Chromatography Sirke

Introduction :-
Chromatography is non destructive procedure for resolving a multi-component mixture of trace,

minor, or major constituents into its individual fraction. While chromatography may be applied both
quantitatively, it is primarily a separation tool.
However final identification usually requires confirmation by some other analytical procedure such
as infrared spectroscopy, nuclear magnetic resonance or mass spectrometry. Chromatography can be
used for the quantitative and qualitative analysis.
Chromatography is relatively new technique which was first invented by M.T.swett , a
botanist 1906 in Warsaw . Few methods of analysis are truly specific to a particular analyte . It
is often found that the analyte of interest must be separated from the myriad of individual
compounds that may be present in a sample . As well as providing the analytical scientist with
methods of separation , chromatography techniques can also provide methods of analysis .
*Chromatography is used to separate mixtures of substances into their components. ... They all
have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a
gas).
*What is it?
Some materials appear homogenous, but are actually a combination of substances. For example, green
plants contain a mixture of different pigments. In addition, the black ink in the pens that are used in
this experiment is a mixture of different colored materials. In many instances, we can separate these
materials by dissolving them in an appropriate liquid and allowing them to move through an
absorbent matrix, like paper. organic and inorganic compounds so that they can be analyzed and
studied. By analyzing a compound, a scientist can figure out what makes up that compound.
Chromatography is a great physical method for observing mixtures and solvents. The word
chromatography means "color writing" which is a way that a chemist can test liquid mixtures. While
studying the coloring materials in plant life, a Russian botanist invented chromatography in 1903. His
name was M.S. Tswett. Chromatography is such an important technique that two nobel prizes have
been awarded to chromatographers. Over 60% of chemical analysis worldwide is currently done with
chromatography or a variation thereon. Chromatography is used in many different ways. Some people
use chromatography to find out what is in a solid or a liquid. It is also used to determine what
unknown substances are. The Police, used to determine what unknown substances are. The Police,
solve a crime. It is also used to determine the presence of cocaine in urine, alcohol in blood, PCB's in
Dr.Shri R.M.S Institute of Sci & Tech ,COP,Bhanpura Page 1

fish, and lead in water. Chromatography is used by many different people in many different ways.
Chromatography is based on differential migration. The solutes in a mobile phase go through a
stationary phase. Solutes with a phase than the solutes that prefer the stationary phase. As the solutes
move through the stationary phase they separate. This is called chromatographic development
*How it works
In all chromatography there is a mobile phase and a stationary phase. The stationary phase is the
phase that doesn't move and the mobile phase is the phase that does move. The mobile phase moves
through the stationary phase picking up the compounds to be tested. As the mobile phase continues to
travel through the stationary phase it takes the compounds with it. At different points in the stationary
phase the different components of the compound are going to be absorbed and are going to stop
moving with the mobile phase. This is how the results of any chromatography are gotten, from the
point at which the different components of the compound stop moving and separate from the other
components. In paper and thin-layer chromatography the mobile phase is the solvent. The stationary
phase in paper chromatography is the strip or piece of paper that is placed in the solvent. In thin-
layer chromatography the stationary phase is the thin-layer cell. Both these kinds of chromatography
use capillary action to move the solvent through the stationary phase.
*What is the Retention Factor ?
The retention factor, Rf is a quantitative indication of how far a particular compound travels in a
particular solvent. In order to make the technique more scientific rather than a mere interpretation by
sight, what is called the Retention Value (Rf value for short) was applied in chromatography. A
particular compound will travel the same distance along the stationary phase by a specific solvent (or
solvent mixture) given that other experimental conditions are kept constant. In other words, every
compound (dye, pigment, organic substance etc) have a specific Rf value for every specific solvent
and solvent concentration. Rf values come very handy for identification because one can compare Rf
values of the unknown sample (or its consituents) with Rf Values of known compounds.
The retention factor, Rf, is defined as ,

the distance moved by the solute (i.e. the dye or pigment under test) and the distance
moved by the the solvent (known as the Solvent front) along the paper, where both distances are
measured from the common Origin or Application Baseline, that is the point where the sample is
initially spotted on the paper.

TYPES OF CHROMATOGRAPHY TECHNIQUES
The following types are:
(1) Column Chromatography.
(2) Paper Chromatography.
(3) Thin Layer Chromatography.
(4) Gas Chromatography.
(5) High Performance Liquid Chromatography.
(6) Supercritical Fluid Chromatography.
(7) Affinity Chromatography.
(8) Two Dimensional Chromatography .
There are different kinds of chromatographic techniques and these are classified according to the
shape of bed, physical state of mobile phase, separation mechanisms.
Apart from these there are certain modified forms of these chromatographic techniques involving
different mechanisms and are hence categorized as modified or specialized chromatographic
techniques.

Type 1. Column Chromatography
In chemistry, Column chromatography is a technique which is used to separate a single chemical

compound from a mixture dissolved in a fluid. It separates substances based on differential
adsorption of compounds to the adsorbent as the compounds move through the column at different
rates which allow them to get separated in fractions. This technique can be used on small scale as
well as large scale to purify materials that can be used in future experiments. This method is a type
of adsorption chromatography technique.
Column Chromatography Principle :
When the mobile phase along with the mixture that needs to be separated is introduced from the top
of the column, the movement of the individual components of the mixture is at different rates. The
components with lower adsorption and affinity to stationary phase travel faster when compared to
the greater adsorption and affinity with the stationary phase. The components that move fast are
removed first whereas the components that move slow are eluted out last.
The adsorption of solute molecules to the column occurs in a reversible manner. The rate of the
movement of the components is expressed as:
Rf = the distance travelled by solute/ the distance travelled by solvent

Rf is the Retardation Factor

Column Chromatography Procedure :
Before starting with the Column Chromatography Experiment let us understand the different phases
involved.
Mobile phase – This phase is made up of solvents and it performs the following functions:
1. It acts as a solvent – sample mixture can be introduced in the column.

2. It acts as a developing agent – helps in the separation of components in the sample to form bands.
3. It acts as an eluting agent – the components that are separated during the experiment are removed
from the column
4. Some examples of solvents used as mobile phase based on their polarity are – ethanol, acetone, water,
acetic acid, pyridine, etc.
Stationary phase – It is a solid material which should have good adsorption property and
meet the conditions given below:
1. Shape and size of particle: Particles should have uniform shape and size in the range of 60 – 200μ
in diameter.

