HIGH PERFORMANCE
LIQUID CHROMATOGRAPHY
What is HPLC?
HPLC is really the automation of traditional liquid
chromatography under conditions which provide for
enhanced separations during shorter periods of time!
Principle of separation of hplc
When a mixture of components are introduced into a HPLC
column, they travel according to their relative affinities towards
the stationary phase.
The components which has more affinity towards the adsorbent,
travels slower.
The component which has less affinity towards the stationary
phase travels faster.
Since no two components have the same affinity towards the
stationary phase, the components are separated.
Types of HPLC Separations
Normal Phase: Separation of polar analytes by
partitioning onto a polar, bonded stationary phase.
Reversed Phase: Separation of non-polar analytes by
partitioning onto a non-polar, bonded stationary phase.
Adsorption: Separation of moderately polar analytes using
adsorption onto a pure stationary phase (e.g. alumina or
silica)
Ion Chromatography: Separation of organic and
inorganic ions by their partitioning onto ionic stationary
phases bonded to a solid support.
Size Exclusion Chromatography: Separation of large
molecules based in the paths they take through a “maze” of
tunnels in the stationary phase.
INSTRUMENTAL REQUIREMENT
Solvent Reservoir
Tubing
Pumps
Injector system
Guard column
Analytical column
Detectors
Recorders & integrators
SOLVENT RESERVOIR
It should be inert to mobile phase.
Made up of stainless steel or glass.
Capacity range200ml- 1000ml.
Mobile phase flow rate: 1-2 ml/min
Degassing: In some cases, aqueous solvents and some organic solvents are
degassed prior to use. This is done to prevent formation of gas bubble in the
column and detector system. To remove dissolve oxygen/nitrogen from the
mobile phase by Sonicator.
It is done by various method:-
- By stirring of the mobile phase under vacuum pumping system
- By sparging with helium gas
-A distillation System
TUBING
The nature of the tubing used to connect all parts of the system deserves some
attention.
The tubing should be
- inert
- have the ability to withstand pressure
- able to carry sufficient volume.
The length of tubing L that can be used with a maximum band width increase
of 5% can be calculated from,
; VR = solute retention in ml
Dm= solute diffusivity in cm2/sec
F= Flow rate in ml/sec
D=diameter of tube in cm
N=column plate no.(4)
PUMPS:
These are used to pass the mobile phases at high
pressure of about 1000 to 10,000 psi into the column, which is required cause of high resistance to the flow offered due to the less particle size of the stationary phase.
Ideal requirement
Produce very high pressure 5000-10000psi.
Produce pulse free output.
Flow rate should be control & reproducible 0.5% relative or better.
Flow rate of mobile phase should be in the range of 0.1-10ml/min.
All material of construction should be corrosion free like Stainless
steel, Teflon.
It should be noted that the high pressure generated by HPLC pumps
do not constitute an explosion hazard because liquids are not very
compressible.
Two types :
1. Constant flow rate.
2. Constant pressure.
1) Constant flow rate:
(a) Reciprocating pump
(b) Syringe drive pump
2) Constant pressure
Mobile phase is held in collapsible
container which is flexible and
pressurized by gas. Since the mobile
phase is directly in contact with gas so
it may be dissolved.
The pneumatic amplifier pump(
Haskell pump) is a modification of
this pump, the gas pressure is applied
to large piston which is connected to the small piston in contact with mobile phase.
Advantage
- Cheap.
- Produce pulse less flow.
Disadvantage:
- For very limited solvent capacity.
- Gradient process can not be applied.
- Flow rate depend on viscosity and back pressure
of the solvent.
- Limited pressure obtained up to 2000 psi.
INJECTOR SYSTEM
These are the devices available for injection of the sample into the
column.
Ideal requirement
Sample should be injected in a narrow plug.
Size of sample should be variable (Few tenth of µL to 500 µL )
Should be reproducible.
System must able to inject against a high pressure without sample loss,
when system to be automated.
It should be convenient to be able to introduce tha sample without
depressurizing the system.
Column packing/stationary phase:
• Particle size for packing have been reduced ,thereby
increasing the efficiency of the column.
• Shape of the particle is also affected.
• Packing material can be irregular or spherical.
• Two types of particles are used:
1.totally porous particle
2.super facially porous particle
Material used for packing is mostly silica.
Rigid, can withstand at pressure 10000psi , available in
wide range of porosity.
Other material used are polystyrene cross linked with
divinylbenzene. It more resistant to chemical attack & may
use at variable PH ranges.
S.p. in liquid solid chromatography
Mobile phase: Non polar
Highly polar material like silica and alumina are used because they contain –
OH (silanol)group on surface which undergo to reaction with solute. This
silanol group have different degree of reactivity.
Tailing -This may be prevented by Glacial acetic acid in acidic comp. and by
triethyleneamine in basic comp.
S.p. in liquid liquid chromatography
Mobile phase: polar.
