HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) NOTES for GPAT, GATE, NET-PART-1-

INTODUCTION :-

High perfomance liquid chromatography is a specific form of a column chromatography generally used in bio chemistry and analysis to separate, identify and quantify the active compound.

PRINCIPLE :-

HPLC is principle is based on adsorption as well as partition chromatography is depending on the nature of stationary phase if stationary phase is solid principle is based on the principle of adsorption chromatography and if the stationary phase is liquid is based on principle of partition chromatography .

It is important for determination of volatile and non volatile compound.

It is important for determination qualitative and quantitative analysis.

It is important for determination of retention time (the time is required , after sample injection maximum angle peak reaches to detector)

TYPES OF HPLC :-

Type of HPLC generally depends on phase system used in the process. following types of HPLC generally used in analysis –

NORMAL PHASE CHROMATOGRAPHY :-

Also known NP-HPLC . This method separates analyte based on polarity. NP-HPLC uses a polar stationary phase and non polar mobile phase. the polar and a non polar mobile phase . the polar analyte interacted with and is retained by the polar stationary phase.

Adsorption strength increase with incrased analyte polarity and the interaction between the polar analyte and the polar stationary phase increases the elution time.

REVERSED PHASE CHROMATOGRAPHY :-

Also known as ion-exchange chromatography of proteins, high-PH anion-exchange chromatography of carbohydrates and oligosaccharides etc.RPC operate on the principle of hydrophobic interactions which result from replusive force between a polar eluent the relatively non-polar analyte and the non polar stationary phase. the binding of the analyte to the stationary phase is proportional to the contact surface area around the non polar segements of the analyte molecules upon association with the   ligand in the aqueous eluents.

SIZE EXCLUSION CHROMATOGRAPHY :-

Size exclusion chromatography also called as gel permeation chromatography or gel filtration chromatography mainly separates particles on the basis of size. it is also useful for determining the teritary structure and quaternary structure of proteins and amino acid. this technique is used for the  molecular weight determination of poly saccharides.

ION EXCHANGE CHROMATOGRAPHY :-

In, ion exchange chromatography retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Ions of the  same charge are excluded. This form of chromatography is widely used in purified water, ligand-exchange chromatography, ion-exchange chromatography of proteins, high-PH anion-exchange chromatography of carbohydrates and oligosaccharides etc.

MCQ :-

1. If the stationary phase is solid principle of HPLC is

A. partition

B.adsorption

C. A and B

D. None of the above

2. If the stationary phase is liquid principle of HPLC is

A. partition

B.adsorption

C. A and B

D. None of the above

3.Which statement is false about NP-HPLC ?

a. NP-HPLC uses a polar stationary phase and non polar mobile phase.

b.the polar analyte interacted with and is retained by the polar stationary phase.

c.Adsorption strength increase with incrased analyte polarity and the interaction between the polar analyte and the polar stationary phase increases the elution time.

d.This form of chromatography is widely used in purified water.

4.Which statement is true about RP-HPLC ?

A.Adsorption strength increase with increased analyte polarity and the interaction between the polar analyte and the polar stationary phase increases the elution time.

B.the polar analyte interacted with and is retained by the polar stationary phase.

C.ion-exchange chromatography of proteins, high-PH anion-exchange chromatography of carbohydrates and oligosaccharides etc.

D.ion-exchange chromatography of proteins, high-PH anion-exchange chromatography of carbohydrates and oligosaccharides etc.

5.Which chromatography is used to determination of structure of   the tertiary structure and quaternary structure of proteins and amino acid ?

a. RP-HPLC

b.NP-HPLC

c. Ion exchange chromatography

d.Size exclusion chromatography

6. which chromatography is used to purified water ?

a. RP-HPLC

b.NP-HPLC

c. Ion exchange chromatography

d.Size exclusion chromatography

ANSWER KEY 

1. b

2.a

3.d

4.b

5.d

6.c

REFERENCE :-

TEXT BOOK OF PHARMACEUTICAL ANALYSIS THIRD EDITION DR.S.RAVI SANKAR.(18.1 TO 18.15)

TEXT BOOK OF DAVID G. WATSON FOURTH EDITION (296 TO 344)