APPLICATION OF

CHROMATOGRAPHY IN
MICROBIOLOGICAL STUDIES

ABHILASH M R
Research Scholar
DOS in Environmental Science
MGM – 06

October - 2016
 Out-line:
 Definition
History
Chromatographically terms
 Classifications in brief
 Applications
 Some advanced applications
from Research articles
 References
 Definition
 Chromatography is a non destructive procedure for
resolving a multi-component mixture of traces, or major
constituents into its individual fractions.

 Different variations may be applied to solid, liquid and

gases.

 While, chromatography may be applied both

qualitative and quantitatively, and it is a primary
separation tool.

 However, one of the disadvantages of chromatography

is the final identification usually requires confirmation by
using some other analytical procedures such as, IR, NMR
UV and Mass Spectroscopy.
 HISTORY
 Chromatography is relatively a new technique which was first
invented by M. Tswett, a botanist in 1906 in Warsaw. In this year
he was successfully doing separations of Chloraphyll, Xanthophyll
and several colored substances by using Calcium carbonate. This
method is also known as Tswett’s Method.

 Followed by 1930 chromatography in the form of Thin layer

chromatography by Izamailove and Shraiber. and Ion-Exchange
chromatography in 1935 by Adams and Holms.

 In 1941, Martin and Synge introduced Partition and Paper

chromatography. 1951 , A. J .P Martin together with A.T. James
was introduced the Gas chromatography in systematic manner.
  Chromatography has application in every
branch of the physical, chemical and biological
sciences
 12 Nobel prizes were awarded between
1937 and 1972 alone for work in which
chromatography played a vital role
 Laboratory technique for the Separation of mixtures

 Chroma -"color" and graphein - "to write”.

 Colour bands - separation of individual compounds

 Measured or analyzed.
  Analytical
 Determine Chemical composition of a sample

 Preparative
 Used to purify sufficient quantities of a substance
Tswett’s Method
 CHROMATOGRAPHY
TERMS
 Chromatograph - equipment that enables a sophisticated
separation
EX. Gas chromatography or Liquid chromatography
 Eluent - Fluid entering column / solvent that carries the analyte.
 Eluate - Mobile phase leaving the column.
 Stationary phase -
 Immobilized on the support particles or on the inner wall of the
column tubing.
 Examples : Silica layer - Thin Layer Chromatography
  Mobile phase
Moves in a definite direction. Liquid (LC), Gas (GC).
 The mobile phase moves through the chromatography
column, where the sample interacts with the stationary
phase and is separated.
 Retention time : Time takes for a particular analyte to
pass through the system.
 Sample(Anylate): Substance analyzed in
chromatography.
 Solvent: Any substance capable of solubilizing another
substance.
  Chromatogram

 Visual output of the chromatograph.

 Separation - Different peaks or patterns on the chromatogram

correspond to different components of the separated mixture.

 Chromatogram - Detector signal vs.
retention time or volume

Detector Signal 1 2

time or volume
X- axis - Retention time

Y-axis - Signal

Signal is proportional to the concentration of the specific analyte

separated.
  Paper Chromatography
 Thin layer Chromatography
 Liquid - Liquid Partition Chromatography
 High Performance (Pressure) Chromatography
 Adsorption Chromatography
 Column Chromatography
 Gel Chromatography
 Ion – Exchange Chromatography
 Gas Chromatography
 DETECTORS

Flame ionization detector

Aerosol-based detector
Flame photometric detector ( FPD).
Atomic-emission detector (AED).
Mass spectrometer ( MS) detector
Nitrogen Phosphorus Detector,
Evaporative Light Scattering Detector (ELD) :
LC. UV detectors
Thermal conductivity Detector, (TCD)
Fluorescence detector
Electron Capture Detector, (ECD)
Photoionization Detector, (PID)
Refractive index Detector (RI or RID)
Radio flow Detector
Chiral Detector
Summary
 Paper Chromatography
 Paper Chromatography has been applied to the
separation of many organic and biomedical products.
 For example, it has been utilized in the
determination of indoles in whole urine and the study
of barbiturates, antibiotics, carbonyl phosphates,
amino acids and microbial degradation determination
processes.
 Thin Layer Chromatography
 Thin layer Chromatography is used extensively for
the quantitative determination of high molecular
weight compounds, particularly in medical and
microbiological research.
The various applications of TLC are as follows;

 Checking of the purification of samples of

microbial degradation, contamination samples and
essential for microbial experiments samples also,
examined.
 Limitations of TLC is that it is used only for small
scale preparative works.
 1. Acids (Acidophilic bacterial culture environment ) – The normal
aliphatic acids with even numbered carbon atoms from decanal to
docosanoic acids have been separated by Kieselguhr ‘G’ by using
cyclohexane.

2. Alcohols – Even numbered alcohal from decanol through

hexacosanol can be separated by Kieselguhr ‘G’

3. Isolation and determination of alkoloides, Vitamines and microbial

by products toxicity in only 30 – 60 min. it takes 12 – 24 hrs. in Paper
Chromatography.

4. Amino acids, Proteins and Peptides was isolated from 34 ninhydrine

and different microbial sources by using silica – gel plates.

5. In the case of antibiotics: tetracyclines have been separated on thin

layers of silica gel G by using 10 % citric acid, n-butanol-methanol
solutions.

