fail to achieve acceptable resolution.
This procedure is mainly used for the separation of Proteins, peptides, oligo-nucleotides etc.
Example of ion-pair reagents.
Alkylsulfonates R-SO3- (R-)

Tetraalkylammonium salts R4N+ (R+)

Strong carboxylic acids (triflouroacetic acid, TFA; heptaflourobutyric acid, HBA)
Chaotropes (BF-, ClO4-, PF6-), SDS, Butanol
 Theory

Retention Mechanism:
Two possible retention process

Partition model

Adsorption model

Partition Model:.

In this model, the ion-pairing agent is present in the

mobile phase. The analyte interacts with the ion-
pairing agent in the mobile phase first. It forms the
ion-pair which is relatively non-polar and partition into
the stationary phase and get retained.

R+A- (mobile Phase) R+A- (stationary Phase)

 Adsorption Model:
The ion-pairing agent present in the mobile phase
gets adsorbed into the non-polar stationary phase
due to its lipophilic alkyl chain. As a result, the ion-
pairing reagent forms a pseudo ion-exchange layer
on the surface of the stationary phase. The analyte
interacts with the ion-pairing agent presented on
the surface to form ion-pair and gets retained.

A- (mobile phase) + R+X- (stationary Phase)

A-R+(stationary Phase) + X-
 COMMON DETECTROS

UV detector: single or dual channel (280 & 364 nm)

RI detector
Photodiode Array detector
Fluorescent detector
Evaporative Light Scattering (ELSD) detector
 Gas Liquid Chromatography vs. High Pressure Liquid
Chromatography
GLC HPLC

1. Samples or its suitable derivatives that 1. Samples that are soluble in the
are volatile at room temperature or at mobile phase (single or mixture of
elevated temperature can only be two or more miscible solvents) can be
analyzed. analyzed.

2. Mainly organic compounds can be 2. Different type of compounds can be

separated. separated

3. Compounds should be stable at high 3. Compounds should be stable under

temperature. experimental condition

4. Molecular weight of compounds is 4. No molecular weight restriction

normally not more than 500.

5. Separation is achieved by partition only 5. HPLC separation can be achieved

between the gas and liquid phase employing various principle of
separation

6. The samples for analysis should not 6. Sample must not have any immiscible
have any non-volatile component compounds/particles
 Gas Liquid Chromatography vs. High Pressure Liquid
Chromatography

GLC HPLC

7. Generally water and oxygen are 7. Water is a common solvent in HPLC

avoided.

8. Detection of compounds are done 8. Detection can be done by UV, RI, DAD,
by FID or by ECD ELSD and many other detectors.

9. Analytical technique. Estimation of 9. Both analytical and preparative.

components is possible Estimation of components is possible.

10. Separated compounds can be 10. Separated compounds can be identified

identified by GLC-MS by LC-MS.

11. EI or CI mass spectrometry is used 11. ESI mass spectrometer is used for
to identify the compounds. detection of compounds

12. Is a destructive procedure. 12. Samples can be recovered in large

quantity.