UNIT-IV

GAS CHROMATOGRAPHY

Pointsto be covered in this topic

INTRODUCTION

’ * THEORY

* INSTRUMENTATION

* DERIVATIZATION
ADVANTAGES

DISADVANTAGES

APPLICATIONS
 GASCHROMATOGRAPHY
INTRODUCTION
A.T. James and P. Martin first time used the gas chromatography
technique in 1952 for separating long chain fatty acids.
The gases and vaporisable substances can also be separated by gas
chromatography based on differential adsorption.
Ingas chromatography, gas is used as the mobile phase and solid or
liquid is used as the stationary phase.
When the stationary phase is solid, it is known as Gas Solid
Chromatography (GSc) and when the stationary phase is liquid, it is
known asGas Liquid Chromatography (GLC).
Gas Solid In gas chromatography, a moving gas phase is passed over a
stationary sorbent to separate the mixture components.
This technique is similar to that of liquid -liquid chromatogra phy with the
only exception that in the former a moving gas is used as the mobile phase
while in the latter it is a liquid. The stationary phase remains the same. i.e.
a solid or a liquid.

Inlet Detector

Column Control Panel

(Touchscreen)

Oven

-PowerSwitch

GAS CHROMATOGRAPHY
OPRINCIPLE
In gas chromatography, the substance to be analysed is partitioned
between the mobile and stationary phases.
During the separation, the sample is vaporised and carried through the
column by the mobile gas phase (i.e., the carrier gas).
The different components get separated based on their vapour pressure
and affinities for the stationary phase.
The affinity of a component towards the stationary phase is termed as
distribution constant (Kc), which is also known as the partition
coefficient.

Kc = [A]s /[A]m
Where.
[A]s = concentration of component Ain the stationary phase
[A]m =concentration of component Ain the mobile phase.
Movement of different components through the column is controlled by the
distribution constant (Kc), thus the chromatographic separation occurs
based on the differences in distribution constant.
Dlrection of mobile-phee flow Detector Chromstogram

Concontrdon of olute in
mobile plheee

Concontraion of olute in
stitlonary pheo

Schematic Representation of the Chromatographic Process

 THEORY
The separation of compounds in a mixture is based on different
polarities in a direct (interaction with stationary phase i.e., solubility) or
indirect way (physical properties i.e., boiling point).
The gas chromatography column consists of solid support that is covered
with ahigh-boiling liquid in a thin capillary tube.
In the example, compound "X" has a higher affinity towards the
stationary phase compared to compound "0".
Compound "0" elutes before compound "X" because it displays a lower
boiling point and aweaker interaction with the stationary phase.
Stationary phase (liquid phase)
Solid Support

comenert miutte

Carrer Gs
(moble pahse) time

D Factor that influences the outcome in the GC

Vapor Pressure of the Compound

The higher the boiling point of the compound is, the lower the vapor
pressure willbe.
Thus, the compound with higher boiling point migrates slower
through the column resulting in a longer retention time and peak
broadening.
" Low boiling solvents like diethylether, dichloromethane, ethyl acetate,
methanol, etc. are usually used to dissolve the sample because they elute
veryearly from the column.
Polarity of the Compound and the Polarity of the Column
The stronger the interaction of the compound with the stationary
phaseis going to be increasing the retention time and peak broadening.
Very polar compounds (i.e., alcohols, amines) often cause tailing on polar
columns (or older columns). Avariety of column polarities is available for
the separation of compounds using gas chromatography.
 INSTRUMENTATION
The following components make up the instrumentation of gas
chromatography;
1. Carrier gas maintained at a high pressure and delivered at a rapid and
reproducible rate,
2. Sample injector,
3. Separation columns,
4. Detectors,
5. Thermostated chambers for regulating the temperature of column and
detectors,
6. Amplifier and recorder system.
Gas Chromatography
Sample
Gas flow Injector
regulator port
Detcctor
Column

