Chromatography is a technique primarily used for separating mixtures and components at micro or nano levels, developed by Russian botanist Michael Tswett in 1904. It operates on the principle of differing affinities of analyte components for stationary and mobile phases, allowing for sensitive quantitative analysis. Modern chromatography utilizes detectors for non-colored analytes, and parameters like temperature, polarity, and molecular weight affect retention times during separation.

1. Chromatography
little to do with colours but
everything with seperating mixtures.

2. Micheal tswett (Russian botanist)
father of chromatography
1904 published his work in seperating
the color pigements of plants.
His Techniques worked only for
substance having native color.

3. Chromatography helps in
• Separate components from complex mixtures.
• Quantitative analysis of a substance.
• Seperation at micro/nano level.
• Providing large temprarure ranges.
• More sensitive than extractions method.
• Separate millionth of a gram material.

4. Working principle
• All types of chromatographic methods work
on same principle.
• Differ in experimental set-up or analytical
details.
• Stationary phase could be
• 1. finely divided solid material.
• 2. viscous liquid
• Within a column of glass, metal or plastic.

5. Continue……..
• Mobile phase could be
• Liquid , liquid solution or gas under pressure.
• MP move through SP carrying analyte mixture.
• Movement of analytes components depends
on their affinity for SP.
• This helps in the seperation of analyte
component from other components along the
way.

6. Continue…….
• This means, progress of each component of
analyte will be affected by SP in different ways
from all others components.
• SP designed to attract or repel certain
member of analyte, each one in different way.
• When MP completed its journey through the
column, various components have been held
by the SP and form color bands at various
points in column.

7. Modern chromatography
• Mostly analyte component do not produce
color.
• Detectors are placed for the identification of
their seperating points on SP.
• Detectors use various properies of analytes to
signal their presence.
• Narrow bore cappilary have been developed
for improved seperations and efficiency.

8. Relationship b/w SP and MP on the
basis of polarity.
Normal phase
chromatography
• Mobile phase is less polar
than the stationary phase.
• Always in GC type
chromatography.
• Where MP is innert gas.
Reverse phase
chromatography
• Mobile phase is more polar
than s phase.
• In TLC and LC both reverse
and normal phases are
used.

9. Lets visualise how chromatography
works
• Group of tourists= analyte mixture
• Bus= mobile phase
• Destination= location on stationary phase

10. Gas chromatography
• Gas-liquid chromatography or cappillary GC.
• SP is in narrow , hollow tube made up of glass
or synthetic polymer coated with plastic to
add strength.
• Stationary phase
Thick, high boiling point viscous liquid form thin
coating inside of the cappilary and stable at 350
degree celcius.
• Column of GC= 30m long or more.

11. Continue……
• Mobile phase
Innert gas: nitrogen ,helium,hydrogen.
• In GC, mobile phase is forced through the
stationary phase under pressure.
• Since analyte carried by MP have to be in vapor
phase.
• Some compounds decompose at high temp and
not suitable for GC analysis e;g. explosives.
• Innert gas purges the oxygen out of system
therefore no chance for analytes to burn when
heated. But leakage in system can cause
explosion.

12. Parts of the gas chromatogram

13. 1. injector
• Heating chamber.
• Analyte mixture introduction.
• Mixture combined with mobile phase.
• MP continually passing through injector under
pressure.
 in GC type, experiment the analyte mixture is
dissolved in a volatile solvent.
For instance, methlyl alcohol is suitable solvent for
seperating coccaine, caffeine and sugar.

14. Injectors configuration
Splitt injector configuration
• Cappillary column are very
thin and easily overloaded
with the analyte so the
mobile phase will splitt so
that some of it vented away
and a part of it goes
through the column.
Splitless injector configuration
• If the analytes are very
dilute or there is very little,
then the entire amount is
sent through the column in
a splitless configuration

15. Stationary phase
• As MP passes through the SP various
components of the analyte mixture exposes to
the surface of SP.
• Two principle determine how long it will take
a substance to traverse the entire column.
1. Molecular weight low MP traverse faster.
2. Polarity polarity b/w the sp and analytes.

16. Retention time and degree of
seperation of analytes depends
1. Varying polarity of stationary phase.
2. Speed of mobile phase.
3. Temprature of mobile and stationary phase.
 retention time is the time, analytes traverse the
stationary phase until reaches the detec tor.
on the basis of differences in structure and
polarities of caffeine and coccaine , they are
seperated.

17. Continue….
• Rule of thumb for GC is that, every 10 degrees
rise in temp of experiment halves the
retention time.
• Therefore temp is powerful factor in designing
an experiment to separate complex mixtures.
• Variation in weights of analytes will be
seperated when oven is set at low temprature
and gradually rises during the run.

18. Detector
• Once a component travers all the way through
the sp it has to be detected.
• Each component is a vapor mixed with innert
mobile phase.
• Detectors are designed so that they donot
respond to the pure mobile phase
• and donot show activity until the mobile
phase contain one of the analytic component.

19. Chromatogram( plot of response of
detector versus retention time)
• Analytes reach the detector
• It convert signal to small electric currents.
• Current is amplified and computerized.
• Each component is displayed as a triangular
peak.
Retention time = top of the peak.
Concentration of component = area under peak.

20. Continue….
• Ideally, each substance in the mixture would
take different amount of time to traverse the
column and reach the detector.
• In every GC, retention time would be same of
same substance maintaining the other
conditions similar.
• It is said that tentative identification of a
substance can be done with retention time.
But it is not reliable.