HPLC

 HPLC stands for high performance/ pressure liquid chromatography

 Definition: It is type of chromatography in which the liquid (M.P) containing
sample passes over the stationary phase under high pressure. M.P is always
liquid and S.P. most of the time solid, sometimes liquid.
 History: The first instrument liquid chromatography was constructed by Csaba
Horvath at Yale University in 1964. And it was described as high pressure liquid
chromatography. Horvath called this high performance liquid chromatography.
So both terms are used but now later one is more widely used.
HPLC
 PRINCIPLE: The principle of separation in normal phase mode and reverse phase
mode is adsorption.
 When a mixture of components is introduced into a HPLC column, they travel
according to their relative affinities towards the stationary phase.
 The component which has more affinity towards the adsorbent, travels slower.
 The component which has less affinity towards the stationary phase travels faster.
 Since no 2 components have the same affinity towards the stationary phase, the
components are separated.
 Uses: It is used in biochemistry and analytical chemistry to identify, quantify and
purify the individual components of a mixture.
HPLC
 Procedure:
 HPLC is a separation technique that involves: The injection of a small volume of
liquid sample into a tube packed with tiny particles (3 to 5 micron (μm) in diameter
called the stationary phase).
 Where individual components of the sample are moved down the packed tube
(column) with a liquid (mobile phase) forced through the column by high pressure
delivered by a pump.
 These components are separated from one another by the column packing that
involves various chemical and/or physical interactions between their molecules and
the packing particles.
 These separated components are detected at the exit of this tube (column) by a
device (detector) that measures their amount. The output from the detector is called
a chromatogram.
Procedure

 In principle, LC and HPLC work the same way except the speed,
efficiency, sensitivity and ease of operation of HPLC is vastly
superior.
 High performance liquid chromatography is a powerful tool in
analysis; it yields high performance and high speed compared to
traditional columns chromatography because of the forcibly
pumped mobile phase
 Uses: It is used in biochemistry and analytical chemistry to
identify, quantify and purify the individual components of a
mixture.
TYPES OF HPLC TECHNIQUES

1. Based on modes of chromatography:

a) Normal phase mode b) Reverse phase mode
2. Based on principle of separation:
a) Adsorption chromatography b) Ion exchange chromatography
c) Size exclusion (or) Gel permeation chromatography
d) Affinity chromatography e) Chiral chromatography
3. Based on elution technique:
a) Isocratic separation b) Gradient separation
TYPES OF HPLC TECHNIQUES

4. Based on the scale of operation:

a) Analytical HPLC b) Preparative HPLC
5. Based on the type of analysis:
a) Qualitative analysis b) Quantitative analysis
BASED ON MODE OF CHROMATOGRAPHY

 a) Normal phase chromatography: In this stationary phase is polar and

mobile face is non-polar.
 Normal phase HPLC was the first kind of HPLC used and it separate
analyte based on polarity.
 It is used when analyte of interest is non-polar in nature.
 Principle: Adsorption of analyte on polar S.P is the principle of Normal
phase. In this non polar M.P. is used so non polar molecules eluted 1st
and polar molecules retained for longer time.
 Use of more polar solvents in the M.P will decrease the retention time of
polar compounds while use of non-polar solvent will increase the
retention time of polar substances.
BASED ON MODE OF chromatography
 Normal Phase HPLC: It takes place by the adsorption of polar group
of the molecule with polar group of S.P. Adsorption strength increases
with the increase in the analyte polarity. And interaction between polar
analyte and the polar S.P increase the elution time.
 The sample binds to polar sites of S.P. It is physical and reversible
process. The sample molecules that had been adsorbed on S.P should
be detached to elute out from the column.
 In order to carry out the process we use different phases having
different polarity index. S.P used in Normal Phase is; Silica,Alumina.
 Mobile phase used in Normal phase is; Organic solvent starting from
low polarity to high polarity. E.g. hexane, chloroform, ethyl acetate,
ethanol etc.
BASED ON MODE OF chromatography
b) Reverse phase chromatography: The term reverse phase HPLC is
derived from the fact that it is reverse of normal phase chromatography
in a sense that M.P is more polar than the S.P in reverse phase.
 The reversal of polarities of M.P and S.P also reverse the order of
elution of analyte.
 In reverse phase HPLC the S.P is non-polar and M.P is polar. It is
most popular mode of chromatography for the analytical and
preparative separation of compounds.
 The retention time is longer for the non-polar molecules allowing the
polar molecules to elute more readily.
 Retention is the result of interaction of non-polar components of
solute and non-polar S.P.
BASED ON MODE OF chromatography
 The introduction of alkyl chains covalently bonded to the solid support such as
silica, created a hydrophobic stationary phase, which has a stronger affinity for
hydrophobic compounds.
 Hydrophobic molecules in the polar mobile phase tend to adsorb to the
hydrophobic stationary phase.
 And hydrophilic molecules in the mobile phase will pass through the column and
are eluted first.
 Hydrophobic molecules can be eluted from the column by decreasing the polarity
of the mobile.
 The more hydrophobic the molecule, the more strongly it will bind to the
stationary phase, and the higher the concentration of organic solvent that will be
required to elute the molecule.
 Stationary Phase may be octadecyl carbon chain (C18)-bonded silica, Octylsilica
(C8),Butyl silica (C4) , Waxy liquid. Mobile Phase may be
Methanol,Acetonitrile,Water, and Buffer.
BASED ON PRINCIPLE OF SEPERATION
a) Adsorption Chromatography:
In the Adsorption Chromatography solute molecules bond directly to the surface of the stationary phase.
 The component which has more affinity towards mobile phase elutes first & the component which has
less affinity towards stationary phase elutes later.
 No two components have same affinity towards mobile phase & stationary phase.