6. Penicillines have been separated on silica gel G by using acetone –

methanol and iso – propanol-methanol (3:7) ratio.
 Liquid - Liquid Partition Chromatography
 Liquid – Liquid chromatography, especially in the
(HPLC), was used for the separation of a very wide
range of biological compounds. In the reverse phase it
was particularly separates the polar compounds.

 Some example of (LLPC), Detergent removal from

protein solutions.

Separation of steroids, bile acids and micotoxines

from microbiological derivatives

 Removal of pesticides, phenols and insecticides

from microbiological related samples.
 High Performance (Pressure) Liquid
Chromatography

 (HPLC), is still infancy, but with further

development of new support materials, as well as
new , more sensitive detectors. it is already replaces
the many gas chromatographic procedures. And it
offers the advantages of speed, resolution and
sensitivity.

 The process have been applied to a wide verity of

natural products extracted by microbes, nucleic
acids, urine serums, carbohydrates, lipids, amino-
acids, bile-acids and antioxidants.
 Determination of Pyruvic acid is produced by
lactic acid bacteria and is a measure of
psychotropic bacteria spoilage by using Aminex
HPX-87H Column.
 Adsorption Chromatography

 A very useful application of this chromatography is

the separation of high molecular weight compounds,
the typical compounds include, micro – organic,
distillation residues. And also, traces of microbial
decompositions and secondary metabolites.
 Column Chromatography
 For, analytic purposes, Column chromatography
finds limited applications:
1. Micro Vestergaard and Sayegh could separate seven
urinary steroids within 5 hrs. which require 36 hrs.
in normal column.
2. Geomaterical isomers compositions like Bixin,
crocetine, certeniodes, and Zechmeister include
microbial decomposition were separated by using
calcium and aluminium oxide other other
absorbents.
3. And most essentially separates the race-mates
(organic solvents achieved by lactose)
 Gel Chromatography
(GEL), has been used great success in the separation
of sugars, polypeptides, proteins, asphalts, and
polystyrenes, The column of Sephadex gel, separated
the hemoglobin. And some other applications as
follows;
1. Purification of Viruses, proteins, enzymes, hormones
are achieved by using Sephadex G-25 (Course material
in this system).
2. Protein building studies can be performed in
microbial by-products using this instruments.
(Such as: Reversible binding of a ligand to a
macromolecule).
 Ion – Exchange Chromatography
 Removal of interfering radicals in media
preparation, softening of hard water and complete
demineralization of water.

Removal of lanthanides and actinides from

radioactive after the microbial degradation processes.

Used to prepare pure reagents for media preparation

and 18 amino acid preparation.
 Gas Chromatography
 The principal application of (GC), are qualitative and
quantitative analysis of liquid gases, and vapors,
particularly of organic compounds.

 any stable compounds that can be vaporized below

300 C can be determined by this method.
 The detection of drug and medicinal properties in
micro-organism.

Analysis of volatile fatty acids produced by bacteria,

particularly anaerobic bacteria, enables fingerprinting of
the particular micro-organisms.
  It is possible to analysis of microbial decomposition of
aldehyde and ketone (responsible for Rancidity).

 Some other example for GC in Environmental Science is

separation and Identification of Polycyclic aromatic hydrocarbons,
Chlorinated pesticides (DDT, BHC), Organ-phosphorus, organo-
tin biocide, organo-lead and organo-mercury for essential
requirements in microbial identification and degradation products
structural elucidation by using (GC-MS).

 And evaluation of microbes pronounced metabolic

disturbances.

 The diagnosis of inborn errors of metabolism especially in the

case of new born microbes.
SOME ADVANCED CASE
STUDIES FROM
RESEARCH ARTICLES
Molecules of interest Mobile phase Stationary phase

Aspergillus niger DEAE- Cellulose

(fungal) extract

Glucose oxidase

Catalase
Molecule of interest Mobile phase Stationary phase
Escherichia coli Sepharose beads coated
(bacterial) lysate with glutathione

Glutathione S-
transferase
Purification of Peptides from Bacillus Strains with Biological
Activity
 REFERENCES:
1. McMurry, John (2011). Organic chemistry: with biological
applications (2nd ed.). Belmont, CA: Brooks/Cole.
p. 395. ISBN 9780495391470.
2. Hostettmann, K; Marston, A; Hostettmann, M (1998). Preparative
Chromatography Techniques Applications in Natural Product
Isolation (Second ed.). 3. Berlin, Heidelberg: Springer Berlin Heidelberg.
p. 50. ISBN 9783662036310.
4. Tswett, M. S. (1905) "О новой категории адсорбционных явлений и о
применении их к биохимическому анализу" (O novoy kategorii
adsorbtsionnykh yavleny i o primenenii ikh k biokkhimicheskomu analizu“
5. Mikhail Tswett (1906) "Physikalisch-Chemische Studien über das
Chlorophyll. Die Adsorption." (Physical-chemical studies of chlorophyll.
Adsorption.) Berichte der Deutschen botanischen Gesellschaft, vol. 24, pp.
316–326. On page 322.
6. L. S.; Sakodynskii, K. I. (March 1993). "M. S. Tswett and the discovery of
chromatography II: Completion of the development of chromatography
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August 2016.
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THANK YOU

ABHILASH M R