Column chromatography was first developed by
the American chemist TSWETT in 1906.
When chromatography is carried out in column it
is called column chromatography.
Otherwise known as gravity chromatography.
4. Column chromatography is a separation technique in
which components of mixture is separated by using a glass
column packed with stationary phase and the liquid mobile
phase flowing continuously through the column.
The technique can be used on scales from micrograms up
The main advantage of column chromatography is the
relatively low cost and disposability of the stationary phase
used in the process.
Column chromatography can be done using gravity to
move the solvent, or using compressed gas to push the
solvent through the column.
5. When a mixture of mobile phase and sample to be separated are
introduced from top of the column, the individual components of mixture
move with different rates.
Those with lower affinity and adsorption to stationary phase move faster
and eluted out first while those with greater adsorption affinity move or
travel slower and get eluted out last.
The solute molecules adsorb to the column in a reversible manner. The
rate of the movement of the components is given as follows
R= Rate of movement of a component / Rate of movement of mobile
phase. i.e. it is the ratio of distance moved by solute to the distance moved
Stationary phase : called adsorbent
It is a solid.
Mobile phase : It is also known as solvent & eluent.
It is a liquid.
Sample : It is known as adsorbate. Which get adsorbs.
Elution : It is a process of removing the components
Eluate : separated component. 6
7. Chromatographic methods can be classified according to the nature of
the stationary and mobile phases.
Ion exchange chromatography
Size exclusion or gel permeation chromatography
The modern instrumental techniques of GLC and HPLC provide excellent
separation and allow accurate assay of very low concentrations of wide
variety of substance in complex mixtures.
CLASSIFICATION OF COLUMN CHROMATOGRAPHY
8. Adsorption chromatography
In adsorption chromatography, the mobile phase containing the dissolved
solutes passes over the surface of the stationary phase.
Retention of the component and their consequent separation depends on
the ability of the atoms on the surface to remove the solutes from the
mobile phase and adsorb them temporarily by means of electrostatic
Usually silica or alumina is utilized as the adsorbent with relatively non
polar solvents such as hexane as the mobile phase in normal phase
adsorption whereas in reversed phase adsorption non polymer beds with
relative polar solvents such as water, acetonitrile methanol as mobile
10. Partition chromatography
In partition chromatography an inert solid material such as silica gel or
diatomaceous earth serves to support a thin layer of liquid which is the
effective stationary phase.
As the mobile phase containing the solutes passes in close proximity to this
liquid phase, retention and separation occur due to the solubility of the
analytes in the two fluids as determined by their partition coefficients.
The method suffers from disadvantage due to some solubility of stationary
phase in the mobile phase.
12. Ion exchange chromatography
In ion exchange chromatography, the stationary phase consists of a
polymeric resin matrix on the surface of which ionic functional groups,
e.g., carboxylic acids or quaternary amines, have been bonded chemically.
As the mobile phase passes over this surface, ionic solutes are retained by
forming electrostatic chemical bonds with the functional groups.
The mobile phase used in this type is always liquid.
14. Size exclusion or gel permeation chromatography
In size exclusion chromatography the stationary phase is a polymeric
substance containing numerous pores of molecular dimensions.
Solutes whose molecular size is sufficiently small will leave the mobile
phase to diffuse into the pores.
Large molecules which will not fit into the pores remain in the mobile
phase and are not retained.
This method is mostly suitable for the separation of mixtures in which the
solutes vary considerably in molecular size.
The mobile phase in this type may be either liquid or gaseous.
16. 1. Column characteristics & selection
2. Stationary phases
3. Mobile phases
4. Preparation of column
5. Introduction of sample
6. Development of column
7. Detection & recovery of components
REQUIREMENTS OF COLUMN CHROMATOGRAPHY
17. (1) COLUMN CHARACTERITIC & SELECTION:
Table:1: selecting a suitable column dimension
Materials of construction Good quality neutral glass, plastic or nylon
Adsorbent(stationary phase)/adsorbate (mixture)
Column length to diameter ratio(cm) 10-15:1 or 30-100:1
Multi component system is present Long column is used
Components with similar affinities for adsorbent are
Long column is used
Components with different affinities for adsorbent are
Short column is used
18. (2) STATIONARY PHASE
Stationary phases used in column adsorption chromatography are also
known as adsorbents.
Requirements of an ideal stationary phase:
1.They should be insoluble in solvents or mobile phases.