Waste
Oven
Computer/
Carricr gas Data analysis

DCarrier Gas
Hydrogen, helium, nitrogen, and air are the most widely used carrier
gases.
Hydrogen in comparison to other gases is more advantageous and also
dangerous touse.
Helium is the next best gas,and is used because of its exceptional thermal
conductivity, inertness, low density, and greater flow rates; but it is
expensive.
Nitrogen is inexpensive but reduces sensitivity.
Air is used only when the atmosphericoxygen is useful to the detector
or separation.
 The following considerations should be kept in mind while selecting a
carrier gas:
It should be inert, i.e., it should not react with the sample, stationary
phase,or contacted hardware.
Should be suitable for the detector used and the type of sample being
analysed.
It should be available in high purity.
It should give best column performance reliable with required speed of
analysis.
It should not be expensive.
It should not cause any fire or explosion hazard.
OSample Injector
The system of sample injector is used for introducing the sample in a
reproducible manner and should vaporise it rapidly so that the sample
enters the column as a single slug.
Liquid samples are introduced into a small inlet chamber using
hypodermicsyringes through a self-sealing rubber septum.
The chamber is heated to cause flash evaporation, and the temperature
should not be very high to avoid sample decomposition.
Solid samples are either dissolved in volatile liquids prior to their
introduction or are directly introduced if they are liquefiable.
Gas samples are introduced into the carrier gas stream using a special gas
sampling valves.
OSeparation Columns
The columns used are made of glassor metal tubing,and have a diameter
of 4.8mm.
They may be of any length ranging from a few centimetres to a hundred
meters. They may be coiled, bent, or straight.
The following six types of analytical columns are used in gas
chromatography:
> Packed Columns:
These columns are prepared by packing metal or glass tubing with
granular stationary phase.
> Open Tubular or Capillary or Golay Columns:
These columns are made of long capillary tubing (30-90m) and have
uniform and narrow internal diameter (0.025-0.075cm).
They are of stainless steel (most popular), copper, nylon, glass, etc.
The liquid phase is coated over the inner wall of capillary tubing as a thin
(0.5-1p)and uniform film.
> Support Coated Open Tubular Columns:
These columns are prepared by coating the inner wall of a capillary
column with a micron size porous layer of support material, followed
by coating with the liquid phase as a thin film.
> WallCoated Open Tubular Columns:
These columns are prepared by coating the unmodified smooth
wall of the tube with the liquid stationary phase.
Porous-Layer Open-Tubular (PLOT) Columns:
These columns are prepared by coating the inner wall with a porous
layer.
Porosity can be achieved either by chemical methods (e.g,, etching) or by
depositing porousparticles on the wall from a suspension.
> Support-Coated Open-Tubular (SCOT) Columns:
In these columns, the porous layer consists of support particles and was
deposited from a suspension
ODetectors
Gas chromatography employs a wide range of detectors, of which Flame
Ionisation Detector (FID) and the Thermal Conductivity Detector
(TCD) are the most commonones.
desirable properties of a detector are:
Its sensitivity should be high and should not show instability at high
sensitivities.
 Its volumeshould be low sothat the compound eluted from the column in
small plug of carrier gas does not undergo further dilution within the
detector.
Its response should be rapid and linear with the concentration of
compound. It should be calibrated to determine the optimum range.
Its response should not be affected by the flow rate of carrier gas and
temperature.
Some commonly used detectors in gas chromatography are discussed
below:

> Katharometer:
This detector relies on the variation in thermal conductivity of the
carrier gas in the presence of an organic compound.
The platinum wires are heated by electric means and equilibrium
conditions of temperature and resistance are attained when the carrier
gas passes over them.
They are mounted in aWheatstone bridge arrangement, and when a
compound emerges, the thermal conductivity of the gas surrounding wire
changes.
Also changing the temperature and resistance of the wire along with
the associated out-of-balance signal, which is amplified and recorded.
Platinum wires connected in
Wheatstone bridge circuit
Carrier
Outlet

To injection point Carrier gas

and column ffom colunn

iagrammatic Representation of Katharometer

Flame lonisation Detector (FID):
This detector is simpler in design and relieson the change inconductivity
of the flame as the compound is burnt.
The change in flame conductivity is not because of simple ionisation of
the compounds emerging from the detector.
The molecule undergoes partial or complete stripping and gives
charged hydrogen-deficient polymers or aggregates of carbon with low
ionisation potential.
The carrier gas used is nitrogen or argon mixed with hydrogen before
passing to the burner tip (made of a platinum capillary).
Collector electrode

Outlet for gases

Silver gauze

= A r filter
Hydrogen Air

Carrier gas from column

Diagrammatic Representation of Flame lonisation Detector

Thermal Conductivity Detector (TCD):
This detector utilises a heated filament placed in the emerging gas
stream.

