1. HPLc
1
INTRODUCTION:
The technique of high performance liquid chromatography is called, because of
its improved performance when compared to classical column chromatography.
It is also called high pressure liquid chromatography as high pressure is used
when compared to classic column chromatography.
The development of HPLC from classic column chromatography can be
attributed to the development of smaller particle size. Smaller particle size is
important since they offer more surface area over the conventional large particle
size.
A porous particle of 5Microns offers a surface area of 100-860 Sq. meters/gram
with an average of 400 meter sq/g. These offer very high plate counts up to
1,00,000/metres.
COMPARISON OF CLASSICAL COLUMN WITH HPLC:
Table no: 1 Comparing Classical Columns with HPLC
PARAMETERS CLASSICAL COLUMN
CHROMATOGRAPHY
HPLC
Stationary phase particle
size
Large 60-200µ Small 3-5µ
Column size
Length × int. Diameter
Large
0.5-5m × 0.5-5cm
Small
5-50 × 1-10mm
Column material Glass Mostly metal
Column packing pressure Slurry packed at low pressure
- often gravity
Slurry packed at high
pressure >4000psi
Operating pressure Low (<20 psi) High (500-3000 psi)
Flow rate Low to very low Medium to high
Column efficiency (low) >500 theoretical plates
per meter
(high) <1,00,000 plates per
meter
Detector flow cell volume Large 300-1000 µl Small 2-5µl
Types of stationary phase Limited range Wide range
Scale of operation Preparative scale Analytical scale
2. HPLc
2
Types of HPLC Techniques:
A. Basedon mode of separation:
There are two different modes normal mode and reverse phase mode. This modes are
based on polarity of stationary and mobile phase. Before explaining into the modes, it
is important to know interactions between solute, stationary and mobile phases.
Polar-polar : interaction affinity is more
Nonpolar-Nonpolar : interaction affinity is more
Polar-Nonpolar: interactions affinity is less
Table no:2 Modes of Separation
As most of the drug substanceare Polar in nature, Reverse phase is used
for separation of pharmaceutical products.
B. Based on principle of separation:
1. Adsorption chromatography:
The principle of separation is adsorption. Separation of components
takes place because difference in affinity of compounds towards
stationary phase. These principle is seen in reverse phase mode and
normal phase mode.
2. Ion exchange chromatography :
Principle of separation is ion exchange, which is reversable
exchange of functional group. In ion exchange chromatography, an
ion exchange resin is used to separate the mixture of similar charged
ions. For cation, cation exchange resin and for anion, anion
exchange resin is used. It is applicable to ion exchange
chromatography by HPLC.
3. Ion pair chromatography :
In ion pair chromatography, a reverse phase column is converted
temporarily into ion exchange column by using ion pairing agent
pentane or hexane or heptane or octane sulphanoic acid sodium
salts, tetramethyl or tetramethyl ammonium hydroxide.
4. Size exclusion or gel permeation chromatography :
Contents Normal phase mode Reverse phase mode
Stationary phase Polar Non polar
Mobile phase Non polar Polar
Separation Non polar Polar
3. HPLc
3
In this type of chromatography, a mixture components with different
partical size separated by using gels. The gel is used as a molecular
sieve and hence mixture of substancewith different molecular size
separated.
Soft gels like dextrin , agarose , polyacrylamide are used. Semi rigid
gel like polystyrene alkyl dextrine in non aqueous medium are also
used.
5. Affinity chromatography:
Affinity chromatography uses the ability of sample with specific
stationary phase.
This technique is mostly used in field of biotechnology,
microbiology, biochemistry.
6. Chiral phase chromatography:
Separation of optical isomers can be done by Chiral stationary
phase. Different principles operate for different types of stationary
phase and different samples. The stationary phases used is made of
silica gel.
C.Based on elution technique:
1. Isocratic separation:
In this technique, same mobile phase used throughout the process
of separation. The same polarity or elution strength is maintained
throughout the process.
2. Gradient separation :
In this technique mobile phase is contain lower polarity or elution
strength is used followed by gradually increasing polarity or elution
strength.
PRINCIPLE:
The principle of separation in normal phase mode and
reverse phase mode is adsorption.
When a mixture of components are introduced in a HPLC
column.
They travel according to their relative affinities towards or
statonary phase.
The component which has more affinity towards
absorbent, travels slower.
The component which has less affinity towards absorbent,
travels faster.
Since no two components have the same affinity towards
the stationary phase, the components are separated.
4. HPLc
4
HPLC DIAGRAMMATIC REPRESENTATION:
Figure no :1
PUMPS:
Solvent or mobile phase used must be passed through column at high pressure
About 1000-3000psi. because of its particle Size of stationary phase is few
microns(5-10u).The resistance to flow of solvent is high hence such high
pressure is recommended.