3.colourless to facilitate observation of zones.
4.should have reproducible properties from batch to batch.
5.The particle should have uniform size distribution and have spherical
6. particle size :60-200µ
19. Type of mesh Size in microns
60/120 mesh 120-250 micron
100/200 mesh 75-150 micron
70/230 mesh 63-200 micron
230/400 mesh 37-63 micron
The most commonly used chromatographic adsorbent is silica or
silicic acid or silica gel (80-100 mesh or 100-200 mesh size, which
has a particle size of 63-200µm).
21. (3) MOBILE PHASE OR SOLVENTS OR ELUENTS
Mobile phase act as a solvent to introduce the mixture into the column as
developer to develop the zones for separation and as an eluent to remove the
pure component out to the column.
Requirements of an ideal mobile phase:
1. The choice of solvent is depend on the solubility characteristic of the
2. It can be used in either pure form or as mixture of solvents.
3. Polarity as seen the most important factor.
4. The solvents should also have sufficiently low boiling points to permit
ready recovery of eluted material.
22. (4) COLUMN PREPARATION
A column is prepared by packing a solid absorbent into a cylindrical glass
or plastic tube.
The size will depend on the amount of compound being isolated. The base
of the tube contains a filter, either a cotton or glass wool plug, or glass frit
to hold the solid phase in place. A solvent reservoir may be attached at the
top of the column.
Two methods are generally used to prepare a column:
1. The dry method and
2. The wet method
23. 1. DRY METHOD
In this method the dry adsorbent is poured to the column directly
Vibration is applied to get rid of air bubbles then the mobile phase is passed
through the adsorbent.
The demerit with this method is that air bubbles are entrapped between the
solvent and the stationary phase. This method cannot be applied in gel
24. 2. WET METHOD
The adsorbent is suspended in the mobile phase and stirred very well to
remove all air bubbles. The resulted slurry is then poured in to the column
At the bottom portion of the column a piece of glass wool or cotton/
whattman filter paper disc must be added before the slurry application.
The top of the silica should be flat, and the top of the silica can be protected
by a layer of sand.
After slurry application, the column must be allowed to settle overnight. This
is the ideal method of column packing.
25. (5) INTRODUCTION OF SAMPLE
Dissolve the sample in the initial mobile phase and apply by pipette to the
top of the column.
This is very good method but in most of cases the samples are not soluble
in the initial mobile phase.
B) Dry loading:
Dissolve sample in any volatile solvent.
The sample solution is then adsorbed an small weight of adsorbent and the
solvent is allowed to evaporate.
The dry adsorbent loaded with the sample is then applied to the column.
27. (6) DEVELOPMENT OF COLUMN
Removal of individual components from a column is called development
Normal phase: Stationary phase (Polar)
Mobile phase (Non-polar)
Non polar Compounds Elutes first.
Reverse phase: Stationary Phase (Non-polar)
Mobile phase (Polar)
Polar compounds Elutes first.
In most of the analysis, Reverse Phase is used as many of the drugs are
polar in nature.
28. Frontal analysis:
This technique was developed by Tiselius in 1940
In this method, the solution of sample mixture is added continuously on the
column. No mobile phase (solvent) is used for development of column.
A mixture containing A,B,C is added on the column.
If component A is least adsorbed, component B is adsorbed to intermediate
extent and component C most strongly to the column adsorbent material.
The mixture flows through the column, the least adsorbed A runs down the
column fast, component B to intermediate extent while ‘c’ is retained at the
top of the column.
A plot of amount of substance against volume of eluate is gives a
29. Displacemental Analysis:
In this method, a small volume of mixture is added to the column and elution is
carried out by a solvent containing a solute which has adsorptivity for column
The adsorbed constituents of mixture are displayed by the solute from mobile
Each solute in the mixture in turn displaces another substance solute which is
less firmly adsorbed.
The least adsorbed constituent is pushed out of the column. The substance used
in mobile phase is called as displacer.
Displacement analysis technique is mainly used in preparative work and is not
suitable for analysis since some overlapping may occur.
The plot of amount of substance (conc. In eluate) against volume of eluate is
gives a chromatogram.
30. Elution analysis:
It is a common method used in column chromatography.
In this method a small volume of mixture to be separated is added on the
top of column & mobile phase is allowed to flow through the column.
The mixture introduced on the column gets separated into individual as the
components of mixture are adsorbed to the column material to different
On other phase of mobile phase, each component of mixture is eluted out
as separated components (called eluate).