Thermal conductivity of the gas phase governs the amount of heat the
filament loses by conduction to the detector walls.
Temperature depends on thermal conductivity (He & H)of surrounding
gas.
Hydrogen and helium have higher thermal conductivity and carrier gas
provide best sensitivity.
 Block

Gas flow out

Gas flow in.

Filament

Cross-SectionalView of a Thermal Conductivity Detector

Thermionic Emission Detector (TED):
This detector utilises fuel-poor hydrogen plasma and a low temperature
flame.
This flame suppresses the normal flame ionisation response of
compounds not containing nitrogen or phosphorus.

Ceramic
insulators

Signal
probe Collector
Ceramic
insulators

Ceramic bead with

Bead probe beater coil

Flame tip

DiagrammaticRepresentation of Thermionic Emission Detector

 Electron Capture Detector (ECD):
This detector relies on the electron affinity of different substances.
It responds to compounds whose molecules have electron affinity, e.g,
chlorinated compounds, alkyl lead, etc.
Itresponds less to hydrocarbons.
ECD is used for detecting trace environmental pollutants.
It is highly sensitive to halogenated compounds and is used for detecting
herbicides, pesticides.
Radioactive ‘ Gas exit
foil Nior H Insulation

Gas Anode
inlet

Diagrammatic Representation of Electron Capture Detector

DERIVATIZATION
Derivatisation is a technique of treatment of the sample to improve the
process of separation by column or detection by detector.
There are two types based upon its need. They are
O Pre-column derivatisation
This is done to improve some properties of the sample for separ.
by column.
By this technique, the components are converted to more volatile nd
thermostable derivatives. Moreover improve separation and less tiling
willbe seen after such convert treatment.
In the following conditions, pre-column derivatisation is done.
1. The component is less volatile.
 The compounds are thermolabile.
3. Toreduce tailing.
4. To improve separation factor.
e.g. Carboxylic acids,sugars, phenols, alcohols etc.can be converted to less
polar compounds by using reagents like Bis trimethyl silyl Acetamide
reagent.

OPost column derivatisation

Post column derivatisation is done to improve the response shown by
detector.
The components may be converted insuch away that their ionisation or
affinity towards electrons is increased.
Normally this is 'on-line' detection technique where the flow rate is
neither stopped nor altered.
Pretreatment of Solid Support is used to hold the stationary phase liquid
as athin film. But sometimes due to some defects, uniformity and
stability of the film of liquidstationary phase may not exist.
In such cases, tailing of peaks and low separation efficiency can be
observed. Therefore to overcome such demerits, it is best to do pre
treatment of the stationary phase.
Generally, while doingthe separation of no polar components like esters,
ethers etc. tailing of peaks are observed.
These problems can be overcome by following techniques:
1. By using more polar liquid stationary phase.
2. Increasing the amount ofliquidphase on the support.
3. By selecting a less active support.
4. Pretreatment of the solid support to remove active sites.

* TEMPERATURE PROGRAMMING
Gas chromatographs are usually capable of performing what is known as
temperature programming gas chromatography (TPGC).
The temperature of the column is changed according preset
temperature isotherm.
TPGC is a very important procedure, which is used for the attainment of
excellent looking chromatograms in the least time possible.
Temperature programming is atechnique in which column temperature
is increased either continuously or in steps as the separation
proceeds.
In general, optimum resolution is associated with minimal
temperature.
Lower temperature results in longer elution times and hence slower
analysis.
Using temperature programming, low boiling point constituents are
separated.
As separation proceeds, column temperature is increased so that the
higher boiling point constituents come off the column with good
resolution andat reasonable length of times.
The elution rate is proportional to the column temperature.
In the beginning, it uses lower temperature that gives a higher
resolution of lighter compounds.
With the increasing temperature, the elution rate of heavier compounds
also increases.
This gives sharper peaks for heavier compounds.
Temperature programmed mode refersto a continualrise of
temperature at predetermined rate during the sample analysis.
This mode of operation offers several advantages such a
Improvement of peak shapes
Improvement of resolution
Completion of analysis in a fraction of time it would take
isothermal operation.
Temperature programming combines the best results of runs at
different temperatures.
In this way, the approximate proper temperature programme can be
estimated.
 ADVANTAGES
Gas chromatography has the following advantages:
It is a reliable technique and provides rapid analysis.
It is highly efficient and leads to high resolution.
It utilises sensitive detectors.
It requires small samples (<1 ml).
It is non-destructive as it enables the coupling to mass spectrometers,
which measures the masses of individual molecules converted into ions, i.e.
molecules that have been electrically charged.
It provides high quantitative accuracy.
It is awell-established techniquewith extensive literature and applications.
DISADVANTAGES
Gaschromatography has the following disadvantages:
It is limited to volatile samples.
It is not suitable for thermolabile samples.
It is not suited to preparative chromatography.
It requires MS detector for structural elucidation of the analyte, since
most of the non-MS detectors are destructive.
APPL.ICATIONS
Qualitative analysis:
It is nothing but identification of a compound, This is done by comparing
the retention time of the sample as well as the standard Under identical
conditions.
Checking the purity of a compound:
By comparing the chromatogram of the standard and that of the
sample.
The purity of the compound can he reported. If additional peaks are
obtained, impurities are present and hence the compound is not pure.
Presence of impurities:
This can be seen by the presence of additional peaks when compared
with astandard or reference material.
 Quantitative analysis:
The quantity of a component can be determined by several methods like
a. Direct comparison method

