Chromatography, Spectroscopy, Electrophoresis, Types of Chromatography

1. Pharmacognosy & phytochemistry
b. Pharma 3rd YeAr UNIT-5th part-ii
Mr. Bulet Kumar Gupta
Assistant Professor
Sai College of Pharmacy, Mau

2. Chromatography
•Chromatography is an important biophysical technique that enables the separation, identification,
and purification of the components of a mixture for qualitative and quantitative analysis.
•The Russian botanist Mikhail Tswett coined the term chromatography in 1906.
•The first analytical use of chromatography was described by James and Martin in 1952, for the
use of gas chromatography for the analysis of fatty acid mixtures.
•A wide range of chromatographic procedures makes use of differences in size, binding affinities,
charge, and other properties to separate materials.
•It is a powerful separation tool that is used in all branches of science and is often the only means
of separating components from complex mixtures.

3. Principle
•Chromatography is based on the principle where molecules in mixture applied onto the surface
or into the solid, and fluid stationary phase (stable phase) is separating from each other while
moving with the aid of a mobile phase.
•The factors effective on this separation process include molecular characteristics related to
adsorption (liquid-solid), partition (liquid-solid), and affinity or differences among their
molecular weights.
•Because of these differences, some components of the mixture stay longer in the stationary
phase, and they move slowly in the chromatography system, while others pass rapidly into the
mobile phase, and leave the system faster.

4. Three components thus form the basis of the chromatography technique.
1.Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid
adsorbed on the surface solid support”.
2.Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
3.Separated molecules

5. Types of Chromatography
1. Paper chromatography
2. Thin-layer chromatography
3. Column chromatography
4. Gas chromatography (GS)
5. Ion-exchange chromatography
6. Gel-permeation (molecular sieve) chromatography
7. Affinity chromatography
8. High-pressure liquid chromatography (HPLC)

6. Paper chromatography (PC)
Paper chromatography (PC) is a type of planar chromatography whereby chromatography
procedures are run on a specialized paper.
PC is considered to be the simplest and most widely used of the chromatographic techniques
because of its applicability to isolation, identification, and quantitative determination of organic
and inorganic compounds.
It was first introduced by German scientist Christian Friedrich Schonbein (1865).

7. Paper Adsorption Chromatography
Paper impregnated with silica or alumina acts as adsorbent (stationary phase) and solvent as
mobile phase.
Paper Partition Chromatography
Moisture / Water present in the pores of cellulose fibers present in filter paper acts as stationary
phase & another mobile phase.

8. Instrumentation of Paper chromatography
1.Stationary phase & papers used
2.Mobile phase
3.Developing Chamber
4.Detecting or Visualizing agents
1. STATIONARY PHASE AND PAPERS
•Whatman filter papers of different grades like No.1, No.2, No.3, No.4, No.20, No.40, No.42 etc
•In general the paper contains 98-99% of α-cellulose, 0.3 – 1% β -cellulose.
PAPER CHROMATOGRAPHY MOBILE PHASE
•Pure solvents, buffer solutions or mixture of solvents can be used.

9. Examples-
Hydrophilic mobile phase
•Isopropanol: ammonia :water 9:1:2
•Methanol : water 4:1
•N-butanol : glacial acetic acid : water 4:1:5
CHROMATOGRAPHIC CHAMBER
•The chromatographic chambers are made up of many materials like glass, plastic or stainless
steel. Glass tanks are preferred most.
Detection
Colorless analytes were detected by staining with reagents such as iodine vapor, ninhydrin, etc.
Radiolabeled and fluorescently labeled analytes were detected by measuring radioactivity and
fluorescence respectively.

11. Application
•To check the control of purity of pharmaceuticals,
•For detection of adulterants,
•Detect the contaminants in foods and drinks,
•In the study of ripening and fermentation,
•For the detection of drugs and dopes in animals & humans
•In analysis of cosmetics
•Analysis of the reaction mixtures in biochemical labs.

12. TLC
Thin Layer Chromatography can be defined as a method of separation or identification of a
mixture of components into individual components by using finely divided adsorbent solid /
(liquid) spread over a plate and liquid as a mobile phase.
Principle of TLC
Thin-layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is
coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide (alumina),
or cellulose. This layer of adsorbent is known as the stationary phase.
After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile
phase) is drawn up the plate via capillary action.
The components with more affinity towards stationary phase travels slower. Components with less
affinity towards stationary phase travels faster.

13. The individual components are visualized as spots at a respective level of travel on the plate.
Their nature or character is identified by means of suitable detection techniques.