31. A plot of amount of substance per ml of eluate against volume of eluate
will gives the following chromatogram.
a) Isocratic elution: (Iso means same or similar)
In this elution technique, the same solvent composition or solvent of
sample polarity is used throughout the process of separation.
e.g.(chloroform only as a solvent or CHCL3:MeOH=1:1)
b) Gradient elution: (gradient means gradual)
In this elution technique, solvents of gradually increasing polarity or
increasing elution length are used during the process of separation.
32. (7) DETECTION & RECOVERY OF COMPONENTS
Fractions are collected by elution analysis
Each fraction is examined by TLC using suitable experimental conditions
Those fractions which give same Rf values in TLC are added as a common
From the column fraction, solvent is evaporated, dried & the materials
collected in container
After spectral analysis (NMR, MS, X-RD etc.)the compound is identified.
34. 1.Column efficiency:
It is expressed by the number of theoretical plates
It is determined by the formula:
The number of theoretical plates is a measure of the “goodness” of the
If the retention time is high and peak width is narrow then it shows
tr is the retention time measured from the instant of injection
w is the peak width
Resolution is the ability to separate two signals
In chromatography it’s the ability to separate two peaks.ie separation of
tr1 and tr2 and w1 and w2 are the times and widths, respectively, of the two
immediately adjacent peaks.
If the peaks are sufficiently close w is nearly the same for both peaks and
resolution may be expressed as
38. 3.Retention time
The rates of migration of substances in chromatographic procedures
depend on the relative affinity of the substances for the stationary and the
It’s the difference in time between the point of injection and the time of
emergence of separation of component from the column.
It is actually the time required for 50% of the component to get eluted.
It is measure in minutes or seconds
40. 4.Retention volume
It is the volume of carrier gas required to elute components from the
column to the time the peak maximum is obtained.
Retention volume depends upon flow rate and retention time
HETP is numerically equal to the column length divided by the number
of theoretical plates in the column
It varies from to one column to another as well as one solute to other
The more efficient the column the better the resolution and the smaller
the HETP. HETP=Length of column / no of theoretical plate
44. 7.Asymetric factor
A chromatographic peak should be symmetrical about its centre to follow
Asymetric factor is the measure of peak tailing or fronting.
It is defined as the distance from the centre line of the peak to the back
slope divided by the distance from the centre line of the peak to the front
46. Any type of mixture can be separated by column chromatography.
Any quantity of the mixture can also be separated.
Wider choice of mobile phase.
In preparative type, the sample can be separated and reused.
Automation is possible.
47. Time consuming method.
More amount of solvents are required which are expensive.
Automation makes the technique more complicated and expensive.
48. In the separation of the mixtures into the pure individual components.
Removal of impurities and in the purification of compounds.
Determination of the homogeneity of chemical substances.
Identification of unknown compounds.
Used in the separation of geometrical isomers, diastereomers, racemates
In the separation and identification of inorganic anions and cations.
The concentrated of substance from dilute solutions such as those obtained
when natural products are extracted with large volumes of the solvents
from the leaves of plants, trees, roots or barks.
49. Column chromatography is a conventional tool for separation of
phytochemicals, removal of impurities and purification of drugs.
Effective separation of constituents from different sources in preparative
scale (milligram to gram) can be achieved by column chromatography.
Availability of wide range of stationary phases makes the technique to be
used for different kinds of mechanisms.
Understanding the basic principles of column chromatography enables us
to find solutions for current research problems.
50. ADVANCEMENT IN COLUMN CHROMATOGRAPHY
High Performance Liquid Chromatography,
Ultra-Performance Liquid Chromatography and
Ultra Performance Convergence Chromatography (Super Critical
The aim of this is to specify the changes that the scientists have made in
each and every step to improve the technique (column chromatography).
Now over 60% of chemical analysis worldwide is currently done with
53. Ultraperformance liquid chromatography (UPLC) is a recent technique in
liquid chromatography, which enables significant reductions in separation
time and solvent consumption.
It improves in three areas: 1.Chromatographic resolution
54. UPLC is a rising chromatographic separation technique whose packing
materials have smaller particle size lesser than 2.5µm.
Reducing these separation times without reducing the quality of the
separation would mean that important analytical information could be
generated more quickly.