Bonded phase HPLC uses as s.p. Consisting of an org. moiety chemically
bonded to the surface of silica through the surface silanol groups. Org-
moieties are long chain Hydrocarbons.
Ion exchange chromatography
Mobile phase: acidic or basic
S.p. that can exchange cation and anions with mobile phase.
May be of cation and anion exchanger and subdivided to strong and weak.
Strong cation-contain sulphonic acid group & PH
2-12.
Weak cation-carboxylic acid-above PH
7.
Strong anion-quaternary ammonium group- PH
2-10.
Weak anion-3 amine-PH
-below 7.
Ion pair chromatography
Based on formation of two oppositely charged analytes.
Mostly used pairing ions are :
Long chain alkylammonium (cation)
Alkyl sulfates(anion)
Size exclusion chromatography
Separate analytes on the basis of molecular size.
Packing should be classified in 3 groups: Rigid, Semi rigid , Soft gels.
The Mobile Phase in HPLC
Must do the following:
solvate the analyte molecules and
be suitable for the analyte to transfer “back and forth” between during
the separation process.
Must be:
compatible with the instrument (pumps, seals, fittings, detector, etc)
compatible with the stationary phase
readily available
of adequate purity.
Not too compressible (causes pump/flow problems)
Free of gases (which cause compressibility problems)
Characteristics
of mobile phase
(1) Viscosity
As viscosity of the mobile phase increases, the efficiency of the
system,as measured by the no. of theoretical plates, decreases.
(2) Compressibility
It is the tendency of a solvent to be compressed in volume
during the pumping cycle. Solvents with a high degree of
compressibility shows greater pulsations with reciprocating
piston pumps.
(3) Refractive index
It is important when a differential refractometer detector is used. As
it measures differences between the R.I. of the mobile phase and the
solute, so greater the difference the greater the sensitivity.
(4) U.V. cut off wavelength
It is the wavelength below which the solvent will absorb more
than 1.0 absorbance unit in a 1cm cell.
It is important when either UV or fluorescence detector are used.
(5)Polarity
It is a measure of the solvent strength or ability to elute a
particular type of compound.
(6)Vapor pressure
It plays an important role in that the mobile phase, in the solvent
reservoir, could easily change in composition due to evaporation of
one of the more volatile constituents.
DETECTORs
Ideal requirement
High sensitivity
Good stability , Reproducible response & give response to all analyte.
Response should be linear over wide range of concentration.
Should not be sensitize to flow rate fluctuation or temp. change or
change in composition.
Capable of withstand at high pressure.
Short response time
Highly reliable and easily used
Should not cause destruction of sample
Types of detectors
(1) Differential detectors or bulk property detectors
They provides a differential measurement of a bulk property that is
possessed by both the solute and the mobile phase.
They are nonspecific and respond to a wide range of compounds. Ex:-
Refractive index detectors, density, dielectric constant.
(2) Selective detectors or solute property detector
They measure a property of the sample which is not possessed by the
mobile phase and respond to some property of solute.
Ex:- U.V. absorbance & fluorescence detectors.
Optimization of Separations in HPLC
Correct choice of column
Correct choice of mobile phase
Decision on the type of mobile phase composition
constant composition = isocratic
varying composition = gradient elution
Determination if flow rate should be constant
usually it is
Decision on heating the column
heating HPLC columns can influence the above
equilibrium….
APPLICATIONS
QUALITATIVE ANALYSIS:-
HPLC is used for identification of compound. Here comparison of
retention time of test sample with the reference compound carried out.
Checking purity of compound
Purity of compound checked by comparison of chromatogram of test
with the reference standard
Presence of impurities
If impurities present into sample we observed additional peak when we
compared the chromatogram of test with the reference standard.
QUANTITATIVE ANALYSIS
HPLC is used for assay of many drugs like cephalosporin, furosemide.
It is also used into drug mixture determination.
Investigation of biological material such as gastric content, blood,
urine sample etc.are done by HPLC.
Many poisonous substance can also be investigated by HPLC
BIOCHEMICAL ANALYSIS
Lipids
Separation of reference mixtures of glycerides, fatty acid can be
done on silica column.
AMINO ACID
HPLC is widely used for analysis and separation of amino acid and protein. For
this size exclusion chromatography is used.
CARBOHYDRATE
HPLC is suitable for analysis of carbohydrates. HPLC is used for determination
of sugar content of soyabean extracts, daily products.
NUCLEIC ACID
HPLC is used into all area of nucleic acid research. HPLC is used for analysis of
nucleic acid, separation of starting material, intermediates, separation and
purification of nucleic acid.
VITAMINS
HPLC is used for analysis of vitamins in variety of food products and animal
feeds.
Isolation of natural pharmaceutical active compounds.
For example morphine,codeine from opium
FOR BIOEQUIVALENT STUDIES
In that, drug or its metabolites can be separated from plasma or
urine, which is then comparable with that of the data of the
standard drug.