14. Components of Thin Layer Chromatography (TLC)
• TLC Plate
• TLC Chamber
• Mobile Phase

15. Some common techniques for visualizing the results of a TLC plate include
1.UV light
2.Iodine Staining: is very useful in detecting carbohydrates since it turns black on contact with
Iodine
3.KMnO4 stain (organic molecules- Alkene, Alkane)
4.Ninhydrin Reagent: often used to detect amino acids and proteins
5.In monitoring the progress of reactions
6.Identify compounds present in a given mixture
7.Determine the purity of a substance.

16. •Analyzing ceramides and fatty acids
•Detection of pesticides or insecticides in food and water
•Analyzing the dye composition of fibers in forensics
•Assaying the radiochemical purity of radiopharmaceuticals
•Identification of medicinal plants and their constituents
Advantage
•It is a simple process with a short development time.
•It helps with the visualization of separated compound spots easily.
•It helps in isolating of most of the compounds.
•The separation process is faster.
•The purity standards of the given sample can be assessed easily.
•It is a cheaper chromatographic technique.

17. HPLC
High-performance liquid chromatography or commonly known as HPLC, is an analytical
technique used to separate, identify or quantify each component in a mixture.
The mixture is separated using the basic principle of column chromatography and then identified
and quantified by spectroscopy.
In the 1960s, the column chromatography LC with its low-pressure suitable glass columns was
further developed to the HPLC with its high-pressure adapted metal columns.
HPLC is thus basically a highly improved form of column liquid chromatography. Instead of a
solvent being allowed to drip through a column under gravity, it is forced through under high
pressures of up to 400 atmospheres.

18. Principle of HPLC
•The purification takes place in a separation column between a stationary and a mobile phase.
•The stationary phase is a granular material with very small porous particles in a separation
column.
•The mobile phase, on the other hand, is a solvent or solvent mixture which is forced at high
pressure through the separation column.
•Via a valve with a connected sample loop, i.e. a small tube or a capillary made of stainless steel,
the sample is injected into the mobile phase flow from the pump to the separation column using a
syringe.
•Subsequently, the individual components of the sample migrate through the column at different
rates because they are retained to a varying degree by interactions with the stationary phase.

19. •After leaving the column, the individual substances are detected by a suitable detector and passed
on as a signal to the HPLC software on the computer.
•At the end of this operation/run, a chromatogram in the HPLC software on the computer is
obtained.
•The chromatogram allows the identification and quantification of the different substances.
•Subsequently, the individual components of the sample migrate through the column at different
rates because they are retained to a varying degree by interactions with the stationary phase.
•After leaving the column, the individual substances are detected by a suitable detector and passed
on as a signal to the HPLC software on the computer.
•At the end of this operation/run, a chromatogram in the HPLC software on the computer is
obtained.
•The chromatogram allows the identification and quantification of the different substances.

20. Instrumentation of HPLC
• The Pump- (High-pressure generation)
• Injector- (Sample is introduced)
• Column- (separation is performed inside the column)
• Detector- (Detect of analyte)
• Recorder- (Record the Result in form of electronic signal form)
1.Normal phase:
Column packing is polar (eg. silica) and the mobile phase is non-polar. It is used for water-
sensitive compounds, geometric isomers, cis-trans isomers, and chiral compounds.
2.Reverse phase:
The column packing is non-polar (e.g C18), the mobile phase is water+ miscible solvent (e.g
methanol). It can be used for polar, non-polar, ionizable, and ionic samples.

22. 3.Ion exchange:
Column packing contains ionic groups and the mobile phase is buffer. It is used to separate anions
and cations.
4.Size exclusion:
Molecules diffuse into pores of a porous medium and are separated according to their relative size
to the pore size. Large molecules elute first and smaller molecules elute later.
Application of HPLC
•Analysis of drugs
•Analysis of synthetic polymers
•Analysis of pollutants in environmental analytics
•Determination of drugs in biological matrices
•Isolation of valuable products

23. •Product purity and quality control of industrial products and fine chemicals
•Separation and purification of biopolymers such as enzymes or nucleic acids
•Water purification
•Pre-concentration of trace components
Advantage of HPLC
1.Speed
2.Efficiency
3.Accuracy

24. High Performance Thin-Layer Chromatography (HPTLC)
HPTLC is an enhanced form of thin-layer chromatography (TLC). A number of enhancements can
be made to the basic method of thin-layer chromatography to automate the different steps, to
increase the resolution achieved, and to allow more accurate quantitative measurements.
HPTLC helps in better resolution of compounds with lower limits of detection and quantifies
separated components with the use of an integrated software platform.
Methodology:
 Stationary Phase Precoated plate (Silica Gel GF254) Particle size- 7 um, Thickness- 100 um
Used to separate- Amino acid, alkaloids, fatty acids, lipids, steroids, terpenoids, etc.
 Mobile Phase:
Similar as TLC
Depends on the polarity of analyte