b. Calibration curve method

C. Internal standard method

> Multicomponent analysis or Determination of mixture of drugs:

Similar to the quantification of a single drug multicomponent analysis
can also be done easily.
Marketed formulations are available which contain several drugs and each
component can be determined quantitatively.
> Isolation and identification of drugs or metabolites in urine, plasma,
serum etc. can be carried out.
Isolation and identification of mixture of components like amino aids,
plant extracts, volatileoils, etc.
 UNIT-IV
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
Points to be covered in this topic
& INTRODUCTION

THEORY

INSTRUMENTATION

* ADVANTAGES

DISADVANTAGES

APPLICATIONS
 HIGH PERFORMANCE
LIQUIDCHROMATOGRAPHY
" INTRODUCTION
High Performance Liquid Chromatography (HPLC) is used to separate
complex chemical mixtures.
Previously, it was known as High Pressure Liquid Chromatography, but
now the term performance is used instead of pressure which indicates
that pressure is not essential for high performance, and also defines
the technique in a better way.
HPLC is a highly rapid process as it involves high resolution and high
speed columns.
Followingare the points showing the advantages of HPLCover gravity
fed (classical) column chromatography:
1. The separated substances show better resolution,
2. Less time is required for separation,
3. separation with more accuracy, precision, and sensitivity,
4. Useful for qualitative as well as quantitative analysis.
The system involves pumping of mobile phase through the packed
column under the influence of high - pressure, therefore the technique
is also named as high-pressure liquid chromatography.
The high-pressure liquid
chromatography
separation method involves
stationary phase
contained in one end of
the column and mobile
phase connected to the
other end of the column.
High performance liquid
chromatography
OPRINCIPLE
HPLC is a form of liquid chromatography used to separate compounds
that are dissolved in solution.

Particles of small diameter is used as stationary phase.

Compounds are separated by injecting a sample mixture onto the
column. The different component in the mixture pass through the column
and differentiates due to differences in their partition behavior
between the mobile phase and the stationary phase.
The following four methods are used for
separating chemical mixtures:
1. Adsorption,
2. Partition,
3. Ion exchange,
4. Exclusion.

Selection of method for the separation process depends on the

stationary phase nature.
HPLC involves separation of mixture compounds on an analytical
column that is packed with small particles of stationary phase (e.g.
silica) by elution with a liquid mobilephase.
Ahigh pressure is applied to pump the mobile or liquidphase through
the packed columns.
The working of HPLC is based on the principle that separation of
molecular forms involves elutionof a sample from a solid inorganic
support by using a mixture of organicsolvents.
In HPLC, capillary columns packed with cross -linked dextran or silica
is used for solid support
Mobile phase
and
Mobile Phase Stationary Phase stationary
phase
& THEORY
HPLC Overcomes the limitations found in standard liquid

