High Performance Liquid Chromatography

           LIQUID CHROMATOGRAPHY

Early liquid chromatography was performed in
glass columns having inside diameters of perhaps
10 to 50 mm. The columns were packed with 50- to
500-cm lengths of solid particles. the particle size of the solid was kept larger than 150 to 200 μm.

                        WHY USE HPLC? 

 Simultaneous analysis
 High resolution
 High sensitivity
 Good repeatability
 Moderate analysis condition
 Easy to fractionate and purify
 Not destructive

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

▪HPLC- It was originally referred to as High Pressure Liquid

Chromatography since high pressure is applied using
pumping system to the column.
▪This pressure works by forcing the mobile phase through, at
much higherrate increasing the resolution power.
▪Due to its high efficiencyand performance High Pressure
Liquid Chromatography is referred to as High Performance
Liquid Chromatography.
Principle High-performance liquid chromatography (HPLC) is the most versatile and widely used type of elution chromatography.

               INSTRUMENTATION OF HPLC

  Solvent storage bottle
 Gradient controller and mixing unit
 De-gassing of solvents
 Pump
 Pressure gauge
 Pre-column
 Sample introduction system
 Column
 Detector
 Recorder

                          TYPES

The types of high performance liquid chromatography are often classified by the separation mechanism or by the type of stationary phase. These include

(1) partition chromatography; 

(2) adsorption chromatography;

(3) ion-exchange, or ion, chromatography; 

(4) size-exclusion chromatography;

(5) affinity chromatography; and 

6) chiral chromatography.

         

             Partition chromatography

The most widely used type of HPLC is partition chromatography in which the stationary phase is a second liquid that is immiscible with the liquid mobile phase.

Partition chromatography can be subdivided into 

1. liquid-liquid and
2. Liquid bonded-phase

In liquid-liquid partition chromatography, the stationary phase is a solvent held in place by adsorption of the surface of the packing particles. In liquid-bonded-phase chromatography, the stationary phase is an organic species that is attached to the surface of the packing particles by chemical bonds.

Bonded-Phase Packings Partition Chromatography Most bonded-phase packings are prepared by reaction of an organochlorosilane with the OH groups formed on the surface of silica particles by hydrolysis in hot dilute hydrochloric acid.
R is often a straight chain octyl- or octyldecyl-group

Two types of partition chromatography are distinguishable based on the relative polarities of the mobile and stationary phases.

 ❖ In normal-phase partition chromatography, the stationary phase is polar and the mobile phase nonpolar.
 ❖ In reversed-phase partition chromatography, the polarity of these phases is reversed.
In normal-phase chromatography, the least polar analyte is eluted first. In reversed-phase chromatography, the least polar analyte is eluted last.

Normal Phase Chromatography: 

 Stationary Phase – Polar nature. Eg: SiO2,Al2O3 ▪ Mobile Phase – Non-Polar nature. Eg:heptane,hexane,cyclohexane,CHCl3,CH3OH
▪ Mechanism:
✓Polar compounds travels slower & eluted slowly due to higher affinity to st.phase
 ✓Non-polar compounds travels faster & eluted 1st due to lower affinity to st.phase. ▪ This technique is not widely used in pharmaceutical separations.

Reverse Phase Chromatography: 

 Stationary Phase – Non-Polar nature. Eg: n-octadecyl, n-octyl, ethyl, phenyl diol, hydrophobic polymers.
 Mobile Phase – Polar nature. Eg: methanol or acetonitrile/water or buffer sometimes with additives of THF or dioxane.
 Mechanism:
✓Polar compounds travels faster & eluted 1st due to lesser affinity to st.phase ✓Non-Polar compounds travels slower & eluted slowly due to higher affinity to st.phase
❖ C18 is has 18 carbon atoms while C8 has 8 carbons in the column packing that are bonded to the silica (Si).
❖ The gel made of silica-base and modifie with octadecyl functional group is called octadecyl silica (ODS) gel.

     ADSORPTION CHROMATOGRAPHY

❑ The principle of separation is adsorption .
❑ Certain solid materials, collectively known as adsorbents, have the ability to hold
molecules at their surface.
❑ This adsorption process, which involves weak, non-ionic attractive forces of the
van der Waals’ and hydrogen-bonding type, occur at specific adsorption sites.
❑ Silica is a typical adsorbent. It has silanol (Si-OH) groups on its surface, which are
slightly acidic, and can interact with polar functional groups of the analyte or
eluent.
❑ Other commonly used adsorbents are alumina and carbon.

      Ion-Exchange Chromatography

 It is the process by which similar charged ions such as cations, anions can be separated.
 By using the suitable ion exchange resin it can be separated.
 It exchanges the ions according to their relative affinities.
 The exchange takes place in a reversible manner between the ions of the solution and the ions present in the ion exchange resin .

    Molecular Exclusion Chromatography

 A mixture of components with different molecular sizes are
separated by using gels.
 The gels used acts as molecular sieve & hence a mixture of
substances with different molecular sizes are separated.
 Soft gels like dextran, agarose or poly acrylamide are used.
 Semi-rigid gels like polystyrene, alkyl dextran in non
aqueous medium are also used.

        Affinity chromatography

Affinity chromatography is based on interactions between an antigen (the analyte) andan antibody.The antibody,which is normally a protein(an immunoglobulin), is boundtothestationaryphaseandcontainsacavityoranotheropenstructurethat fits a part of the antigen, called the epitope . Whenthesamplecontainingtheantigenpassesthroughthecolumn,theantigenis “grabbed” by the antibody, while all other components are eluted from the column. The assumption is that the antibody is selective only for the antigen, like a key to a lock.
Affinity chromatography is mainly used for on/off purposes,retaining the antigen for selective purification and concentration.

     Chiral Chromatography

Configurationalisomers(cis–trans isomers etc.)as well as diastereomers(containing two optical centers) can be separated in ordinary HPLC columns. Optical enantiomers (mirror images) need the assistance of a chiral selector in the stationary phase or as an additive to the mobile phase. Thus, separation of enantiomers can take place by either of the following:
1) reaction with an optically active reagent to form diastereomers, 2) addition of an optically active ingredient to the mobile phase for complexation to diastereomers, and 3) stationary phases with built-in stereoisomeric (chiral) functions