25. Application
Forensic Analysis: A challenge in forensic toxicology is the identification of unknown poisonous
substances in intoxication cases. HPTLC offers rapid identification as well as qualitative and
quantitative analysis for toxic substances.
Herbal Applications: HPTLC fingerprint technology can be used in the identification of botanical
materials that are very complex in nature.
Food Industry: To evaluate nutrients, beverages, vitamins, and pesticides in fruit, vegetables, and
other foodstuffs.
Pharmaceutical Industry: Used in post-production quality control. analysis o forced degradation
studies, stability testing, and to check the prese impurities in the drug.

26. Difference between HPLC & HPTLC
High Performance/Pressure Liquid Chromatography (HPLC) High Performance/Pressure Thin Layer Chromatography
(HPTLC)
It is a Type of column Chromatography It is a type of Planar Chromatography
The Stationary Phase filled into the column The Stationary Phase fixed onto the column
It is a closed system It is a Open System
Used High Pressure Operate at Atmospheric Pressure
Does not allow parallel analysis Allow Parallel Analysis

27. Column chromatography
Column chromatography is a technique which is used to separate a single chemical compound
from a mixture dissolved in a fluid.
Column chromatography separates substances based on differential adsorption of compounds to
the adsorbent as the compounds move through the column at different rates which allows them to
get separated in fractions. This technique can be used on a 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.
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

28. components of the mixture is at different rates. The components with lower adsorption and
affinity to the 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 slowly are eluted out last.

29. Plate Theory of Chromatography
The theory was integrated into the chromatography technique in 1941 by Martin and
Synge.
According to the model, the chromatographic column consists of separate layers known
as the theoretical plates.
These plates provide separation equilibrium of the sample between the mobile and
stationary phase.
As the mobile phase passes through the stationary phase in a column, the analytes in
the mobile phase are distributed between the two phases establishing an equilibrium.
The number of the theoretical plate in a column is represented by N. The efficiency of the
column is represented by HETP (Height Equivalent to a Theoretical Plate). A measure of
the efficiency of a chromatography column is the height equivalent to a theoretical plate

30. The rate theory of chromatography is expressed mathematically by the van
Deemter equation.

31. 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 isolate metabolites from biological fluids.

32. Gas chromatography
•Gas chromatography differs from other forms of chromatography in that the mobile
phase is a gas and the components are separated as vapors.
Principle
The Principle for gas chromatography is partitioning, and the components of the sample
will partition (i.e. distribute) between the two phases: the stationary phase and the mobile
phase.
Compounds that have a greater affinity for the stationary phase spend more time in the
column and thus elute later and have a longer retention time (Rt) than samples that
have a higher affinity for the mobile phase.
Instrumentation of Gas Chromatography
1 Carrier gas in a high-pressure cylinder with attendant pressure regulators and

33. 2.Sample injection system- Liquid samples are injected by a micro syringe with a needle
3.The separation column- The heart of the gas chromatography is the column which is made of
metals bent in U shape or coiled into an open spiral. Several sizes of columns are used depending
upon the requirements.
4.Liquid phases- Liquid phases are available limited only by their volatility, thermal stability.
5.Detector
6.Recorder
Procedure of Gas Chromatography
Step 1: Sample Injection and Vaporization
1.A small amount of liquid sample to be analyzed is drawn up into a syringe.
2.The syringe needle is positioned in the hot injection port of the gas chromatograph and
the sample is injected quickly.

35. Sepration is depending on the interaction
of molecules towards stationary phase.
Less interaction travel first
More interaction travel last.

36. 3 The injection of the sample is considered to be a “point” in time, that is, it is assumed
that the entire sample enters the gas chromatograph at the same time, so the sample
must be injected quickly.
4 The temperature is set to be higher than the boiling points of the components of the
mixture so that the components will vaporize.
5 The vaporized components then mix with the inert gas mobile phase to be carried to
the gas chromatography column to be separated.
Step 2: Separation in the Column
•Components in the mixture are separated based on their abilities to adsorb on or bind
to, the stationary phase.
•A component that adsorbs most strongly to the stationary phase will spend the most
time in the column (will be retained in the column for the longest time) and will, therefore,

37. retention time (Rt). It will emerge from the gas chromatograph last.
Step 3: Detecting and Recording Results
1.The components of the mixture reach the detector at different times due to differences
in the time they are retained in the column.
2.The component that is retained the shortest time in the column is detected first. The
component that is retained the longest time in the column is detected last.
Application
(a) Air-borne pollutants
(b) Performance-enhancing drugs in athlete’s urine samples
(c) Oil spills
(d) Essential oils in perfume preparation