chromatography.
Inclassic liquid chromatography, the separation process is very slo
the movement of solvent occurs under gravity.
The limiting factor is the size of column packing in liquid
chromatography.
In the HPLC setup, the apparatus should operate efficiently under high
pressure and should be specialised at low tolerances.
Thus, HPLC is highly expensive than other chromatography techniques.
Amixture of polar and non -polar liquid components forms the mobile
phase of HPLC.
The concentration of these liquids depends on the sample composition.
The solvent should not have dissolved gases, which might hinder solution
mid -separation and particulates.
In HPLC column, the components are separated according to their
differing interactions with the column packing.
If the interaction between species and stationary phase is weak, it spends
a comparatively less time in the column and reduces the retentio
time.
For homogeneous columns, silica or alumina can be used as stationary
phase, while a liquid stationary phaseis considered as a bonded column.
HPLC pump isused to introduce solvent and sample in the column.
Use of HPLCpump also helps to maintain a constant, pulse free flow rate.
The HPLC pump can be a multi - piston pump or syringe pump.
Atthe column end, HPLC detector is present that registers the presence
of components in the sample but not the solvent.
In the HPLCsystem, aUV absorption detector or an NMR detector is
preferred.
* INSTRUMENTATION
Modern HPLCInstrument includes the following components:
1) Solvent reservoir anddegassing system,
2) Pumping system,
3) Sample injection system,
4) Columns,
5) Detectors,
6) Stripchart recorder, and
7) Data handling system
Solvent reservoir Flow Sample
splitting valve injectjon port

Degassers
Separation
column
TGuard
çolum
Mixing valve Constant
pump and Equilibration
3 coil temperature
gradient system chamber

To waste
Vacuum Recorder
pump
Data processing Detector
Time

Schematic Representation ofHPLC Instrument

OSolvent Reservoir and Degassing System
High pressure liquid chromatography makesuse of asingle solvent or a
mixture of solvent as amobile phase, which contained in a reservoir.
Selection of mobile phase depends on the chromatographic method
and the detector to be used.
Commercially available special grades of solvents that have been
refined to completely remove the UV -absorbing impurities and any
particulate matter are also used in HPLC
 Prior to using other grades of solvents, purification should be performed.
This is because the separation may get influenced if the impurities are
strongly UV-absorbing, affect the detector, or are of high polarity ( e.g.
traces of H,0 or EtOH, commonly included as a stabiliser, in CHCI,).
Liquid entering the pump should be free from any impurity (dust and
particulate matter), or else these impurities will result in irregular
pumping action, irregular behaviour of column owing to its
contamination, damage the seals and valves, and ultimately block the
column.
Degassing
Generally, liquids dissolve some amounts of atmospheric gases ( e.g., air or
suspended air-bubbles) that cause some major practical problems in
HPLC, specifically affecting the working of pump and the detector.
These problems can be avoided by degassing the mobile phase.
Degassing isperformed by:
1. Subjecting the mobile-phase under vacuum,
2. Distillation,

3. Spurging with a fine spray of an inert gas of low solubility (argon or

helium), or
4. Heating and ultrasonic stirring.
OPumping System
The types of pumps that are used in HPLC are:
1. Screw-driven syringe pump,
2. Reciprocatingpump, and
3. Pneumatic or constant-pressure pump.
> Screw-Driven Syringe Pump
Avariable speed stepper motor turns a screw that drives a piston, which
displaces the mobile phase from a chamber (of 200 -500cm³ volume).
The mobile phase capacity depends on the solvent chamber volume.
This volume is quite large for running numerouschromatograms before the
chamber is required to be refilled.
 Mobile phase outlet

Seals

Motor and Check

valves
gearbox

Screw driven Solvent chamber

piston

Mobile phase inlet

Diagrammatic Representationof Syringe Pump

Reciprocating Pump
In this pump an gear drives the piston in and out of a solvent chamber.
Upon moving forward, the inlet check valve closes, the outlet check valve
opens, thus pumping the mobile phase into the column.
Upon moving back, the outlet valve closes and the chamber is refilled.
In comparison to syringe pumps, the reciprocating pumps have unlimited
capacity, and their internal volume can be adjusted to very low, ranging
from 10-100ul.
Theflow rate can be altered by varying the length of piston stroke or the
motor speed.
Access tothe valves and seals is direct.
Mobile phase outlet

Seals

Check Diagrammatic
valves
Piston Representation of
Reciprocating Pump
Eccentric cam
Solvent
chamber