Different types of pumps are available, they are:
1. Mechanicalpumps:
Operate with constant flow rate and uses a sapphire piston.These type of
pumps use at analytical scale.
2.Pneumatic Pumps
Operate with constant pressure and use highly compressed gas. solvent used
must be of high purity and preferably HPLC grade and filtered through
0.45micron.
3.Receprocating pump:
Which are currently used 90% of commercially available HPLC systems.
Hydrolically pumped by a reciprocating piston.
5. HPLc
5
Figure no:2
Solvent is pumped backed by motor driven piston. Two ball check valves
Which Opens and closes Which control the flow. The piston is in direct contact
with the Solvent. Small internal volume 35-45micro litres high output pressure
upto 10,000 Psi.
CHECK VALVES:
These are present to controlthe flow rate of solvent and back pressure.
PULSE DAMPNERS:
These are used to control the pulses observed from wavy baseline caused by
the Pumps
MIXING UNIT:
Mixing unit is used to mix the solvents in different proportions and pass
through the column.
There are two types, they are:
6. HPLc
6
GRADIENT CONTROLLER:
In an isocratic separation, mobile phase is prepared by using pure solvent or
mixture of solvent.
Solvent of same eluting power or polarity is used.
But in gradient elution technique, the polarity of the solvent is gradually
increased and hence the solvent composition has to be changed.
Hence a gradient controller is used when two or more solvents pumps are used
for separation.
SOLVENT DEGASSING:
Several gas are soluble in organic solvents.When solvents pumped under high pressure
air or gas bubbles are formed which will interfere with separation process, steady baseline
and shape of the peak.
Hence degassing solvent is important.
Table no:4 types of solvent degassing
Vaccum filters
Remove air
bubbles. Not
always reliable and
complete
Helium purging
Passing Helium
through the solvent.
This is very
effective but
expensive
Ultra sonication
By using ultrasonicator,
which convert ultra
high frequency to
mechanical vibration
7. HPLc
7
Flow controland programming system:
As a part of their pump system, many commercial instruments are equipped with
computer controlled devices for measuring the flow rate by determining the pressure
drop across a restrict or located at pump outlet.
Any difference in signal from present value is then used to increase or decrease the
speed of pump motor.
SAMPLE INJECTORS:
Several Devices are available either for manual orauto or injection of sample.
Different devices are:
Table no:5 Types of Sample Injection.
Septum
injectors
Sample injection
through rubber
Septum
Stop flow (on line)
Flow of mobile phase
is stopped for while
and sample injected
through valve
Rheodyne
injector (loop
valve type)
Fixed volume loop like
20 to 50Microlitres.It
has two modes lode
position and inject
position
8. HPLc
8
Figure no :3
COLUMN:
The column is made up of stainless steel and manufactured. So that they
can withstand pressure up to 5.5×10⁴ pa (8000 psi)
The straight column length of 20-50cm and diameter 1-4mm are generally
used through smaller capillary columns are available.
Best columns are precision bored with an internal mirror finish which allows efficient
packing of column.
The porous plugs of stainless steelor Teflon are used in ends of column to retaining the
packing materials.
COLUMN PACKING:
There are two types of column packing pellicular and porous particle.
Pellicular former consists of a spherical, non porous, glass or polymer beads with
typical diameters of 30-40 Microns.
A thin layer of silica, alumina, polystyrene-di-vinyl benzene synthetic resin or an ion
exchange resin is deposited on the surface of these beads.
For some applications, an additional coating is applied. Which consists of a liquid
stationary phase that is held in place by adsorption.
A typical porous packing liquid chromatography consists of porous micro particles
having diameter of ranging from 3-10Microns for given particle Size.
Silica particles are prepared by agglomerating of sub micron particles of silica under
condition that lead to the large particle having high uniform diameter resulting thin
organic film chemical or physical bonding to surface.
10. HPLc
10
DETECTORS:
1)U. V absorbancedetectors:
Filters of photo meters with a mercury lamp as the source. At intensity line of 245nm Is
isolated by filters. Some instruments lines 250,313,334,315 also be employed by Substitution of
filters. Deuterium or tungsten filament is used as source ,provide simple means of Detecting
absorbing species as they eluted from column filter wheel which contains severalfilters that can be
rapidly switched detect various Species as they are eluted. These detector are used in repetitive and
quantitative analysis.
Fixed wavelength : Measures at one wavelength, usually 254nm
Variable wavelength: Measuresone wave length at a time, but can detect over
A wide range of wavelengths.
Diode array:Measures a spectrum of wavelengths stimultaneously. The
Measures the ability of absorbance of sample.
This can be established at one or severalwavelengths.
2) IR ABSORBANCE DETECTORS:
Two types of IR detectors
220-320 wavelength scanning being provided by three semicircular filter wedges.