38. Methods of Identification
1. Ultra violet & Visible Spectroscopy
2. Infrared Spectroscopy
3. Mass Spectroscopy
4. Nuclear Magnetic Resonance Spectroscopy
Ultra violet & Visible Spectroscopy
Spectrophotometric techniques utilized for the structural elucidation of any isolated unknown
chemical constituents.
UV-visible spectroscopy is applicable to know degree of conjugation in that isolated compounds.
The absorption spectra of plant constituents can be measured in the very dilute against a blank
solvent by using automatic recording spectrophotometer.

39. If the isolated compound is colorless then it absorb in the range of 200 to 400nm, i.e. UV range
and if the isolated compound is colored then it absorbs in the range of 400 to 700 nm, i.e. Visible
range.
The wavelength of the maxima and minima from the absorption spectrum of the compound is
recorded in nm which can be further compared with the standard peaks for their identification.

40. Instrumentation of UV & Visible Spectroscopy

41. IR Spectroscopy
It is one of the most common and widely used spectroscopic techniques employed mainly by
inorganic and organic chemists due to its usefulness in determining the structures of compounds
and identifying them.
The major use of infrared spectroscopy is to determine the functional groups of molecules.

42. Principle of IR Spectroscopy
The principle of infrared spectroscopy is based on the vibrations of atoms and the dipole moment
of the compounds. When infrared radiation passes through the sample, a fraction of incident
radiation of particular energy is absorbed by the vibrating atoms. The energy of the vibratory
bonds corresponds to the energy of absorption. This way infrared spectrum is obtained.
Instrumentation of IR Spectroscopy
The main parts of the IR spectrometer are as follows:
1.Radiation source
2.Sample cells and sampling of substances
3.Monochromators
4.Detectors
5.Recorder

45. Application of IR Spectroscopy
•Identification of an organic compound: Functional groups, for example, two compounds with
similar overlay able spectra must have the same functional groups. Interestingly, an FT-IR
spectrophotometer is used at airports to detect drug addicts.
•Purity of chemical compound: Although thorough FTIR is not commonly used as a quantitative
analysis, it is useful for determining the purity of chemical compounds.

46. Mass Spectroscopy
Mass spectrometry is an analytical method useful for calculating the mass-to-charge ratio ( m / z)
of one or more molecules in the sample. Such measurements may also often be used to determine
the precise molecular weight of the sample components. Mass spectrometry is an analytical
method to find the molecular mass of a compound and indirectly helped to prove the identity of
isotopes.
Principle of Mass Spectroscopy
Mass spectroscopy is the most accurate method for determining the molecular mass of the
compound and its elemental composition.
The basic principle of mass spectrometry (MS) is to generate ions from either inorganic or organic
compounds by any suitable method, to separate these ions by their mass-to-charge ratio (m/z) and
to detect them qualitatively and quantitatively by their respective m/z and abundance.

47. • Ionization
• Acceleration
• Deflection
• Detection

49. Electrophoresis
•Electrophoresis involves the migration of charged particle or molecules under the
influence of an applied electric field.
•Various important biomolecules such as peptides, amino acids, proteins, nucleic acid
and nucleotides has ionizable groups and they exist in solution as electrically charged
particles either as cations or as anions at any given pH.
•These charged particles will move towards the cathode or to the anode end based on
their net charge in a mixture under the influence of an applied electric field.
ELECTROPHORESIS IS BASICALLY OF TWO TYPES
1.Free boundary or moving boundary electrophoresis.
2.Zone electrophoresis.

50. Moving Boundary Electrophoresis
•It is a type of electrophoresis without supporting media, in a free solution.
•Tiselius developed this type of electrophoresis in 1937.
•For the separation of different charged molecules in a mixture, sample is placed in
glass, which is connected to the electrodes. On applying electric potential across the
tube, charged molecule migrates towards one or another electrode.
Zone Electrophoresis
•It involves the separation of charged particles on inert matrix, or supporting or stabilizing
media.

51. ON THE BASIS OF SUPPORTING MEDIA, IT IS OF FOLLOWING TYPES
1.Paper electrophoresis
2.Cellulose acetate electrophoresis
3.Capillary electrophoresis
4.Gel electrophoresis.
Type Of Gel Electrophoresis
1.Agarose gel electrophoresis
2.SDS-PAGE
3.Pulse field gel electrophoresis (PFGE)
4.2D gel electrophoresis