b) Ion-exchange chromatography: Ion exchange chromatography is a process that allows the separation
of ions and polar molecules based on their charge.
 It can be used for almost any kind of charged molecule including large proteins, small nucleotides and
amino acids.
 Retention is based on the attraction between solute ions and charged sites bound to the stationary
phase. Ions of the same charge are excluded.
 The use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it.
 Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic
forces.
 c) Gel permeation chromatography: This type of chromatography lacks an
attractive interaction between the stationary phase and solute.
 The liquid or gaseous phase passes through a porous gel which separates the
molecules according to its size.
 The pores are normally small and exclude the larger solute molecules, but
allow smaller molecules to enter the gel, causing them to flow through a
larger volume.
 This causes the larger molecules to pass through the column at a faster rate
than the smaller ones.
 d) Affinity chromatography:
 This is the most selective type of chromatography employed. It utilizes the
specific interaction between one kind of solute molecule and a second
molecule that is immobilized on a stationary phase.
 For example, the immobilized molecule may be an antibody to some specific
protein.
 When solutes containing a mixture of proteins are passed by this molecule,
only the specific protein is reacted to this antibody, binding it to the
stationary phase. This protein is later extracted by changing the ionic
strength or pH.
 e) Chiral chromatography: It involves the separation of stereoisomers. In the
case of enantiomers, these have no chemical or physical differences apart
from being three-dimensional mirror images.
 Conventional chromatography or other separation processes are incapable of
separating them.
 To enable chiral separations to take place, either the mobile phase or the
stationary phase must themselves be made chiral, giving differing affinities
between the analytes.
BASED ON ELUTION TECHNIQUE:

 a) Isocraticelution: A separation in which the mobile phase composition

remains constant throughout the procedure is termed isocratic elution. It
isbest for simple separations.
 Often used in quality control applications that support and are in close
proximity to a manufacturing process.

 b) Gradient elution: A separation in which the mobile phase composition is

changed during the separation process is described as a gradient elution. It is
best for the analysis of complex samples.
 Often used in method development for unknown mixtures. It is most popular.
BASED ON SCALE OF OPERATION:

 a) Analytical HPLC: No recovery of individual components of substance. We

can separate individual components but cannot assess the quantity in this
analysis.
 Analytical chromatography is used to determine the existence and possibly
also the concentration of analyte(s) in a sample.
 b) Preparative HPLC: Individual components of substance can be recovered.
Quantity of the substance and individual components can be assessed.
 Preparative chromatography is used to purify sufficient quantities of a
substance for further use, rather than analysis.
BASED ON TYPE OF ANALYSIS:

 a) Qualitative analysis: Analysis of a substance in order to ascertain the

nature of its chemical constituents. We can separate individual components
but cannot assess the quantity in this analysis.
 b) Quantitativeanalysis: Determining the amounts and proportions of its
chemical constituents. Quantity of the impurity and individual components
can be assessed.