Gas chromatography by Mr. Vinayak Bodhankar

Introduction
Principle & theory
Instrumentation
Derivatization
Temperature programming

1. Mr. Vinayak R. Bodhankar—
M. Pharm. Ph.D* I

2. Content

> Introduction

> Principle & theory
> Instrumentation

> Derivatization

> Temperature programming

3. QI Introduction & History

> It is process of separating components from the mixture by using gaseous
mobile phase.

> German chemist Erica Cremer developed the theoretical foundation of GC in
1947.

> Archer Martin & Richard Synge received a Nobel Prize in 1952 for the
invention of partition chromatography.

> John Martin: Father of modern chromatography

< Types of Gas Chromatography

Stationary phase may be solid or liquid.

1. Gas solid chromatography (GSC)

Tnert gases are used (light weight). e.g. H, He, Ar
2. Gas liquid chromatography (GLC)

Nitrogen is commonly used.

4. Q Principle & Theory

Y” It involves separation of components of sample due to partition in between
gaseous mobile phase and liquid/solid stationary phase.

v Mixture of components to be separated are converted into vapors and mixed
with gaseous mobile phase.

v Components separated by Partition phenomenon.

2 Ne ale —— Blute faster i.e, Less time |
Components
> Less soluble ———-|Elute slower ie: More time)

Y” No two components having the same partition coefficient so the components
are separated according to their partition coefficient.

y” Elution rate depends on solubility in mobile phase i.e. partition phenomenon.

5. Instrumentation

Gas Chromatogr

flow control
valve

o

chromatography
column

Carrier
gas

computer data

6. 1. CARRIER GAS

The cylinder/ gas tank is fitted with a pressure controller to control the pressure of
gas, a pressure gauge that indicates the pressure, a molecular sieve to transfer filtered
dry gas and a flow regulator to ensure a constant rate of flow of mobile phase to the
column.

It should meet the following criteria:

Y” Should be chemically inert

Y Should be cheap and readily available

v Should be of high quality and not cause any fire accidents
v Should give best possible results

Y Should be suitable for the sample to be analyzed and for the detector

7. sit
|

> Hydrogen, helium, nitrogen blo dioxide
> Hydrogen has low deny ande < ( ity. However, it reacts
plosive.in.nature.

I

> Nitrogen is igexpensivabpt IO
e ai |

> He is the.most prefer

8. 2. SAMPLING UNIT

> Sampling unit or injection port is attached to the column head.

> Since the sample should be in vaporized state, the injection port is provided
with an oven that helps to maintain its temperature at about 20-50” C above
the boiling point of the sample.

> Gaseous samples may be introduced by use of a gastight hypodermic needle
of 0.5-10 ml capacity.

> For Liquid samples, micro syringes of 0.1-100uL capacity may be used.

Se
Carrier gas => (Eim

‘|

Glass insert

=> Split

Heater = cage

0.25 mml.D.x30 m df=0.25 um " Column

WS

9. Injections of samples into capillary columns

a. Split injectors

> It splits the volume of sample stream into two unequal flows by means of a needle
valve, and allow the smaller flow to pass on to the columns and the bigger part is
allowed to be vented to the atmosphere.

This technique is not suitable when highest sensitivity is required.

. Splitless injectors

They allow all of the sample to pass through the column for loading.

Vv S&S Vv

Sample should be very dilute to avoid overloading of the column & high capas
column such as SCOT or heavily coated WCOT column should be used.
c. On column injectors

> A syringe with a very fine quartz needle is used.
> Air cooled to -20% below the B.P. of sample.

> After that warmer air is circulated to vaporize the sample.

10. d. Automatic injectors:

> For improving the reproducibility and if a large number of samples are to
be analyzed, then automatic injectors are used.

> The solid samples are introduced as a solution or in a sealed glass ampoule,
crushed in the gas stream with the help of a gas tight plunger, and the
sample gets vaporized and flows into column under the influence of carrier

gas.