2. Stability and inertness of particles: high mechanical stability and chemically inert. Also, no
reaction with acids or bases or any other solvents used during the experiment.
3. It should be colourless, inexpensive and readily available.
4. Should allow free flow of mobile phase
5. It should be suitable for the separation of mixtures of various compounds.
Column Chromatography Experiment :
 The stationary phase is made wet with the help of solvent as the upper level of the mobile phase and
the stationary phase should match. The mobile phase or eluent is either solvent or mixture of solvents.
In the first step the compound mixture that needs to be separated, is added from the top of the column
without disturbing the top level. The tap is turned on and the adsorption process on the surface of
silica begins.
 Without disturbing the stationary phase solvent mixture is added slowly by touching the sides of the
glass column. The solvent is added throughout the experiment as per the requirement.
 The tap is turned on to initiate the movement of compounds in the mixture. The movement is based
on the polarity of molecules in the sample. The non-polar components move at a greater speed when
compared to the polar components.
 For example, a compound mixture consists of three different compounds viz red, blue, green then
their order based on polarity will be as follows blue>red>green
 As the +
 of the green compound is less, it will move first. When it arrives at the end of the column it is collected
in a clean test tube. After this, the red compound is collected and at last blue compound is collected. All
these are collected in separate test tubes.
Column Chromatography Applications :
 Column Chromatography is used to isolate active ingredients.
 It is very helpful in Separating compound mixtures.
 It is used to determine drug estimation from drug formulations
 It is used to remove impurities.
 Used to isolation metabolites from biological fluids.
Types of Column Chromatography:

1. Adsorption column chromatography – Adsorption chromatography is a technique of separation, in
which the components of the mixture are adsorbed on the surface of the adsorbent.

2. Partition column chromatography – The stationary phase, as well as mobile phase, are liquid
in partition chromatography.
3. Gel column chromatography – In this method of chromatography, the separation takes place
through a column packed with gel. The stationary phase is a solvent held in the gap of a solvent.
4. Ion exchange column chromatography – A chromatography technique in which the stationary
phase is always ion exchange resin.
Type 2. Paper Chromatography
Chromatography technique that uses paper sheets or strips as the adsorbent being the stationary
phase through which a solution is made to pass is called paper chromatography. It is an inexpensive
method of separating dissolved chemical substances by their different migration rates across the
sheets of paper. It is a powerful analytical tool that uses very small quantities of material. Paper
chromatography was discovered by Synge and Martin in the year 1943.

Paper Chromatography Principle :
The principle involved can be partition chromatography or adsorption chromatography. Partition

chromatography because the substances are partitioned or distributed between liquid phases. The two
phases are water held in pores of the filter paper and the other phase is a mobile phase which passes
through the paper. When the mobile phase moves, the separation of mixture takes place. The
compounds in the mixture separate themselves based on the differences in their affinity towards
stationary and mobile phase solvents under the capillary action of pores in the paper. Adsorption
chromatography between solid and liquid phases, wherein the solid surface of the paper is the
stationary phase and the liquid phase is the mobile phase.
Paper Chromatography Procedure:

Below we have explained the procedure to conduct Paper Chromatography Experiment for easy
understanding of students.
1. Selecting a suitable type of development: It is decided based on the complexity of the solvent,
paper, mixture, etc. Usually ascending type or radial paper chromatography is used as they are easy to
perform. Also, it is easy to handle, the chromatogram obtained is faster and the process is less time-
consuming.
2. Selecting a suitable filter paper: Selection of filter paper is done based on the size of the pores,
and the sample quality.
3. Prepare the sample: Sample preparation includes the dissolution of the sample in a suitable
solvent (inert with the sample under analysis) used in making the mobile phase.
4. Spot the sample on the paper: Samples should be spotted at a proper position on the paper by
using a capillary tube.
5. Chromatogram development: Chromatogram development is spotted by immersing the paper in
the mobile phase. Due to the capillary action of paper, the mobile phase moves over the sample on the
paper.
6. Paper drying and compound detection: Once the chromatogram is developed, the paper is dried
using an air drier. Also, detecting solution can be sprayed on the chromatogram developed paper and
dried to identify the sample chromatogram spots.
E Paper Chromatography Applications :

There are various applications of paper chromatography. Some of the uses of Paper Chromatography
in different fields are discussed below:
 To study the process of fermentation and ripening.
 To check the purity of pharmaceuticals.
 To inspect cosmetics.
 To detect the adulterants.
 To detect the contaminants in drinks and foods.
 To examine the reaction mixtures in biochemical laboratories.
 To determine dopes and drugs in humans and animals.
Types of paper chromatography:
1. Ascending Paper Chromatography – The techniques goes with its name as the solvent moves in an
upward direction.
2. Descending Paper Chromatography – The movement of the flow of solvent due to gravitational pull
and capillary action is downwards hence the name descending paper chromatography.

3. Ascending – Descending Paper Chromatography – In this version of paper chromatography
movement of solvent occurs in two directions after a particular point. Initially, the solvent travels
upwards on the paper which is folded over a rod and after crossing the rod it continues with its travel
in the downward direction.
4. Radial or Circular Paper Chromatography – The sample is deposited at the center of the circular
filter paper. Once the spot is dried, the filter paper is tied horizontally on a Petri dish which contains
the solvent.
5. Two Dimensional Paper Chromatography – Substances which have the same r f values can be
resolved with the help of two-dimensional paper chromatography.
Type 3. Thin Layer Chromatography
Thin Layer Chromatography is a technique used to isolate non-volatile mixtures. The experiment is
conducted on a sheet of aluminium foil, plastic, or glass which is coated with a thin layer of adsorbent
material. The material usually used is aluminium oxide, cellulose, or silica gel.

On completion of the separation, each component appears as spots separated vertically. Each spot has a
retention factor (Rf) expressed as:
Rf = dist. travelled by sample / dist. travelled by solvent
The factors affecting retardation factor are the solvent system, amount of material spotted, absorbent and
temperature. TLC is one of the fastest, least expensive, simplest and easiest chromatography technique.
Thin Layer Chromatography Principle

Like other chromatographic techniques, thin layer chromatography (TLC) depends on the separation
principle. The separation relies on the relative affinity of compounds towards both the phases. The
compounds in the mobile phase move over the surface of the stationary phase. The movement occurs in
such a way that the compounds which have a higher affinity to the stationary phase move slowly while
the other compounds travel fast. Therefore, the separation of the mixture is attained. On completion of
the separation process, the individual components from the mixture appear as spots at respective levels
on the plates. Their character and nature are identified by suitable detection techniques.
Thin Layer Chromatography Procedure

Before starting with the Thin Layer Chromatography Experiment let us understand the different
components required to conduct the procedure along with the phases involved.
1. Thin Layer Chromatography Plates – ready-made plates are used which are chemically inert and
stable. The stationary phase is applied on its surface in the form of a thin layer. The stationary phase on
the plate has a fine particle size and also has a uniform thickness.