Range of instrument is 2.5-14.5micrometres or 4000-690cm-1
Based upon FTIR , cell length 2.0-10microns volume of 1.5microlitres.
One or more single wavelength settings. Alternative spectra for peaks can be scanned
by stopping flow at time of elution.
FTIR used analogues to diode-aray instrument And u.v absorbance measurement
3) REFRACTIVE INDEX;:
Solvent passes through one half of cell on its way to column eluted then flow
to another chamber.
Two compartments are separated by glass plate mounted at an angle.
Such that bending of incident beam occurs if two solutions differ in refractive
index.
Resulting in displacement of beam with respect to photosensitive surface of
detector. it causes variation in output signal which, when amplified and
recording provides chromatography.
11. HPLc
11
Figure no:5
FLURIMETRIC DETECTORS:
This detectors is based on the fluorescent radiation emitted by some class of
compounds.
The excitation wavelength and emissions wavelength can be selected for each
compound.
This detectors have more specifications and sensitivity.
The disadvantage is that some compounds are not fluorescent.
CONDUCTIVITY DETECTORS:
Based upon electrical conductivity, the response is Recorded.
This detector is used when the sample has conducting ions like anions and cations.
12. HPLc
12
APPLICATIONS:
QUALITATIVE ANALYSIS:
It is nothing but identification of a compound.
This is done by comparing the retention time of sample as well as the standard.
Under identical conditions, the retention time of the standard and the sample are same.
If there is a deviation, then they are not the same compounds.
1)CHECKING PURITYOF A COMPOUND:
By comparing the chromatogram of standard and rhat of the sample, the purity
of the compound can be inferred.
If addition of peaks are obtained, the impurities are present and hence compoud
is not pure. From percentage area of peak obtained, the percentage purity can
also be known.
2)PRESENCE OF IMPURITIES:
This can be seen by the presence of additional peaks when compared with a reference
standard or reference material.
The percentage of impurities may also be calculated from peak areas.
QUANTITATIVE ANALYSIS:
The quantity of a compound can be determined by several methods like
1)DIRECT COMPARISONMETHOD:
By injecting a sample and standard separately and comparing peak areas,the
quantity of the sample can be determined.
Area of peak=peak height×width of peak at the half Height
A1/A2=W1/W2
Where A1 and A2 are peak area of sample and standard
W1 andW2 are weight or concentration of sample and
standard
2) CALIBRATION CURVE METHOD:
A series of standards are used to determine their peak areas.
A calibration Curve of peak area vs concentration of the drug is plotted.
From the peak area of the unknown sample, by interpolation, the
concentration of the sample can be determined. This method has the
advantage that errors,if any, are minimised.
3)INTERNAL STANDARD METHOD:
13. HPLc
13
In this method, a compound with similar retention characteristics is used. A
known concentration of the internal standard is added to the sample solution
whose concentration is not known.
The chromatogram is Recorded and their peak areas are determined. By using
formula, the concentration of the unknown solution is determined.
4) MULTI COMPONENT ANALYSIS OR DETERMININGOF
MIXTURE OF DRUGS:
Similar to the quantification of a single drug, multi component analysis
can be done easily. The quantity of each component is determined by
using any one of the above methods.
Marketed formulations which contain severaldrugs, can be determined
quantitatively for each component.
6) Isolation and identication of mixture of drugs or metabolites in urine, plasma, serum e.t.c can be
carried out.
7) isolation and identication of mixture of components of natural or synthetic
origin.
8) Biopharmaceutical and pharmacokinetics studies.
9) stability studies.
10) purification of some compounds of natural or synthetic origin on p
reparative scale.
REFERENCE:
1)Principle of instrumental analysis, 5th
edition , by Skoog. Holder. Nieman
Page no: 726-738
14. HPLc
14
2) Text book of pharmaceutical analysis, 4th
edition, by Dr. S. Ravi Sankar
page no:18-1 to 18-13
3) Instrumental method of chemical analysis by Gurdeep R. ChatwaL,Sham k .Anand
4)Quantitative chemical analysis, 7th
edition, By Harris, chapter 25.
5) Text book of chemical analysis, 2nd
edition, By Francis Rouessac and Annick Rouessac.
6)Text book of modern analytical chemistry, By David Harvey.
7)Journal of Global pharma technology, June-2010, By Rishabh malivya, Vipin Bansal, Om
Prakash pal.
8)International journal of Trend in scientific research and development,june/2011, yogesh kumar,
sayad MD. Mumtaz, mustq Ahmed.
9)International research journal of pharmacy, March 2013,By Azim MD Shabbir, mitra moloy,
Bhasin. S
10)Journal of chromatography, January 2011,By Alexander k. Shilkov, Voldimar I. Ossipov, Olaga N.
Pozharikaya, Svetala N. Ivanova, Valery G. Markov.
