liquid chromatography LC / HPLC

LIQUID CHROMATOGRAPHY (LC/HPLC)  

Principle:  Early liquid chromatography  was carried out in long glass columns with wide 

diameter. The diameters of the stacked particles inside the column were of the order of 150- 

200 microns range. Even then, the flow rates (eluent time) of the mobile phase with the 

analyte were very slow and separation times were long - often several hours!. With the 

advent of latest technology the  particle diameters were reduced as small as to 10 microns 

with replacement of glass columns with steel ones. The flow rate of the mobile phase was 

improved by applying high pressure to the column using pumps and hence the performance 

was improved. This development led the  instrument to be mostly called as  “High-

Performance Liquid  Chromatography” or  “High-Pressure Liquid Chromatography” 

(HPLC). Though HPLC retains major of the credits to the analytical side, the earlier one of 

simple Liquid Chromatography still finds applications in the preparative purposes.  

The HPLC technique can be divided into four main categories depending on the nature of the 

processes that occur at the columns as follow:

High-Performance Adsorption Chromatography: Here the analyte species (components

to be analysed) are adsorbed onto the surface of a polar packing. The stationary phase

consists of finely divided solid particles packed inside a steel tube. If the component mixture

is eluted through this tube with the mobile phase, different components present in the mixture

adsorb to different degrees of strength and they become separated as the mobile phase moves

steadily through the column. The nature of the  adsorption involves the interaction of polar

molecules with a very polar solid stationary phase. The stationary phase could be silica gel or

alumina. This method is extensively used for the separations of relatively non-polar, water-

insoluble organic compounds (since polar molecules will be adsorbed on to the column

momentarily). One particular application is in resolving isomeric mixtures such as meta- and

para-substituted benzene derivatives.

 High-Performance Partition Chromatography:  It is the most widely used liquid

chromatographic procedures to separate most kinds of organic molecules. Here the

components present in the analyte mixture distribute (or partition) themselves between the

mobile phase and stationary phase as the  mobile phase moves through the column. The

stationary phase actually consists of a thin liquid film either adsorbed or chemically bonded

to the surface of finely divided solid particles. Of these the latter is considered more

important and has a distinct stability advantage. It is not removed from the solid phase either

by reaction or by heat and hence it is more popular. It finds wide applications in various

fields, viz., pharmaceuticals, bio-chemicals, food products, industrial chemicals, pollutants,

forensic chemistry, clinical medicine, etc.

High-Performance Ion-Exchange Chromatography:  This method is used to separate

mixtures of ions (organic or inorganic),  and finds its application mostly in protein

separations. The stationary phase consists of very small polymer resin “beads” which have

many ionic bonding sites on their surface, termed as Ion Exchange Resins. This resin can be

either an anion exchange resin, which possesses positively charged sites to attract negative

ions, or a cation exchange resin, which possesses negatively charge sites to attract positive

ions. If the analyte mixture which contains mixture of ions is introduced into the column

packed with suitable ion-exchange resin, selected ions will be attached or bonded on to the

resin, thus being separated from other species that do not bond. Later, these attached ions can

be dislodged from the column by repeated elution with a solution that contains an ion that

competes for the charged groups on the resin surface, in other words, which has high affinity

for the charged sites on the resin than the analyte ions. Thus the analyte ions get exchanged

and separated from the column.

High Performance Size Exclusion Chromatography:  This technique is for separating

dissolved species on the basis of their size and particularly applicable to high-molecular-

weight species like oligomers and polymers to determine their  relative sizes and molecular

weight distributions. Here, the stationary phase is polymer resin, which contains small pores.

If the components to be separated are passed through the column the small sized particles can

easily enter into these pores and their mobility is retarded. Whereas the large sized particles,

which can’t enter into these pores can come out of the column fast and elude first. Thus the

separation of various sized particles is possible  through variations in the elution time. It is

classified into two categories based on the nature of the columns and their packing as:

Gel Filtration Chromatography - which uses hydrophilic packing  to separate polar species

and uses mostly aqueous mobile phases. This  technique is mostly used to identify the

molecular weights of large sized proteins & bio-molecules.

Gel Permeation Chromatography -  which uses hydrophobic packing to separate nonpolar

species and uses nonpolar organic solvents. This technique is used to identify the molecular

weights of polymers.

Instrumentation:  The basic HPLC system consists of  a solvent (mobile phase) reservoir,

pump, degasser, injection device, column and  detector. The pump draws the mobile phase

from the reservoir and pumps it to the column through the injector. At the end of the column

(effluent end), a detector is positioned. Mostly UV absorption detector is used. In the case of

analytical studies, after the detection the eluents are collected in waste bottles. In the case of

preparative studies the eluents are fractionally collected for further studies. Most of the

HPLC design will be the same as described for all the four main groups previously described.

However, there can be differences in selecting the specific detectors for particular type of

analysis, say for example, with ion-exchange chromatography, detectors commonly used are

conductivity detectors for obvious reasons. Other important detectors for HPLC separations

include refractive index detector, fluorescence  detector and mass selective detector. The

following is the most generalised outlay of the HPLC system:

Disadvantages:  Column performance is very sensitive, which depends on the method of

packing. Further, no universal and sensitive detection system is available.