2. Thin Layer Chromatography Chamber – Chamber is used to develop plates. It is responsible to keep
a steady environment inside which will help in developing spots. Also, it prevents the solvent evaporation
and keeps the entire process dust-free.
3. Thin Layer Chromatography Mobile phase – Mobile phase is the one that moves and consists of a
solvent mixture or a solvent. This phase should be particulate-free. The higher the quality of purity the
development of spots is better.
4. Thin Layer Chromatography Filter Paper – It has to be placed inside the chamber. It is moistened in
the mobile phase.
Thin Layer Chromatography Experiment

The stationary phase that is applied to the plate is made to dry and stabilize.
 To apply sample spots, thin marks are made at the bottom of the plate with the help of a pencil.
 Apply sample solutions to the marked spots.
 Pour the mobile phase into the TLC chamber and to maintain equal humidity, place a moistened filter paper
in the mobile phase.
 Place the plate in the TLC chamber and close it with a lid. It is kept in such a way that the sample faces the
mobile phase.
 Immerse the plate for development. Remember to keep the sample spots well above the level of the mobile
phase. Do not immerse it in the solvent.
 Wait till the development of spots. Once the spots are developed, take out the plates and dry them. The
sample spots can be observed under a UV light chamber.
Thin Layer Chromatography Applications
 The qualitative testing of Various medicines such as sedatives, local anaesthetics, anticonvulsant
tranquilisers, analgesics, antihistamines, steroids, hypnotics is done by TLC.
 TLC is extremely useful in Biochemical analysis such as separation or isolation of biochemical

metabolites from its blood plasma, urine, body fluids, serum, etc.
 Thin layer chromatography can be used to identify natural products like essential oils or volatile oil, fixed
oil, glycosides, waxes, alkaloids, etc
 It is widely used in separating multicomponent pharmaceutical formulations.
 It is used to purify of any sample and direct comparison is done between the sample and the authentic
sample
 It is used in the food industry, to separate and identify colours, sweetening agent, and preservatives
 It is used in the cosmetic industry.

 It is used to study if a reaction is complete.
Disadvantages Of Thin Layer Chromatography:
1. Thin Layer Chromatography plates do not have longer stationary phase.

2. When compared to other chromatographic techniques the length of separation is limited.
3. The results generated from TLC are difficult to reproduce.
4. Since TLC operates as an open system, some factors such as humidity and temperature can be
consequences to the final outcome of the chromatogram.
5. The detection limit is high and therefore if you want a lower detection limit, you cannot use TLC.
6. It is only a qualitative analysis technique and not quantitative.
Type 4. Gas-Liquid Chromatography (GLC) or Simply Gas Chromatography

(GC)

It is a type of chromatography in which the mobile phase is a carrier gas, usually an inert gas such as helium
or an unreactive gas such as nitrogen, and the stationary phase is a microscopic layer of liquid or polymer
on an inert solid support, inside glass or metal tubing, called a column. The instrument used to perform gas
chromatographic separations is called a gas chromatograph (also: aerograph, gas separator).
Fig. Gas Chromatography

A gas chromatograph is a chemical analysis instrument for separating chemicals in a complex sample. A gas
chromatograph uses a flow-through narrow tube known as the column, through which different chemical
constituents of a sample pass in a gas stream (carrier gas, mobile phase) at different rates depending on their
various chemical and physical properties and their interaction with a specific column filling, called the
stationary phase.
As the chemicals exit the end of the column, they are detected and identified electronically. The function of
the stationary phase in the column is to separate different components, causing each one to exit the column at
a different time (retention time). Other parameters that can be used to alter the order or time of retention are
the carrier gas flow rate, and the temperature.
In a GC analysis, a known volume of gaseous or liquid analyte is injected into the “entrance” (head) of the
column, usually using a micro syringe (or, solid phase micro-extraction fibers, or a gas source switching
system). As the carrier gas sweeps the analyte molecules through the column, this motion is inhibited by the
adsorption of the analyte molecules either onto the column walls or onto packing materials in the column.
The rate at which the molecules progress along the column depends on the strength of adsorption, which in
turn depends on the type of molecule and on the stationary phase materials. Since each type of molecule has
a different rate of progression, the various components of the analyte mixture are separated as they progress
along the column and reach the end of the column at different times (retention time).
A detector is used to monitor the outlet stream from the column; thus, the time at which each component
reaches the outlet and the amount of that component can be determined. Generally, substances are
identified (qualitatively) by the order in which they emerge (elute) from the column and by the retention
time of the analyte in the column.

Auto Samplers:
The auto sampler provides the means to introduce automatically a sample into the inlets. Manual insertion
of the sample is possible but very rare nowadays. Automatic insertion provides better reproducibility and
time-optimization.
i. Liquid
ii. Static head-space by syringe technology
iii. Dynamic head-space by transfer-line technology
iv. Solid phase micro extraction (SPME).
Inlets:
The column inlet (or injector) provides the means to introduce a sample into a continuous flow of carrier
gas. The inlet is a piece of hardware attached to the column head.
Common inlet types are:

1. S/SL (Split/Split less) injector
2. On-column inlet
3. PTV injector
4. Gas source inlet or gas switching valve
5. P/T (Purge-and-Trap) system
6. SPME (solid phase micro extraction)
Columns:
Two types of columns are used in GC:
1. Packed columns are 1.5-10 m in length and have an internal diameter of 2-4 mm. The tubing is usually
made of stainless steel or glass and contains a packing of finely divided, inert, solid support material (e.g.,
diatomaceous earth) that is coated with a liquid or solid stationary phase. The nature of the coating material
determines what type of materials will be most strongly adsorbed. Thus numerous columns are available
that are designed to separate specific types of compounds.
2. Capillary columns have a very small internal diameter, on the order of a few tenths of millimeters, and
lengths between 25-60 meters are common. The inner column walls are coated with the active materials
(WCOT columns), some columns are quasi-solid filled with many parallel microspores (PLOT columns).
Most capillary columns are made of fused-silica with a polyimide outer coating. These columns are
flexible, so a very long column can be wound into a small coil.
Among these new developments are:
(i) Internally heated micro FAST columns, where two columns, an internal heating wire and a temperature
sensor are combined within a common column sheath (micro FAST);

(ii) Micro packed columns (1/16″ OD) are column-in-column packed columns where the outer column
space has a packing different from the inner column space, thus providing the separation behavior of two
columns in one. They can be easily fit to inlets and detectors of a capillary column instrument.
For some cases, temperature is ramped either continuously or in steps to provide the desired separation.
This is referred to as a temperature program. Electronic pressure control can also be used to modify flow
rate during the analysis, aiding in faster run times while keeping acceptable levels of separation.
Detectors:
A number of detectors are used in gas chromatography. The most common are the flame ionization
detector (FID) and the thermal conductivity detector (TCD). Both are sensitive to a wide range of
components, and both work over a wide range of concentrations.
They include:
i. Discharge ionization detector (DID)
ii. Electron capture detector (ECD)
iii. Flame photometric detector (FPD)
iv. Hall electrolytic conductivity detector (E1CD)
v. Helium ionization detector (HID)
vi. Nitrogen phosphorus detector (NPD)
vii. Mass selective detector (MSD)
viii. Photo-ionization detector (PID)
ix. Pulsed discharge ionization detector (PDD)
Some gas chromatographs are connected to a mass spectrometer which acts as the detector. The
combination is known as GC-MS. Some GC-MS are connected to an Nuclear Magnetic Resonance
Spectrometer which acts as a back-up detector. This combination is known as GC-MS- NMR. Some GC-
MS-NMR are connected to an infrared spectra which acts as a back-up detector. This combination is
known as GC-MS-NMR-IR. It must, however, be stressed that this is very rare as most analysis needed can
be concluded via purely GC-MS.
Methods:
The method is the collection of conditions in which the GC operates for a given analysis. Method
development is the process of determining what conditions are adequate and/or ideal for the analysis
required. Conditions which can be varied to accommodate a required analysis include inlet temperature,
detector temperature, column temperature and temperature program, carrier gas and carrier gas flow rates,
the column’s stationary phase, diameter and length, inlet type and flow rates, sample size and injection
technique.

