GAS CHROMATOGRAPHY-DHANYA

8/7/2019 GAS CHROMATOGRAPHY-DHANYA

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Seminar on

Derivatization technique in gaschromatography

&

Application of gas chromatography

1



Derivatization

Derivatisation is a technique of treatment of the sample toimprove the process of separation by column or detectionby detector

Two types:

Precolumn derivatisation : For improve separation bycolumn

postcolumn derivatisatisation : For improve response bydetector.

2



Precolumn derivatisation:

Here the compound is converted into more volatile

and thermostable derivatives.

Improve the separation and less tailing will be seen.

In the following condition, pre derivatization is done:

The compound is less volatile

The compounds are thermolabile

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To reduce tailing

To improve separation factor

Post column derivatization:

Improve the response shown by detector

The compound may be converted in such a way thattheir ionisation or affinity towards electrons is

increased.

4



GC samples are usually derivatized to render highly polar

materials sufficiently volatile so that they can be eluted at

reasonable temperatures without thermal decomposition or

molecular rearrangement.

Derivatization method in GC.

acylation

alkylation.

Esterification.

5



The ideal derivatization procedure will:

Accomplish the desired modification.

Proceed quantitatively, or at least reproducibly.

Produce products that are readily distinguishable and separable

from the starting materials.

Proceed rapidly with simple and straightforward laboratory

techniques, and will be both selective and applicable to a

number of similar compounds.

6



Involve reagents and reactions that present no unusual hazards.

Compounds containing functional groups with active hydrogen

(-COOH, -OH, -NH and -SH) are usually derivatized for

analysis by gas chromatography.

These functional groups have a tendency to form

intermolecular hydrogen bonds that affect the volatility, their

tendency to interact deleteriously with column packing

materials and their thermal stability.

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Esterification:

Acids can be esterified by treating them with an

appropriate alcohol using an inorganic acid as

catalyst.

Hydrochloric acid was popular for this purpose.

Derivative must be involatile not to allow loss whenremoving the excess alcohol

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Method: Treat one or two milligrams of the acid contained

in a 125 µl of either methanol or ethanol that contains 3M hydrochloric acid and heat at 65ÛC for about 35 minutes.

A stream of nitrogen is bubbled through the reaction mixture to

remove the alcohol.

Amino acids are more difficult to derivatize but can also be

esterified in a comparable manner.

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Method:

A few milligrams of the amino acid mixture is mixed with 2

ml of 4M alcoholic methanol and heated at 70ÛC for 2

hours. excess methanol is then removed by evaporation in a

stream of nitrogen.

Any remaining water is removed by adding a little

dichloromethane (ca 150 l) and repeating the evaporation

process.

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Esterifying reagent is diazomethane.

yellow gas used in the form of an ethereal solution.

Its reacts with an organic acid in the following manner,

R² COOH + CH2N2 R² COO±CH3 + N2

When the reaction is complete, the yellow color

persists .

The reagent acts as its own indicator.

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Apparatus for Generating Diazomethane for Esterification12



Procedure:

Nitrogen is bubbled through ether so that the gas is

saturated with ether and then passed through a vesselcontaining a solution of N± methyl± N± Nitroso± p

toluene sulfonamide.

When potassium hydroxide, is added through a

dropping funnel, diazomethane is generated.

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The nitrogen containing the diazomethane is passed

into a solution of the sample until an yellow color

persists

The sample solution consists of 0.5 to 30 mg of acid

dissolved in 2 ml of a 10% solution of methanol in diethyl

ether.

About 4 ml/ min. of nitrogen is used and after the

reaction is complete, the solvent is removed by

evaporation.14



Acylation Reactions

Acylation is also a popular reaction for the production of

volatile derivatives of highly polar and involatile organic

materials.

Advantages.:

Reduces the polarity of the substance

Improve the peak shape and reduce peak tailing.

By inserting protecting groups into the molecule, acylation

also improves the stability of those compounds that are

thermally labile.15



Acylation can render extremely polar materials such as

sugars amenable to separation by GC

Acylation is frequently used to derivatize amines, amides,

alcohols, thiols, phenols, enols, and glycols etc««.

Example of acylation is the reaction between acetic

anhydride and an alcohol

R ±CO±CO-R + R'±O±H = R ±CO±O± R' + R-CO-O-H

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Method:

About 5 mg the acid is dissolved in 5 ml of

chloroform together with 0.5 ml of acetic anhydride

and 1 ml of acetic acid and is heated for 2-16 hours at

50ÛC.

The excess reagents are removed under vacuum and

the residue dissolved in chloroform and injecteddirectly onto the column.

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Application of Gas Chromatography

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Principle application:

Qualitative

Quantitative analysis

1.Qualitative analysis:

By comparing the retention times or volume of the unknown to

the retension time or volume of a serious of std.

By collecting the individual components as they emerge from

the chromatography & subsequently identifing these

component by other method.

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2.Quantitative analysis:

Analysis of individual component in a mixture.

GCD: Size of the peak to the amount of compound which has

passed through the detector .

Method involved:

External standardization or calibration curve method.

Internal standardization.

Direct comparison method.

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Internal standardization.

It involves addition of a compound called internal standard

Internal standard have following requirements:

It should be structurally similar to the compound to be

analyzed.

It should be separable from test compound

Internal std peak should be similar to the sample peak.

Area of the peak to the amount of compound in a sample.

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Direct comparison method.

By injecting a sample and std separately and compare

the peak area.

Area of the peak= peak height× width of peak at half

height.

A1/A2= W1/W2

= Response factor

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3.Checking the purity of a compound

4.Presence of impurities.

5.Isolation and identification of drugs or metabolites in

urine, plasma, serum etc can be carried out.

6.Isolation and identification of mixture of compoundlike aminoacid, plant extract, volatile oils etc.

7.Multicomponent analysis

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Gasoline

a multi component mixture containing a large number of hydrocarbons, with very similar molecular weights

Exclusively dispersive in interactive character.

The structure of many of the hydrocarbons very similar andmany isomers present.

Due to their interactive similarity the separation factorsbetween individual components is very small.

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Chromatogram of

Gasoline

1. Isobutane2. n-Butane3. Isopentane4. n-Pentane

5. 2,3-Dimethylbutane6. 2-Methylpentane7. 3-Methylpentane8. n-Hexane9. 2,4-Dimethylpentane10. Benzene11. 2-Methylhexane12. 3-Methylhexane13. 2,2,4-Trimethylpentane14. n-Heptane15. 2,5-Dimethylhexane16. 2,4-Dimethylhexane

17. 2,3,4-Trimethylpentane18. Toluene19. 2,3-Dimethylhexane20. Ethylbenzene21. m-Xylene22. p-Xylene

23. o-Xylene25



REFERANCE

Practical pharmaceutical chPractical pharmaceuticalchPrinciples of instrumental analysis,5th

edition,Skoog,Holler.

Vogel¶s textbook of chemical analysis

Practical pharmaceuticalchemistry,A.H.Beckett,J.B.Stenlake

Text book of pharnaceutical analysis by Dr.S. RaviSanker.

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About 5 mg the acid is dissolved in 5 ml of chloroform

together with 0.5 ml of acetic anhydride and 1 ml of acetic

acid and is heated for 2-16 hours at 50ÛC. The excess reagents

are removed under vacuum and

the residue dissolved in chloroform and injected directly

onto the column.

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