The technology takes full advantage of chromatographic principles to run
separations using columns packed with smaller particles and high flow
55. The principle of UPLC is based on Van Deemter equation which describes
the relationship between flow rate and HETP or column efficiency
H=A+B/v + Cv
A = Eddy diffusion
B = Longitudinal diffusion
C = Equilibrium mass transfer
v = flow rate
van Deemter equation, that describes the relationship between linear
velocity (flow rate) and plate height (HETP or column efficiency)
56. Decreases run time and increases sensitivity.
Reducing analysis time so that more product can be produced with
Provides the selectivity, sensitivity, and dynamic range of LC analysis
Maintains resolution performance.
Fast resolving power quickly quantifies related and unrelated compounds.
Operation cost is reduced.
Less solvent consumption.
57. Due to increased pressure requires more maintenance and reduces the life
of the columns of this type.
61. 1. Pumping system
Achieving small particle, high peak capacity separations requires a greater
Both the gradient and isocratic separation modes are used.
The binary solvent manager uses two individual serial flow pumps to
deliver a parallel binary gradient.
There are built-in solvent select valves to choose from up to four solvents.
There is a 15,000-psi pressure limit (about 1000 bar) to take full
advantage of the sub-2μm particles
62. 2.Sample injection
In UPLC, sample introduction is critical. Conventional injection valves,
either automated or manual, are not designed and hardened to work at
To protect the column from extreme pressure fluctuations, the injection
process must be relatively pulse-free and the swept volume of the device
also needs to be minimal to reduce potential band spreading.
Low volume injections with minimal carry over required to increase
63. 3.UPLC columns
Resolution is increased in a 1.7 μm particle packed column because efficiency is
Separation of the components of a sample requires a bonded phase that provides
both retention and selectivity.
Four bonded phases are available for UPLC separations:
1. ACQUITY UPLCTM BEH C18 & C8 (straight chain alkyl columns),
2. ACQUITY UPLC BEH Shield RP18 (embedded polar group column)
3. ACQUITY UPLC BEH Phenyl (phenyl group tethered to the silyl
functionality with a C6 alkyl)
4. ACQUITY UPLC BEH Amide columns (trifunctionally bonded amide
64. 1.ACQUITY UPLCTM BEH C18 & C8
These are considered as the universal columns of choice for most UPLC
separations by providing the widest pH range.
They incorporate trifunctional ligand bonding chemistries which produce
superior low pH stability.
This low pH stability is combined with the high pH stability of the 1.7μm
BEH particle to deliver the widest usable pH operating range.
2.ACQUITY UPLC BEH Shield RP 18
These are designed to provide selectivity's that complement the ACQUITY
UPLC BEH T M C18 and C8 Columns.
65. 3.ACQUITY UPLC BEH Phenyl columns
These utilize a trifunctional C6 alkyl ethyl between the phenyl ring.
4.ACQUITY UPLC BEH Amide columns
1. BEH particle technology, in combination with a trifunctionally bonded
amide phase, provides exceptional column life time, thus improving assay
2. BEH Amide columns facilitate the use of a wide range of phase pH [2 –
67. Requirements of an ideal detector:-
It should give quantitative response.
It should have high sensitivity and low noise level.
It should have a short response time.
It should provide signal or response quantitative to wide spectrum of
It should generate sufficient signal or electrical current, which can be
measured or easily amplified for detection by meter.
68. 1. UV detectors
The majority of organic compounds can be analyzed by UV detectors, and
almost 70% of published HPLC analyses were performed with UV
Measures the ability of solutes to absorb light at a particular wavelength(s)
in the ultraviolet (UV) or visible wavelength range.
When light of certain wavelength is directed at flow cell, the substance
inside the flow cell absorb the light. As a result the intensity of the light
that leaves the flow cell is less than that of the light that enters it.
An absorbance detector measure the extent to which the light intensity
decreases ( i.e. the absorbance).
69. Three common 3 types of this detectors:
Fixed wavelength detectors
Variable wavelength detectors
Photodiode array detectors
Fixed wavelength detector absorbance of only one given wavelength is
monitored by the system at all times ( usually 254nm)
Simplest and cheapest of the UV/VIS detectors
Limited in flexibility
Limited in types of compounds that can be monitored.
Variable wavelength detector a singal wavelength is monitored at any given
time, but any wavelength in a wide spectral range can be selected
Wavelength very from 190-900nm
More expensive, requires more advanced optics 69
70. More versatile, used for a wide range of compounds
More sensitive due to photomultiplier tube or amplification circuitry.