Depending on the detector(s) (see below) installed on the GC, there may be a number of detector
conditions that can also be varied. Some GCs also include valves which can change the route of sample and
carrier flow, and the timing of the turning of these valves can be important to method development.
Carrier Gas Selection and Flow Rates:
Typical carrier gases include helium, nitrogen, argon, hydrogen and air. Which gas to use is usually
determined by the detector being used, for example, a DID requires helium as the carrier gas. When
analyzing gas samples, however, the carrier is sometimes selected based on the sample’s matrix, for
example, when analyzing a mixture in argon, an argon carrier is preferred, because the argon in the sample
does not show upon the chromatogram. Safety and availability can also influence carrier selection, for
example, hydrogen is flammable, and high-purity helium can be difficult to obtain in some areas of the
world.
The purity of the carrier gas is also frequently determined by the detector, though the level of sensitivity
needed can also play a significant role. Typically, purities of 99.995% or higher are used. Trade names for
typical purities include “Zero Grade”, “Ultra-High Purity (UHP) Grade”, “4.5 Grade” and “5.0 Grade”.
The carrier gas flow rate affects the analysis in the same way that temperature does (see above). The higher
the flow rates the faster the analysis, but the lower the separation between analytes. Selecting the flow rate
is, therefore, the same compromise between the level of separation and length of analysis as selecting the
column temperature.
Inlet Types and Flow Rates:
The choice of inlet type and injection technique depends on if the sample is in liquid, gas, adsorbed, or solid
form, and on whether a solvent matrix is present that has to be vaporized. Dissolved samples can be
introduced directly onto the column via a COC injector, if the conditions are well known; if a solvent
matrix has to be vaporized and partially removed, a S/SL injector is used (most common injection
technique); gaseous samples (e.g., air cylinders) are usually injected using a gas switching valve system;
adsorbed samples (e.g., on adsorbent tubes) are introduced using either an external (on-line or off-line)
desorption apparatus such as a purge-and-trap system, or are desorbed in the S/SL injector (SPME
applications).
Sample size and injection technique:

The real chromatographic analysis starts with the introduction of the sample onto the column. The
development of capillary gas chromatography resulted in many practical problems with the injection
technique. The technique of on-column injection, often used with packed columns, is usually not possible
with capillary columns.

The injection system, in the capillary gas chromatograph, should fulfill the following two
requirements:
1. The amount injected should not overload the column.

2. The width of the injected plug should be small compared to the spreading due to the chromatographic
process. Failure to comply with this requirement will reduce the separation capability of the column. As a
general rule, the volume injected, Vinj, and the volume of the detecor cell, V det, should be about 1/10 of the
volume occupied by the portion of sample containing the molecules of interest (analytes) when they exit the
column.
Some general requirements, which a good injection technique should fulfill, are:
(i) It should be possible to obtain the column’s optimum separation efficiency.
(ii) It should allow accurate and reproducible injections of small amounts of representative samples.
(iii) It should induce no change in sample composition. It should not exhibit discrimination based on
differences in boiling point, polarity, concentration or thermal/catalytic stability.
(iv) It should be applicable for trace analysis as well as for undiluted samples.
Data Reduction and Analysis:
Qualitative analysis: Generally chromatographic data is presented as a graph of detector response (y-axis)
against retention time (x-axis). This provides a spectrum of peaks for a sample representing the analytes
present in a sample eluting from the column at different times. Retention time can be used to identify
analytes if the method conditions are constant.
Also, the pattern of peaks will be constant for a sample under constant conditions and can identify complex
mixtures of analytes. In most modern applications, however, the GC is connected to a mass spectrometer or
similar detector that is capable of identifying the analytes represented by the peaks.
Quantitive analysis: The area under a peak is proportional to the amount of analyte present. By calculating
the area of the peak using the mathematical function of integration, the concentration of an analyte in the
original sample can be determined. Concentration can be calculated using a calibration curve created by
finding the response for a series of concentrations of analyte, or by determining the response factor of an
analyte.

Application:
The major success of the application of GC in pharmaceutical quantitative analysis is firstly due to the very
high efficiencies of separation power, secondly to the extreme sensitivity of the detection of even very small
amounts of separated species and finally to the precision and accuracy of the data from quantitative analyses
of very complex mixtures. GC analyses are also easy to automate from sample introduction to separation.
Because of the above main advantages and its short analysis time and reliable results GC is used as quality
control purposes in the pharmaceutical industry. In fact pharmaceutical analysis generally involves two
steps; separation of the compound of interest and quantitation of the compounds. The better the separation
the easier the quantitation. GC detectors have different responses to each compound. In order to determine
quantitative amounts of various compounds in a separation the detector must be calibrated using standards.
Standard solutions of sample are injected and the detector response recorded. Comparison of the standard
and sample retention times allows qualitative analysis of the sample. Comparison of the peak area of the
standards with that of the sample allows quantitation of analyte. Due to this fact, GC is widely used as a
routine analytical technique in pharmaceutical quantitative analysis mostly used in for the determination of
organic volatile impurities and nicotine level during drugs formulation.
 Checking the purity of compound.

 Presence of impurity.
 Quantitative analysis.
 Multicomponant analysis or determination of mixture of drug.
 Isolation and identification of drug.
Limitations Associated with Gas Chromatography:

In fact, a GC analysis takes much more time; sometimes a single sample must be run more than an hour
according to the chosen program; and even more time is needed to “heat out” the column; so it is free from
the first sample and can be used for the next. Equally, several runs are needed to confirm the results of a
study—a GC analysis of a single sample may simply yield a result per chance.
Also, GC does not positively identify most samples; and not all substances in a sample will necessarily be
detected. All a GC truly tells is at which relative time a component eluted from the column and that the
detector was sensitive to it.
To make results meaningful, analysts need to know which components at which concentrations are to be
expected; and even then a small amount of a substance can hide itself behind a substance having both a
higher concentration and the same relative elution time. Last but not least it is often needed to check the
results of the sample against a GC analysis of a reference sample containing only the suspected substance.
A GC-MS can remove much of this ambiguity, since the mass spectrometer will identify the component’s
molecular weight. But this still takes time and skill to do properly.