Mobile phase inlet

 Pneumatic or Constant-Pressure Pump
This is the simplest form of pump, consisting of collapsible solvent
container inside a vessel pressurised by a compressed gas.
These pumps are inexpensive and pulseless.
However, they suffer from limited capacity, pressure output, and their
pumping rates rely on solvent viscosity.
They also cannot be operated in gradient elution mode.
OSample Injection System
The following three modes of sample injection system are used in HPLC:
Septum Injectors:
In this system, the sample is introduced through a high pressure syringe
aself -sealing septum of elastometer.
The major shortcoming of this system is that the mobile phase in
immediate contact with the septum , gives rise to a leaching effect that
results in ghost or pseudo peaks.
Stop-Flow Septum-Less Injection:
In this system, the flow of mobile phase through the column is stopped
for a few moments,
When the column attains ambient pressure, the column top is opened
and the sample is introduced at the top of the packing.
The first two methods are inexpensive.
Micro-Volume Sampling Valves:
In highly sophisticated modern HPLC apparatus, micro-volume sampl
valves, having goodprecision and adaptable for automatic injection
used.
These valves allow sampling to be done reproducibly into pressuri
columns with minimum interruption of the mobile phase flow.
describes the operation of asample loop in two varied modes:
i) Sampling mode,
ii) Injection mode.
 Mobile phase To column
Mobile phase To column

Adjustable Sample in Sample out

length Sample in Sample out
sample loop

(a) Sampling mode (b) Injcction mode

DColumns
The following two types of columns are used in HPLC:
1)Guard columns,
2) Column thermostats.
Guard Columns
It is ashort column present between the injector and analytical column.
Although the packing composition of guard columns and analytical
columns is similar;, but particle size is larger in guard columns to aid in
the reduction of pressure drop.
The benefitsof guard columns are:
1. They eliminate foreign particles and contaminants from the solvents,
thereby improving the life of analytical columns.
2. In liquid-liquid chromatography, they minimise the loss of stationary
phase from the analytical columns since the mobile phase is saturated
with the stationary phase.
Injector Defector

Deactivated fixed
silica tubing

Union
Column
Guard Column
 Column Thermostats

Chromatographic operations are done at room temperature without the

requirement of sharp control of column temperature.
" Improved chromatograms are obtained if the column temperature is
maintained constant to the few tenths of a degree Celsius.
To achieve a constant and precise temperature control, water jackets are
fitted in the columns.
The modern commercial instruments contain heaters for controlling the
column temperature to a few tenths of a degree from near ambient to
150°C.

Detectors
1 In HPLC, the detector monitors the mobile phase passing out of the
column, which further releases electrical signals directy proportional
tothe characteristics of the solute or the mobile phase.

The commonly used detectors in HPLCare:

1)Bulk-Property Detectors
2) Solute-Property Detectors
3) Multipurpose Detectors
4) Electrochemical Detectors
> UV-Detector
Principle - An UV -detector works on the principle of absorption of UV
visible light from the effluent emerging out of the column and passing
through a photocell positioned in the radiation beam.
Movable calibrated
filter
Sample photocell

Xeference
Source Hg-laip Quartz lens Dual channel Compound UV-filter
cell
photocell

Schematic Representation of aDouble-Beanm UV Detector

Fluorescence Detector
Many compounds (solutes) are present in the mobile phase. When they
allowed to pass ascolumn effluent through a cell irradiated with xe
or deuterium source,
first UV radiation is absorbed and subsequently radiation of a longer
wavelength is emitted in the following two ways:
1) If instantly, named as 'Fluorescence, and
2) If after a time-gap, named as 'Phosphorescence!
Xenon radiation
or
Deuterium sourcc
-Slit

Slit Filtcr
Filter

Flowcel1 Photomultiplicr
detector

Schematic Representation of aFluorescence Detector

Refractive Index Detector or RI-Detector or Refractometer
In refractive index detector, light emitted from the source is
concentrated intothe cellcontaining the sample and reference sample.
The light passes through the cell and reaches the beam splitter that
diverts the light towards two photocells.
A change in the observed refractive index of the sample results in a
difference in their relative output, and this difference is amplified and
recorded. Sample

- Amplifier Recordcr

Referencc
sample
Block Diagram of a Refractive Index Detector
 Multipurpose Detector
Amultipurpose detector includes three detectors that are combined and
kept together in a single unit.
An example of this type of detector is Perkin -Elmer 3D System,
developed by Perkin-Elmer.
Thethree different detectors perform the following functions:
1. Fluorescence Function:They monitor emission above 280nm, based on
excitation at 254nm.
2. UV-Function: It is a fixed wavelength (254nm) detector.
3. Conductance-Function: The metal inlet and outlet tubes function like
electrodes that measure the conductance of ions.

