derivatization technique

1. DEREVETIZATION TECHNIQUE IN GC
Presented by
Sumit Kumar kar
Redg.no:170523886013
M .Pharm
Pharmaceutics

2. INTRODUCTION:
 Derivatization is the process of chemically modifying a
compound to produce a new compound which has properties
that are suitable for analysis using a GC or HPLC.
 The chemical structure of the compound remains the same and
just modifies the specific functional groups of reacting
compound to a derivative of deviating chemical and physical
properties in order to make them detectable and analyzable.
 The compound which is chemically modified is known as the
Derivative.
 Derivatization is needed in GC, HPLC, UV-Visible spectroscopy
etc.

3. WHY DERIVETIZE IN GC?
 To permit analysis of compounds which are not directly amenable to
analysis due to inadequate stability and volatility.
 To improve chromatographic detectability.
 The main reason for derivatizing is to impart volatility to non-volatile
compounds.
 Derivatization is useful to allow the use of GC & GC/MS on sample that
would other wise be not possible in various areas of chemistry such as
medical, forensic & environmental.

4. IMPORTANCE OF DERIVETIZATION
 Increases volatility
 Eliminates the presence of polar OH, NH & SH groups.
 Derivatization targets O, N, S and P functional groups (with hydrogen available).
 Enhances sensitivity for ECD
 The introduction of ECD detectable groups, such as halogenated acyl groups, allows detection of
previously undetectable compounds. In general, halogen substituent increases the sensitivity in
electron capture detector (ECD) in the order I > Br > Cl > F.
 For aromatic compounds, the substituents affect the sensitivity of the ECD according to their
electron withdrawing capability. Strong electron withdrawing groups such as NO2 increase the
sensitivity of the detection, while electron donating groups reduce it.
 Increases detectability, i.e. steroids
 Increases stability (thermostability)

5. CONDITION FOR CHOOSING A DERIVETIZING AGENT
 The derivatizing agent must be stable.
 The derivatizing agent and its products formed during derivatization should not
be detectable or must be separable from analyte.
 The analyte should be reactive with the derivatizing agent under convenient
conditions.
 Derivatizing agent should be non-toxic.

6. TYPES OF DERIVETIZATION
1. SILYLATION
2. ALKYLATION
3. ACYLATION
4. CHIRAL DERIVATIZATION

7. 1. SILYLATION
 Most prevalent method, readily volatizes the sample.
 Mechanism:
 This process produces silyl derivatives which are more volatile
and thermally stable.
 Replaces active hydrogen with TMS (trimethyl silyl groups)
 Silylation occurs the nucleophilic attack (SN2). The better the
leaving group, the better the silylation.

8.  SN2 REACTION MECHANISM:
 Order of reactivity of functional group towards silylation:
 1° Alcohol > 2°alcohol > 3°alcohol > phenol > Carboxyl > Amines >
Amides > Hydroxyl
NOTE:
 Column, which have active hydrogen in the stationary phase cannot
be used in silylation process, because it derivatize the silylating
agent.

9. ADVANTAGES
 Ability to silylate a wide
variety of compounds.
 Easy to prepare.
DISADVANTAGES
 Silylating agents are
moisture sensitive.
 Must use aprotic solvent.

10. MECHANISM OF SILYLATION

11. SILYLATING REAGENTS AND THEIR MECHANISM

14. 2.ALKYLATION
 Alkylation reduces molecular polarity by replacing active
hydrogens with an alkyl group. These reagents are used
to modify compounds with acidic hydrogens, such as
carboxylic acids and phenols. These reagents make
esters, ethers, alkyl amines and alkyl amides.
 The principal reaction employed for preparation of these
derivatives is nucleophilic displacement.

15. ADVANTAGES
 Alkylation derivatives are
stable.
 Reaction condition can vary
from strongly acidic to
strongly basic.
 Limited to amines and acidic
hydroxyls.
 Reagents are often toxic.
DISADVANTAGES

16. MECHANISM OF ALKYLATING AGENT

19. 3.ACYLATION
• Acylation reduces the polarity of amino, hydroxyl and thiol
groups and adds halogenated functionalities for ECD. In
comparison to silylating reagents, the acylating reagents target
highly polar, multifunctional compounds, such as carbohydrates
and amino acids.
 Acylation converts these compounds with active hydrogens into
ester, thioesters and activated acyl amide reagents.
 The anhydrides and acyl halides reagents form acid by-products,
which must be removed before GC analysis.
 Acylation is normally carried out in pyridine, tetrahydrofuran or
other solvent capable of accepting the acid by-product.

20. ADVANTAGES
 Addition of halogenated
carbons increased
detectability by ECD.
 Derivatives are hydrolytically
stable.
 Increases sensitivity by
increasing molecular weight.
DISADVANTAGES
 Acylation derivatives are
difficult to prepare.
 Acid by-products needs to be
removed before analysis.
 Acylation reagents are
moisture sensitive.
 Reagents are hazardous.

21. ACYLATING REAGENTS AND THEIR
MECHANISM

23. 4.CHIRAL DERIVETIZATION
 These reagents target one specific functional group and
produce individual diastereomers of each of the
enantiomers.
 There are two ways of separating enantiomers by
chromatography:
1. Separation on an optically active stationary phase.
2. Preparation of diastereomeric derivatives that can be
separated on a non-chiral stationary phase.

24. REAGENTS:
1. TPC (N-trifluoroacetyl-L-prolyl chloride)
 Used for optically active amines, most probably amphetamines.
2.MCF ((-) menthylchloroformate)
 Used for optically active alcohols.

25. FUNCTIONAL GROUP AND THEIR DERIVETIZING
METHOD