Type 5. High Performance Liquid Chromatography
High-performance liquid chromatography (HPLC) is a form of column chromatography used frequently in
biochemistry and analytical chemistry. It is also sometimes referred to as high-pressure liquid
chromatography. HPLC is used to separate components of a mixture by using a variety of chemical
interactions between the substance being analyzed (analyte) and the chromatography column.

In isocratic HPLC, the analyte is forced through a column of the stationary phase (usually a tube packed
with small round particles with a certain surface chemistry) by pumping a liquid (mobile phase) at high
pressure through the column. The sample to be analyzed is introduced in a small volume to the stream of
mobile phase and is retarded by specific chemical or physical interactions with the stationary phase as it
traverses the length of the column.
The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase
composition. The time at which a specific analyte elutes (comes out of the end of the column) is called the
retention time and is considered a reasonably unique identifying characteristic of a given analyte. The use
of pressure increases the linear velocity (speed) giving the components less time to diffuse within the
column, leading to improved resolution in the resulting chromatogram.
Common solvents used include any miscible combinations of water or various organic liquids (the most
common are methanol and acetonitrile). Water may contain buffers or salts to assist in the separation of the
analyte components, or compounds such as Trifluoroacetic acid which acts as an ion pairing agent.
A further refinement to HPLC has been to vary the mobile phase composition during the analysis; this is
known as gradient elution. A normal gradient for reverse phase chromatography might start at 5% methanol
and progress linearly to 50% methanol over 25 minutes, depending on how hydrophobic the analyte is.
The choice of solvents, additives and gradient, depends on the nature of the stationary phase and the
analyte. Often a series of tests are performed on the analyte and a number of generic runs may be processed
in order to find the optimum HPLC method for the analyte — the method which gives the best separation of
peaks.
Instrumentation of HPLC :
 Solvent delivery system (mobile phase).
 Pumps (a) Reciprocating piston pump.
(b)Syringe type pump.
(c) Constant pressure pump.
 Injector.
 Column.
 Detector.
 Data processing unit (computer).
Types of HPLC:
(1.)Normal phase chromatography:

Normal phase HPLC (NP-HPLC) was the first kind of HPLC chemistry used, and separates analytes based
on polarity. This method uses a polar stationary phase and a non-polar mobile phase, and is used when the
analyte of interest is fairly polar in nature. The polar analyte associates with and is retained by the polar
stationary phase.
Adsorption strengths increase with increase in analyte polarity, and the interaction between the polar
analyte and the polar stationary phase (relative to the mobile phase) increases the elution time. The
interaction strength not only depends on the functional groups in the analyte molecule, but also on steric
factors, and structural isomers are often resolved from one another.
Use of more polar solvents in the mobile phase will decrease the retention time of the analytes while more
hydrophobic solvents tend to increase retention times. Particularly polar solvents in a mixture tend to
deactivate the column by occupying the stationary phase surface. This is somewhat particular to normal
phase because it is most purely an adsorptive mechanism (the interactions are with a hard surface rather
than a soft layer on a surface).
NP-HPLC had fallen out of favour in the 1970s with the development of reversed-phase HPLC because of a
lack of reproducibility of retention times as water or protic organic solvents changed the hydration state of
the silica or alumina chromatographic media. Recently it has become useful again with the development of
HILIC bonded phases which utilize a partition mechanism which provides reproducibility.
(2.) Reversed phase chromatography:

Reversed phase HPLC (RP-HPLC) consists of a non-polar stationary phase and a moderately polar
mobile phase. One common stationary phase is a silica which has been treated with RMe 2SiCl, where R
is a straight chain alkyl group such as C 18H37 or C8H17. The retention time is, therefore, longer for
molecules which are more non-polar in nature, allowing polar molecules to elute more readily.
Retention time is increased by the addition of polar solvent to the mobile phase and decreased by the
addition of more hydrophobic solvent. Reversed phase chromatography is so commonly used that it is
not uncommon for it to be incorrectly referred to as “HPLC” without further specification.
RP-HPLC operates on the principle of hydrophobic interactions which result from repulsive forces
between a relatively polar solvent, the relatively non-polar analyte, and the non-polar stationary phase.
The driving force in the binding of the analyte to the stationary phase is the decrease in the area of the
non-polar segment of the analyte molecule exposed to the solvent.
This hydrophobic effect is dominated by the decrease in free energy from entropy associated with the
minimization of the ordered molecule-polar solvent interface. The hydrophobic effect is decreased by
adding more non-polar solvent into the mobile phase. This shifts the partition coefficient such that the
analyte spends some portion of time moving down the column in the mobile phase, eventually eluting
from the column.

The characteristics of the analyte molecule play an important role in its retention characteristics. In
general, an analyte with a longer alkyl chain length results in a longer retention time because it increases
the molecule’s hydrophobicity.
Very large molecules, however, can result in incomplete interaction between the large analyte surface
and the alkyl chain. Retention time increases with hydrophobic surface area which is roughly inversely
proportional to solute size. Branched chain compounds elute more rapidly than their corresponding
isomers because the overall surface area is decreased.
Another important component is pH since this can change the hydrophobicity of the analyte. For this
reason most methods use a buffering agent, such as sodium phosphate, to control the pH. An organic
acid such as formic acid or most commonly trifluoro-acetic acid is often added to the mobile phase.
These serve multiple purposes: they control pH, neutralize the charge on any residual exposed silica on
the stationary phase and act as ion pairing agents to neutralize charge on the analyte. The effect varies
depending on use but generally improves the chromatography.
Reversed phase columns are quite difficult to damage compared with normal silica columns; however,
many reverse phase columns consist of alkyl derivatized silica particles and should never be used with
aqueous bases as these will destroy the underlying silica backbone. They can be used with aqueous acid
but the column should not be exposed to the acid for too long, as it can corrode the metal parts of the
HPLC equipment.
Reverse-phase chromatography (RPC) includes any chromatographic method that uses a non-polar stationary
phase. All of the mathematical and experimental considerations used in other chromatographic methods
apply (i.e., separation resolution proportional to the column length and inversely proportional to the column
width).
Reversed-phase column chromatography is widely used in the pharmaceutical, chemical, and biochemical
industry for separating molecules of small molecular weight. In more recent years RPC has been used to
separate larger molecules. Any inert non-polar substance that achieves sufficient packing can be used for
reversed-phase chromatography. Common examples include larger hydrocarbons, diphenyl, and
divinylbenzene.
Typically mobile phases are made of water or a polar organic compound such as acetonitrile or the lighter
alcohols. Buffer solutions are used to control the protonation of functional groups. For example, a buffer of
high pH will encourage elution of alcohols because they are charged in basic solutions and, therefore, prefer
to be in the mobile phase. For the same reason, very acidic solutions encourage the elution of nitrogen
containing molecules. Using multiple buffers allows for selective elution of a wide variety of chemicals.
Reversed-phase high performance liquid chromatography (RP-HPLC) has become a powerful tool for the
analysis of peptides and proteins for the following reasons:

 Tested stability of the matrix under a variety of mobile phase scenarios
 Reproducibility of separations
 Excellent resolutions achievable for molecules of interest (either closely related or structurally
distinct)
 Appreciable recoveries and throughput
 Ease of separations provided by gradient elution
(3.) Size exclusion chromatography:

Size exclusion chromatography (SEC) is a chromatographic method in which particles are separated based
on their size, or in more technical terms, their hydrodynamic volume. It is usually applied to large
molecules or macromolecular complexes such as proteins and industrial polymers. When an aqueous
solution is used to transport the sample through the column, the technique is known as gel filtration
chromatography.
The name gel permeation chromatography is used when an organic solvent is used as a mobile phase. The
main application of gel filtration chromatography is the fractionation of proteins and other water-soluble
polymers, while gel permeation chromatography is used to analyze the molecular weight distribution of
organic-soluble polymers. Either technique should not be confused with gel electrophoresis, where an
electric field is used to “pull” or “push” molecules through the gel depending on their electrical charges.
SEC is a widely used technique for the purification and analysis of synthetic and biological polymers,
such as proteins, polysaccharides and nucleic acids. Biologists and biochemists typically use a gel
medium—usually polyacrylamide, dextran or agarose—and filter under low pressure. Polymer chemists
typically use either a silica or cross-linked polystyrene medium under a higher pressure. These media are
known as the stationary phase.
The advantage of this method is that the various solutions can be applied without interfering with the
filtration process, while preserving the biological activity of the particles to be separated. The technique
is generally combined with others that further separate molecules by other characteristics, such as
acidity, basicity, charge, and affinity for certain compounds.
The underlying principle of SEC is that particles of different sizes will elute (filter) through a stationary
phase at different rates. This result in the separation of a solution of particles based on size, provided
that all the particles are loaded simultaneously or near simultaneously, particles of the same size should
elute together.
Applications:
1. Proteomics :
SEC can also assay protein tertiary structure as it measures the hydrodynamic volume (not molecular
weight), allowing folded and unfolded versions of the same protein to be distinguished. For example,
the apparent hydrodynamic radius of a typical protein domain might be 14a and 36A for the folded
and unfolded forms respectively.
SEC allows the separation of these two forms as the folded form will elute much later due to its
smaller size. Alternatively, folded and unfolded versions of the same metalloproteinase can be
separated according to their different isoelectric points by using quantitative preparative native
continuous polyacrylamide gel electrophoresis (QPNC-PAGE).
2. Polymer synthesis:
SEC can be used as a measure of both the size and the polydispersity of a synthesised polymer; that one
is able to find distribution of sizes of polymer molecules. If standards of a known size are run
previously, then a calibration curve can be created to determine the sizes of polymer molecules of
interest.
Alternatively, techniques such as light scattering and/or viscometry can be used online with SEC to
yield absolute molecular weights that do not rely on calibration with standards of known molecular
weight. Due to the difference in size of two polymers with identical molecular weights, the absolute
determination methods are generally more desirable. A typical SEC system can quickly (in about half an
hour) give polymer chemists information on the size and polydispersity of the sample.
(4.) Ion exchange chromatography:

Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of ions and
polar molecules based on the charge properties of the molecules. It can be used for almost any kind of

charged molecule including large proteins, small nucleotides and amino acids, with the experimental
solution to be separated collectively known as the analyte. It is often used as a first step in protein
purification. Ion exchange chromatography retains analyte molecules based on coulombic (ionic) interac-
tions. The stationary phase surface displays ionic functional groups that interact with analyte ions of
opposite charge.
This type of chromatography is further subdivided into cation exchange chromatography and
anion exchange chromatography:
i. Cation exchange chromatography retains positively charged cations because the stationary phase
displays a negatively charged functional group such as a phosphoric acid.
ii. Anion exchange chromatography retains negatively charged anions using positively charged functional
group such as a quaternary ammonium cation.
Proteins have numerous functional groups that can have both positive and negative charges. Ion exchange
chromatography separates proteins according to their net charge, which is dependent on the composition
of the mobile phase. By adjusting the pH or the ionic concentration of the mobile phase, various protein
molecules can be separated.
For example, if a protein has a net positive charge at pH 7, then it will bind to a column of negatively-
charged beads, whereas a negatively charged protein would not. By changing the pH so that the net
charge on the protein is negative, it too will be eluted.
Elution by changing the ionic strength of the mobile phase is a more subtle effect — it works as ion from
the mobile phase and will interact with the immobilized ion in preference over those on the stationary
phase.
This “shields” the stationary phase from the protein (and vice versa) and allows the protein to elute.
A sample is introduced, either manually or with an auto sampler, into a sample loop of known volume.
A buffered aqueous solution known as the mobile phase carries the sample from the loop onto a column
that contains some form of stationary phase material. This is typically a resin or gel matrix consisting of
agarose or cellulose beads with covalently bonded charged functional groups.
The target analytes (anions or cations) are retained on the stationary phase but can be eluted by
increasing the concentration of a similarly charged species that will displace the analyte ions from the
stationary phase. For example, in cation exchange chromatography, the positively charged analyte could
be displaced by the addition of positively charged sodium ions.
The analytes of interest must then be detected by some means, typically by conductivity or UV/Visible
light absorbance. In order to control an IC system, a Chromatography Data System (CDS) is usually
needed. In addition to IC systems, some of these CDSs can also control Gas Chromatography (GC) and
HPLC systems.

(5. ) Bio-affinity chromatography:
This chromatographic process relies on the property of biologically active substances to form stable,
specific, and reversible complexes. The formation of these complexes involves the participation of
common molecular forces such as the van der Waals interaction, electrostatic interaction, dipole- dipole
interaction, hydrophobic interaction, and the hydrogen bond. An efficient, bio-specific bond is formed
by a simultaneous and concerted action of several of these forces in the complementary binding sites.
Parameters:
There are different parameters upon which the separation by HPLC relies.
Some of the important parameters are discussed below:
1. Internal diameter:
The internal diameter (ID) of an HPLC column is a critical aspect that determines quantity of analyte
that can be loaded onto the column and also influences sensitivity. Larger columns are usually seen in
industrial applications such as the purification of a drug product for later use. Low ID columns have
improved sensitivity and lower solvent consumption at the expense of loading capacity.
i. Larger ID columns (over 10 mm) are used to purify usable amounts of material because of their large
loading capacity.