Fluorescence photo cel

Filter

Source Lens Lens UV-photocell

Hg-lamp Flow cell
2.4 uL

Inlet Electrodes for Outlct

conductancc ccll

Block Diagram of Perkin-Elmer Detector

> Electrochemical Detectors
At the present time, amperometric detector is considered the best
electrochemical detector and it possesses the following distinguished
features:
1) Small internalcell-volume,
2) High sensitivity,
3) Limited range of applications, and
4) Best for trace analyses as UV-detector lacks adequate sensitivity.
Practically, it is difficult to utilise the functions of electro chemical
reduction as a mean of detection of HPLC.
 OStrip Chart Recorder
The signals emerging from the HPLC detector are continuously recorded as
function of time.
For these purposes generally, a potentiometric recorder is used.
The most efficient recorder is that which records 1 -10mV full -scale
deflection over a stretch of approximately ten inches and having a
response -time of one second or less.
" Therefore, the most preferred recorder is a strip -chart recorder with
variable chart speeds ranging between 5-5mm/min.
Afeedback signal arrangement (device) using a servomechanism is used to
balance the input signal of a potentiometric -recorder continuously.
With pre - adjusted attenuation a pen is attached with this device, which
moves proportionately along the width of the chart paper so that signals
can be accurately recorded.
OData Handling System
InHPLC, there is a tremendousdevelopment in the data handling devices
that ranges from a strip chart recorder, an electricintegrator, and a PC
-based workstation to aclient -server network system (the latest one).
The automation and sophistication has also advanced with the time.

Chart recorder PC workstation

Integrator

Ethernct
Terminal server

Clicnts

Networked interfaces Networked interfaces

Data handling network
Diagrammatic Presentation of Chromatography Data Handling System
 ADVANTAGES
HPLChas the following advantages:
1. It isa simple, rapid, and reproducible technique.
2. It is highly sensitive.
3. It shows a better performance.
4. It is a rapid process and is less time consuming.
5. Its resolution and separation capacity is high.
6. It is accurate and precise.
7. It utilises a chemically inert mobile and stationary phases.
8. It needs a small amount of mobile phase for developing chamber:
9. It involves early recovery of separated components.
10. It enables easy visualisation of separated components.
11. It shows a good reproducibility and repeatability.
12. It is useful in qualitative and quantitative analysis.
13. It is used for analytical and preparative purposes.
14. It is used forvalidation and quality control studies of product.
DISADVANTAGES
The disadvantages of HPLC focus on the detection systems available, and
include:

1. The most commonly used detectors in HPLC are UV spectrometers;

however, the compound to be analysed should have aUV absorbing
chromophore.
2. Variable wavelength UV spectrometers offer versatility but some steroids
and other drugs must be derivatized before UV detection.
3. Another slight disadvantage is that the chemicaly bonded stationary
phases applicable in drug analysis should be used with in 3 -7 pH range
toensure long term stability.
 APPILICATIONS
Following are some common applications of HPLC:
Stability Studies Stability of various pharmaceutical compounds,
degradation products ( e,g., stability studies of atropine), and other
chemical substances can be studied using the technique of HPLC.
> Bioassays and its Complementation HPLC is used in the bioassay test of
many complex molecules ( e.g., peptide hormones and antibiotic
molecules).
Design of Dosage Form Biopharmaceutics of the dosage form and the
pharmacokinetic properties of the drugs are studied with the help of
HPLC. These properties are involved in dosage form designing.
In Cosmetic Industry In this industry, HPLC is used for analysing the
quality of various cosmetic products such as lipsticks, gels, creams, etc.
> Isolation of Natural Pharmaceuticaly Active Compounds HPLC is the
most speific and sensitive method used for the separation of different
therapeutically active components present in plant extracts. HPLC
method is used in the isolation of different types of alkaloids and
glycosides.
Controlof Microbiological Processes HPLC is used to analyse antibiotics (
e.g, tetracyclines, chloramphenicol, streptomycin, and penicillins)
produced by various microbiological processes.
Assay of Cephalosporins Many derivatives of cephalosporin class of
antibiotics can be precisely separated by HPLC.
Assay of Furosemide The study of furosemide and its decomposition
products is performed using fluorescence and UV detection methods
during the HPLC analysis.
Assay of Corticosteroids The mixture of s0x corticosteroids
(hydrocortisone
tisone acetate, cortisone, deoxycortisone, hydrocortisone,
prednisolone, and prednisone)can be assayed by HPLC.