Chromatography- (Gas chromatography and HPLC- High Performance Liquid Chromatography)

1. Department of Microbiology
Seminar topic on
GAS CHROMATOGRAPHY
&
HPLC
presented by
Vivek kumar
M.Sc 1st year(sem-1)
Bangalore university

2. INTRODUCTION
 separation technique
 Discovered by Russian botanist- Mikhail Tswett
in 1903.
 Derived from Greek word- “chroma” and
“graphein”.
 Used in qualitative and quantitative analysis of
molecules.
 Two categories- (a) analytical (b)preparative
 Partition or distribution coefficient Kd.

3. Classification of
Chromatography
chromatography
Interaction of solute
to stationary phase
adsorption partition Ion exchange
Chromatographic
bed shape
2-D
chromatography
Thin layer
chromatography
Paper
chromatography
3-D
chromatography
Column
chromatography
Physical state of
mobile phase
Liquid
chromatography
Gas
chromatography
Super critical fluid
chromatography

4. GAS CHROMATOGRAPHY
It is a process of separating
components from the given sample
by using a gaseous mobile phase.

5. Working principle
 The sample solution injected into the
instruments enters a gas stream which
transport the sample into a separation tube
known as the “column”.
 The various components are separated inside
the column.
 The detector measures the quantity of the
components that exit the column.

6. Instrumentation

7. Application
 Analysis of foods like carbohydrates, proteins,
lipids, vitamins, steroids, drug and pesticide
residues, trace elements.
 Pollutants like formaldehyde, carbon monoxide,
benzene, DDT etc.
 Dairy product analysis- rancidity
 Separation and identification of volatile materials,
plastics, natural and synthetic polymers, paints
and microbiological samples.
 Inorganic compound analysis.

8.  This method has high
sensitivity when used
with thermal detectors
 This techniques gives
relatively good
accuracy and
precision.
 Separation and
analysis of sample
very quickly
 Sample with less
quantity is also
 Only volatile sample or
that sample which can
be made volatile are
separated by this
method.
 During injection of the
gaseous sample proper
attention is required.
 The sample of gas which
is about to inject must be
thermally stable so that it
does not get degraded
when heated.
Advantages Disadvantages

9. High performance liquid
chromatography
 HPLC is a product of the scientific effort
towards optimization of the conventional
column chromatography.
 This method uses an extremely high pressure.
The flow rate therefore is high and the
experimental time is shortened considerably.
 This technique is suitable for both analytical
and preparative purposes.

10.  The technique may be used with small
amounts of sample(pico or even femtogram)
 It is popular for the separation of polar
compounds such as drug metabolites, which ,
in general are poorly resolved by other
techniques.
 Thus, it may apply the principle of adsorption,
partition, ion-exchange, exclusion, and affinity
chromatography.

11. Working principle
 It is a technique for separation identification
and quantification of components in a mixture.
 It is especially suitable for compounds which
are not easily volatalised, thermally unstable
and have high molecular weights.
 The liquid phase is pumped at a constant rate
to the column packed with the stationary
phase.
 Before entering the column the analysis
sample is injected into the carrier stream.

12.  On reaching the column the sample
components are selectively retained on the
basis of physico-chemical interactions
betweeen the analyte molecules and the
stationary phase.
 The mobile phase moving at the steady rate
elutes the component based on the operating
conditions.
 Detection techniques are employed for
detection and quantification of the eluted
components.

13. Instrumentation

14. Application
 Pharmaceutical applications.
 Environmental applications.
 Applications in forensics.
 Food and flavor.
 Application in clinical tests.

15.  It offers a quick, automated
and highly accurate method
to identify certain chemical
components in a sample
 It is highly efficient and uses
a pump rather than gravity,
to force a liquid solvent
through a solid adsorbent
material
 It is accurate and highly
reproducible.
 HPLC run cans can be
performed with minimal
training.
 HPLC is costly,
complex and doesn’t
work for all samples
 HPLC does have low
sensitivity for certain
compounds, and
some cannot be
detected as they are
irreversibly adsorbed.
Advantage Disadvantage

16. REFERENCES
 VASANTHA PATTABHI,
N.GAUTHAM.2002.BIOPHYSICS.SEPARATIO
N TECHNIQUES.PAGE NUMBER-24-31.