ii. Analytical scale columns (4.6 mm) have been the most common type of columns, though smaller
columns are rapidly gaining popularity. They are used in traditional quantitative analysis of samples
and often use a UV-Vis absorbance detector.
iii. Narrow-bore columns (1-2 mm) are used for applications when more sensitivity is desired either
with special UV-Vis detectors, fluorescence detection or with other detection methods like liquid
chromatography-mass spectrometry
iv. Capillary columns (under 0.3 mm) which are used almost exclusively with alternative detection
means such as mass spectrometry. They are usually made from fused silica capillaries, rather than the
stainless steel tubing that larger columns employ.
2. Particle size:
Most traditional HPLC is performed with the stationary phase attached to the outside of small spherical
silica particles (very small beads). These particles come in a variety of sizes with 5 µm beads being the
most common. Smaller particles generally provide more surface area and better separations, but the
pressure required for optimum linear velocity increases by the inverse of the particle diameter squared.
This means that changing to particles that are half as big in the same size of column will double the
performance, but increase the required pressure by a factor of four. Larger particles are more often used
in non-HPLC applications such as solid-phase extraction.
3. Pore size:
Many stationary phases are porous to provide greater surface area. Small pores provide greater surface
area while larger pore size has better kinetics especially for larger analytes. For example, a protein
which is only slightly smaller than a pore might enter the pore but not easily leave once inside.
4. Pump pressure:
Pumps vary in pressure capacity, but their performance is measured on their ability to yield a consistent
and reproducible flow rate. Pressure may reach as high as 6000 lbf/in 2 (~40 MPa, or about 400
atmospheres). Modern HPLC systems have been improved to work at much higher pressures, and
therefore, be able to use much smaller particle sizes in the columns (< 2 micrometres).
These “Ultra High Performance Liquid Chromatography” systems or UHPLCs can work at up to 15,000
lbf/in2 (-100 MPa or about 1000 atmospheres). Note that the term “UPLC”, sometimes found instead is
a trademark of Waters Corporation and not the name for the technique in general.
ADVANTAGES of HPLC :
 Speed.
 High Resolution .
 Accuracy.
 Automation.
 Higher resolution and speed of analysis.

 HPLC column can be reused without repacking of regeneration.
 Greater reproducibility due to close control of the parameters affecting the efficiency of
separation.
 Easy automation of instrument operation and data analysis.
 Adapatability of large scale preparative procedures.
 Accurate quantitative measurement.
DISADVANTAGES of HPLC :
 Cost.
 Complexity.
Type 6. Supercritical Fluid Chromatography (SFC)

Supercritical fluid chromatography (SFC) is a form of normal phase chromatography that uses
a supercritical fluid such as carbon dioxide as the mobile phase.[1][2] It is used for the analysis and
purification of low to moderate molecular weight, thermally labile molecules and can also be used for the
separation of chiral compounds. Principles are similar to those of high performance liquid
chromatography (HPLC), however SFC typically utilizes carbon dioxide as the mobile phase; therefore
the entire chromatographic flow path must be pressurized. Because the supercritical phase represents a
state in which liquid and gas properties converge, supercritical fluid chromatography is sometimes called
convergence chromatography.
It is a robust and easy-to-use form of normal phase chromatography ideally suited to the analysis and
purification of low to moderate molecular weight, thermally labile molecules. It is especially suited to the

separation of chiral compounds. Similar to high performance liquid chromatography (HPLC), SFC
typically utilizes carbon dioxide as the mobile phase; therefore, the entire chromatographic flow path must
be pressurized.
In addition, SFC metering pumps require that the pump head be kept cold in order to maintain the carbon
dioxide in a supercritical state, where it can be effectively metered at some specified flow rate. The
chemist sets mobile phase flow rate, composition, and column temperature. In addition, SFC provides an
additional control parameter, pressure, which the chemist similarly sets through the keyboard. From an
operational standpoint, SFC is as simple and robust as HPLC.
Any molecule that will dissolve in methanol or a less polar solvent is an ideal candidate for SFC. SFC can
even separate polar solutes. Many strong bases that are difficult to separate by other techniques separate
rapidly and efficiently with good peak shapes.
Application:
SFC is used in industry primarily for separation of chiral molecules, and uses the same columns as standard
HPLC systems. SFC is now commonly used for achiral separations and purifications in
the pharmaceutical industry.[3]
Type 7. Affinity Chromatography

It is a chromatographic method of separating biochemical mixtures, based on a highly specific biologic
interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.
Affinity chromatography combines the size fractionation capability of gel permeation chromatography with
the ability to design a stationary phase that reversibly binds to a known subset of molecules.
Affinity chromatography can be used to:
i. Purify and concentrate a molecule from a mixture into a buffering solution.
ii. Reduce the amount of a molecule in a mixture.
iii. Discern what biological compounds bind to a particular molecule, such as drugs.

Usually the starting point is an undefined heterogeneous group of molecules in solution, such as a cell
lysate, growth medium or blood serum. The molecule of interest will have a well known and defined
property which can be exploited during the affinity purification process. The process itself can be thought
of as an entrapment, with the target molecule becoming trapped on a solid or stationary phase or medium.
The other molecules in solution will not become trapped as they do not possess this property. The solid
medium can then be removed from the mixture, washed and the target molecule released from the
entrapment in a process known as elution.
Binding to the solid phase may be achieved by column chromatography, whereby the solid medium is
packed onto a chromatography column, the initial mixture run through the column to allow binding, a wash
buffer run through the column and the elution buffer subsequently applied to the column and collected.
These steps are usually done at ambient pressure (as opposed to HPLC or FPLC).
Alternatively binding may be achieved using a batch treatment, by adding the initial mixture to the solid
phase in a vessel, mixing, separating the solid phase (by centrifugation, for example), removing the liquid
phase, washing, re-centrifuging, adding the elution buffer, re-centrifuging and removing the eluate.
Sometimes a hybrid method is employed, the binding is done by the batch method, then the solid phase with
the target molecule bound is packed onto a column and washing and elution are done on the column.
A third method, expanded bed adsorption, which combines the advantages of the two methods mentioned
above, has also been developed. The solid phase particles are placed in a column where liquid phase is
pumped in from the bottom and exits at the top. The gravity of the particles ensures that the solid phase
does not exit the column with the liquid phase.

Application:
 Affinity chromatography is one of the most useful methods for the separation and purification of
specific products.
 It is essentially a sample purification technique, used primarily for biological molecules such as
proteins.
Its major application includes:
 Separation of mixture of compounds.
 Removal of impurities or in purification process.
 In enzyme assays
 Detection of substrates
 Investigation of binding sites of enzymes
 In in vitro antigen-antibody reactions
 Detection of Single Nuceotide polymorphisms and mutations in nucleic acids
Advantages of Affinity Chromatography:

 High specificity
 Target molecules can be obtained in a highly pure state
 Single step purification
 The matrix can be reused rapidly.
 The matrix is a solid, can be easily washed and dried.
 Give purified product with high yield.
 Affinity chromatography can also be used to remove specific contaminants, such as proteases.