Photodiode array detector operates by simultaneously monitoring
absorbance of solutes at several different wavelength.
Light from the broad emission source such as a deuterium lamp is
collimated by an achromatic lens system so that the total light passes
through the detector cell onto a holographic grating.
In this way , the sample is subjected to light of all wavelengths
generated by lamp. The dispersed light from the grating is allowed to fall
on to a diode array .
The array may contain many 100 of diodes and the output from is diode
is regularly sampled by a computer and stored on a hard disc.
72. 2. Fluorescent detector
The light from an excitation source passes through a filter or
monochromator, and strikes the sample.
A proportion of the incident light is absorbed by the sample, and some of
the molecules in the sample fluoresce. The fluorescent light is emitted in
Some of this fluorescent light passes through a second filter or
monochromator and reaches a detector, which is usually placed at 90° to
the incident light beam to minimize the risk of transmitted or reflected
incident light reaching the detector.
74. 3. Refractive index detector
(ADD AFTER 73 NO. SLIDE)
This detector based on the deflection principle of refractometry, where the
deflection of a light beam is changed when the composition in the sample
flow-cell changes in relation to the reference side (as eluting sample moves
through the system).
As sample elutes through one side, the changing angle of refraction moves
This results in a change in the photon current falling on the detector which
The extent of unbalance (which can be related to the sample concentration)
is recorded on a strip chart recorder.
76. 4. Light scattering detector
Universal , distractive
Useful for large molecules and wide linear range.
Analytes are de-solvated in the detector.
The reduction in light intensity detected (due to scattering by the analytes)
There are 3 steps involved in detection:
Mobile phase evaporation
The flow from the column is nebulized with a stream of inert gas.
77. The mobile phase, which must
be volatile, is evaporated,
leaving tiny particles of the
The particles are passed
through a laser beam and they
scatter the laser light.
The scattered light is
measured at right angles to the
laser beam by a photodiode
78. 5.Electrochemical detector
Most sensitive detector
The level of current is directly proportional to the analyte concentration.
Respond to substances that are oxidizable or reductable.
3 electrodes are employed
79. 6. Mass spectrometric detector
It is a method that combines separation power of HPLC with detection
power of mass spectrometry.
Mass spectrometry (MS) is a powerful analytical tool that can supply both
structural information about compounds and quantitative data relating to
80. 1. Columns: ACQUITY UPLC BEM C18, BEH Shield RP18, BEH C8 OR BEH
2. Dimensions: 2.1 X 50mm 1.7μm.
3. Mobile Phase A1: 20mM NH4COOH in H2O, pH 3.0.
4. Mobile Phase A2: 20mM NH4HCOOH in H2O, pH 10.0.
5. Mobile Phase B1: Acetonitrile .
6. Mobile Phase B2: Methanol.
7. Flow rate: 0.5ml/min.
8. Injection Volume: 10.0μl.
9. Week needle wash: 3% methanol.
10. Strong needle wash: 90% acetonitrile.
11. Temperature: 30°C.
12. Detection: UV 254 nm.
13. Sampling rate: 20pts/sec
Common UPLC chromatographic conditions
81. Analysis of natural products and traditional herbal medicine.
Identification of metabolite.
Study of metabonomics/metabolomics.
Bio analysis/bioequivalence studies.
Forced Degradation Studies.
82. A.H. BECKETT & J.B. STENLAKE, Practical pharmaceutical chemistry, 4th
edition, part two, page no: 86-105.
ASHUTOSH KAR, Pharmaceutical analysis – II, page no: 161-181. DAVID
G.WATSON, Pharmaceutical analysis, page no: 270 - 271.
B.K. SHARMA, Instrumental methods of chemical analysis, page no : C-8 to
C-15. Dr. A.V. KASTURE, Dr. K.R. MAHADIK, Dr. S.G. WADODKAR, Dr.
H.N. MORE, Pharmaceutical analysis volume – II, page no: 10-17.
VOGEL’S, Text book of quantitative analysis, page no:289- 314.
G.DEVALA RAO, A text book of advanced pharmaceutical analysis, page
Review article -Ranjith Reddy Kondeti et al., World J Pharm Sci 2014; 2(9):
Lucie Novakova, Ludmila Matysova, Petr Solich. Advantages of application of
UPLC in pharmaceutical analysis. Talanta: 2006. P. 908-918.
H KAUR Instrumental methods of chemical analysis ninth edition 2013 ;1091-