11. 3. LIQUID PHASE / STATIONARY PHASE
It should have the following requirements:

> It should be non-volatile

> Should have high decomposition temperature

> Should be chemically inert

> Should posses low vapor pressure at column temperature

> Should be chemically and structurally similar to that of the solute i.e., polar

for polar solute.

12. 4. COLUMN

> Columns are of different shapes and sizes that includes: "U" tube type or
coiled helix type.

> They are mainly made of copper, stainless steel, aluminium, glass, nylon

and other synthetic plastics.

Support material
> It's main function is to provide mechanical support to the liquid phase.
> An ideal support should have a large surface area, chemically inert, should

be thermostable.

> Commonly used solid phases are: diatomaceous earth, glass beds, porous

polymers, sand, etc.

13. Polyimide Coating

Fused Silica

Stationary Phase

1 = I
, Inner Diameter .

<— Outer Diameter —>'

14. Types of columns

> There are two general types of columns:

1. Packed columns

In GLC, they are densely packed with finely divided, inert, solid support

material (diatomaceous earth) coated with liquid stationary phase.

> In GSC, the columns are packed with adsorbents or porous polymers.
= Length: 1.5-10m

= Internal diameter: 2 - 4 mm.
2. Capillary columns

= Length ranges from 10-100 m

= Inner diameter is usually 0.1-0.5mm.

15. Q Types of Capillary column

< Wall coated open tubular columns (WCOT)

It consist of a capillary tube whose walls are coated with liquid stationary
phase.

< Support coated open tubular columns (SCOT)

The inner wall of the capillary is lined with a thin layer of support material
such as diatomaceous earth, onto which the stationary phase has been
adsorbed.

> It is also known as PLOT (porous layer open tubular columns).

+ SCOT columns are generally less efficient than WCOT columns.

> Both types of capillary column are more efficient than packed columns.

16. Solid support coated
with liquid phase

Porous
Adsorbent

Liquid phase

Wall-coated Open Tubular Support-coated Open Tubular Porous Layer Open Tubular
(WCOT) (SCOT) (PLOT)

Fig. Types of Capillary Column

17. Q Equilibrium of the column

> The packed columns are equilibrated before introduction of the sample.

> This is done by allowing continuous flow of heated carrier gas through the
columns for a specific duration of time (24hrs) at prescribed temperature.

> Ideally, prepared and conditioned columns show a zero base line on the

recorder upon passage of carrier gas alone.

U Column temperature

> This can be controlled by jackets equipped with vapors of a boiling li
electrically heated metal blocks or circulating air baths.

> Compounds of low B.P eluted at lower temperature

> Compounds of high B.P boils at higher temperature resulting broader

peaks, require temperature programming.

18. 5. DETECTORS

> The eluted solute particles along with the carrier gas exit from the column and
enter the detector.

> The detector then produces electrical signals proportional to the concentration
of the components of solute.

> The signals are amplified and recorded as peaks at intervals on the

chromatograph.

Q Properties of an ideal detector

y” Sensitive, Stable and reproducible

Y” Should operate at high temp. (0-400 °C)
Y” Linear response

v Wide dynamic range

v Fast response time

Y” Non-destructive

19. I. Thermal conductivity detector
"TCD is based upon the fact that the heat lost from a filament depends upon the thermal

conductivity of the stream of surrounding gas as well as its specific heat.”

Carrier

Sample y gas
gas

(exiting the column)

20. > When only carrier gas flows heat loss to metal block is constant, filament
temperature remains constant.

> When an analyte species flows past the filament generally thermal conductivity
changes, thus resistance changes which is sensed by Wheatstone bridge
arrangement.

> The imbalance between control and sample filament temperature is measured and
a signal is recorded.

«+ Advantages

> Simple and inexpensive

> Durable and posses long life

> Accurate results

> Non-selective, hence known as universal detectors

+ Disadvantages

> Low sensitivity

> Affected by fluctuations in temperature and flow rate.

21. II. Electron capture detector

> Molecules of compounds, which posses affinity for electrons, differ in their electron
absorbing capacities.

> This difference is utilized in this detector for identification of the compounds.
Working: A foil made up of a radioactive metal like Ni63 is placed inside a Teflon
coated cell which also contains a cathode and an anode.

> In the absence of organic species, the produced electrons migrate towards positive
electrode and produce a certain constant standing current.

> When a sample/eluent is present it captures the electrons, elutes from column, there
is a drop in this constant current.

> The potential across two electrodes is adjusted to collect all the ions and a steady

saturation current, is recorded.

anode (+)

carrier — carrier

cathode (-) B-emitter

22. O Advantages

= Highly selective

= Highly sensitive for the detection of compounds like halogens, quinones, peroxides,
nitrites, etc.

= Itis non-destructive

= More sensitive than TCD and FID.

U Disadvantages
= Least sensitive to compounds whose molecules have negligible affinity for electro:

= Carrier gas used should be of pure form like pure nitrogen.

23. TIT. Flame ionization detector

> This employs hydrogen flame that is
maintained in a small cylindrical jet made up
of platinum or quartz.

> Effluent from the column with helium or
nitrogen as carrier gas are fed into the
hydrogen flame, gets ignited and undergoes
pyrolysis to produce ions.

> For detection of these ions, two electrodes are
used that provide a potential difference.

> The ions produced are repelled by the positive
electrode which hit the collector plate.

> The current produced in doing so is amplified

and fed to an appropriate recorder.

Collector —}

Nozzle —}

Column

Electrical signal

Data processor

Hydrogen flame

24. IV. Flame photometric detector
> It is a selective detector that is responsive to

compounds containing sulphur or phosphorous Exhaust

Light Optical

Reflector | Filter

> The detection principle is the formation of
excited sulphur (S*) and excited hydrogen he
phosphorous oxide species (HPO*) in a
reducing flame.

> A photomultiplier tube measures the Era
characteristic chemiluminescent emission from oii
these species.

> The optical filter can be changed to allow the
photomultiplier to view light of 394 nm for
sulphur measurement or 526 nm for

phosphorus.

25. Q Derivatization

Y” It is the process of chemically modifying a compound to produce a new
compound which has properties that are suitable for GC analysis.

Y” The chemical structure of the compound remains the same and just
modifies the functional groups of the reacting compound in order to make
them detectable and analyzable.

Y” Derivatization is needed in GC, HPLC, UV-Vis spectroscopy.

+ Objective of Derivatization

Y” To permit analysis of compounds which are not directly amenable to
analysis due to inadequate stability and volatility.

v To improve chromatographic detectability.

In many cases, sample of interest is undetected, as a result it may be necessary

to derivatize the compound before GC analysis. The main reason for

derivatization is to impart volatility for non-volatile compounds.

26. « Ideal Characters & Disadvantages of Derivatization

> A derivatization reaction should be rapid, quantitative.

> Excess reagent should not interfere with the analysis and should be easily removed.

> Introduction of reaction pre or post column increases complexity, chance of error
and total analysis time.

> Care should be taken that the reaction is quantitative and no additional impurities
are introduced into analysis.

Benefits of Derivatization

1. Increases volatility

v Eliminates the presence of polar groups (OH, NH)

Y” It targets Hetero atoms

2. Enhances sensitivity for ECD

3. Increases detectability

4. Increases stability

27. Q Conditions for selection of derivatizing agent

= Jt must be stable

= Jt should be non toxic

= The procedure should be adaptable to automation.

= The analyte should be reactive with it under convenient conditions

Q Types of Derivatization

1.
2. Alkylation
3.
4

. Chemical derivatization

Silylation

Acylation

28. Q Temperature Programming

Y” It refers to changing the temperature of the column during chromatography
analysis.

Y Its done using a gas chromatography oven, which provides a stable thermal
environment for the analysis.

Y Changing the temperature during the analytical run has an effect on analysis

mainly on RT and selectivity.
Retention time
Y” Increasing the temp. affect the RT that’s because less volatile components ar,
earlier.
Y” Its estimated that an increase of 30% will cut retention time by half.
Selectivity
Y” Changing the temp. alter components relative position to improve

v With adsorbent, for example SP can becomes more polar with