Limitations of Affinity Chromatography:
 Time consuming method.
 More amounts of solvents are required which may be expensive.
 Intense labour
 Non-specific adsorption cannot be totally eliminated, it can only be minimized.
 Limited availability and high cost of immobilized ligands.
 Proteins get denatured if required pH is not adjusted.

Type 8. Two-Dimensional Chromatography
By using an additional physicochemical (Chemical classification) criterion for separation of the mixture of
analytes (sample), the resolution and quality of chromatographic separation can be increased. As a result,
higher specificity concerning the separational capability of the chromatographic technique is obtained,
allowing separation and preparation or analysis of compounds indistinguishable by one-dimensional
chromatography.
In Gas-Phase Chromatography, two-dimensional separation is achieved by coupling a second, short column
to the first long column. Coupling is achieved by different techniques, for example, shock-freezing the
elutes in order of elution from the first column at fixed time-intervals, and then reheating them in order of
elution, releasing them into the second column. The time of traversal through the second column needs to
be shorter than the time remaining until the next sample is reheated to prevent compound build-up and to
fully exploit the separational capability.
Application:
The purpose of employing this technique is to separate mixtures that one dimensional liquid
chromatography otherwise cannot separate effectively. Two dimensional liquid chromatography is better
suited to analyzing complex mixtures samples such as urine, environmental substances and forensic
evidence such as blood.

1. Wilson, K., Walker, J. (2018). Principles and Techniques of Biochemistry and Molecular Biology (8 eds.).
Cambridge University Press: New York.
2. https://www.med.unc.edu/pharm/sondeklab/files/resource-files/protein-purification-handbooks/Affinity
%20chromatography.pdf
3. https://www.slideshare.net/poojakamble1609/affinity-chromatography-ppt
4. https://www.sigmaaldrich.com/content/dam/sigmaaldrich/docs/promo_NOT_INDEXED/General_Informatio
n/1/ge-affinity-chromatography.pdf
5. https://www.slideshare.net/sagarsavale1/affinity-chromatography-56328075
6. https://www.shodex.com/en/da1/09/0201.html
7. https://www.slideshare.net/asimsami/affinity-chromatography-73800707
8. wolfson.huji.ac.il/purification/PDF/ReversePhase/AmershamRPCManual.pdf
9. www.google.com/url
10. www.waters.com/.../nav.htm?cid=10049076&locale=en_IN
11. www.silicycle.com/.../reversed-phase-chromatography-general-introduction-for-improved-method-
development
12. "Rapid Chromatographic Techniques for Preparative Separation with Moderate Resolution." Still, W. C.;
Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43 (14), 2923-5.
13. "Preparative Reversed-Phase Flash Chromatography, a Convenient Method for the Workup of Reaction
Mixtures." Kuhler, T. C.; Lindsten, G. R. J. Org. Chem. 1983, 48 (20), 3589-3591.
14. "Reverse Phase Flash Chromatography: A Convenient Method for the Large Scale Separation of Polar
Compounds." O'Neil, I. A. Synlet 1991, 661-662.
15. "Ion Exchange Chromatography, Principles and Methods." by Pharmacia, Laboratory Separation Division.
16."Gel Filtration, theory and Practice." by Pharmacia, Laboratory Separation Division.


Chromatography Sirke

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Chromatography Sirke

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Introduction :-

Chromatography is non destructive procedure for resolving a multi-component mixture of trace,

Dr.Shri R.M.S Institute of Sci & Tech ,COP,Bhanpura Page 1

*What is the Retention Factor ?

The retention factor, Rf, is defined as ,

Dr.Shri R.M.S Institute of Sci & Tech ,COP,Bhanpura Page 2

(2) Paper Chromatography.

(3) Thin Layer Chromatography.

(4) Gas Chromatography.

(5) High Performance Liquid Chromatography.

(6) Supercritical Fluid Chromatography.

(7) Affinity Chromatography.

(8) Two Dimensional Chromatography .

Dr.Shri R.M.S Institute of Sci & Tech ,COP,Bhanpura Page 3

In chemistry, Column chromatography is a technique which is used to separate a single chemical

Column Chromatography Principle :

Rf = the distance travelled by solute/ the distance travelled by solvent

Dr.Shri R.M.S Institute of Sci & Tech ,COP,Bhanpura Page 4

1. It acts as a solvent – sample mixture can be introduced in the column.

Dr.Shri R.M.S Institute of Sci & Tech ,COP,Bhanpura Page 5

Column Chromatography Applications :

 Column Chromatography is used to isolate active ingredients.

 It is very helpful in Separating compound mixtures.

 It is used to determine drug estimation from drug formulations

 It is used to remove impurities.

 Used to isolation metabolites from biological fluids.

Types of Column Chromatography:

Dr.Shri R.M.S Institute of Sci & Tech ,COP,Bhanpura Page 6

Type 2. Paper Chromatography

Dr.Shri R.M.S Institute of Sci & Tech ,COP,Bhanpura Page 7

The principle involved can be partition chromatography or adsorption chromatography. Partition

Paper Chromatography Procedure:

E Paper Chromatography Applications :

 To study the process of fermentation and ripening.

 To check the purity of pharmaceuticals.

 To detect the adulterants.

 To detect the contaminants in drinks and foods.

 To examine the reaction mixtures in biochemical laboratories.

 To determine dopes and drugs in humans and animals.

Types of paper chromatography:

Dr.Shri R.M.S Institute of Sci & Tech ,COP,Bhanpura Page 9

Type 3. Thin Layer Chromatography

Dr.Shri R.M.S Institute of Sci & Tech ,COP,Bhanpura Page 10

Thin Layer Chromatography Principle

Thin Layer Chromatography Procedure

Dr.Shri R.M.S Institute of Sci & Tech ,COP,Bhanpura Page 11

Thin Layer Chromatography Experiment

 Apply sample solutions to the marked spots.

Thin Layer Chromatography Applications

 TLC is extremely useful in Biochemical analysis such as separation or isolation of biochemical

 It is widely used in separating multicomponent pharmaceutical formulations.

 It is used in the cosmetic industry.

Disadvantages Of Thin Layer Chromatography:

1. Thin Layer Chromatography plates do not have longer stationary phase.

Type 4. Gas-Liquid Chromatography (GLC) or Simply Gas Chromatography

Dr.Shri R.M.S Institute of Sci & Tech ,COP,Bhanpura Page 13

Fig. Gas Chromatography

Dr.Shri R.M.S Institute of Sci & Tech ,COP,Bhanpura Page 14

Common inlet types are:

Dr.Shri R.M.S Institute of Sci & Tech ,COP,Bhanpura Page 15

Dr.Shri R.M.S Institute of Sci & Tech ,COP,Bhanpura Page 16

Sample size and injection technique: