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S U B : - I N S TRU M E NTA L M E T HO D O F A N A LYS I S
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R A I GA D C O L LE GE O F P H A R M A C Y, M AHA D
 GC is a valuable technique for analyzing compounds of pharmaceutical interest.
 GC is probably the most utilized of all the Chromatographic technique.
 Derivatization:-
 It 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.
 .A modified analyte in this case will be the product, which is known as the
derivative.
 The derivative may have similar or closely related structure, but not the same as
the original non-modified chemical compound.
 Derivatization is done due to:-
 To permit analysis of compounds which cannot be directly amenable analyzed (for
eg; inadequate stability and volatility.)
 To improve chromatographic behavior or detectability.
 The main reason for derivatizing is to impart volatility to otherwise non-volatile
compounds.
 Derivatization invariably involves a chemical reaction.
 Hence, it is important to remember that one can obtain an appropriate derivative
by selecting suitable reaction conditions; however, the derivative should be
sufficiently volatile for GC analysis.
 A Derivatization reaction should be rapid, quantitative, and produce
minimal by product.
 Excess reagent should not interfere with the analysis and should be easily
removed
 Advantages:
i. It increases volatility.
ii. It enhances sensitivity for detectors.
iii. Increases detectability.
iv. It increases stability.
 Disadvantages:
i. Derivatization often is a last resort when developing a method.
ii. Introduction of a reaction pre or post column, increases complexity, chances of
error and total analysis time
There are four types such as:-
i. Silylation
ii. Alkylation
iii. Acylation
iv. Chiral derivatization
 Mechanism:-
 This process produces sily derivatives which are more volatile, more thermally
stable.
 Replaces active hydrogens with TMS (trimethyl silyl groups).
 Note:
a. Solvent used should be as pure and as little as possible as it will eliminate the
excessive peaks and prevent a large solvent peak.
b. Ease of reactivity functional group:-
Alcohol > Phenol > Carboxyl > Amine > Amide/Hydroxyl.

 Mechanism: N,O-bis (trimethylsilyl)acetamide (BSA).

 Advantages:
 Ability to silylate a wide variety of compounds.
 Large number of silylating reagents available and easily prepared.
 Disadvantages:
 Silylation reagents are moisture sensitive.
 Must use aprotic (no proton available) organic solvents
 Alkylation reduces molecular polarity by replacing active hydrogens with an alkyl
group
 It represent the replacement of active hydrogen by
an aliphatic or aliphatic-aromatic(eg; benzyl)
group in process referred as Esterification.
 The principal reaction employed for preparation
of these derivatives is Nucleophilic displacement.
 Eg. Trimethylanilinium hydroxide (TMAH).
 Advantages:-
i. Wide range of alkylation reagents available.
ii. Reaction conditions can vary from strongly acidic to strongly basic.
iii. Some reactions can be done in aqueous solutions.
iv. Alkylation derivatives are generally stable.
 Disadvantages:
i. Limited to amines and acidic hydroxyls
ii. Reaction conditions are frequently severe.
iii. Reagents are often toxic
 An acyl group is introduced to an organic compound.
 Acylation reduces the polarity of amino, hydroxyl, and thiol groups and adds
halogenated functionalities for Electron Capture Detector.
 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 esters, thioesters and amides.
 Advantages:
i. Derivatives are hydrolytically stable.
ii. Increased sensitivity by adding molecular weight.
 Disadvantages:
i. These derivatives can be difficult to prepare.
ii. Reaction products often need to be removed before analysis.
iii. Acylation reagents are moisture sensitive
 Pre-Column Derivatization:-
 This is done to improve some properties of the sample for separation by column.
 By this derivatization technique, the components are converted to more volatile and thermostable
derivatives.
 The conditions for pre-column derivatization is as follows:
a. The component is less volatile.
b. The compounds are thermolabile.
c. To improve separation factor.

 Post-Column Derivatization:-
 It is done to improve the response shown by the detector.
 The components may not be detected by detectors unless derivatization is done.
 The components may be converted in such a way that their ionization or affinity towards electrons
is increased.
 A temperature programming facilitates controlled increase of even temperature
during an Analysis.
 Thus, the peaks also become sharp and emerge quickly.
 Therefore, in Temperature programming the components of a wide boiling range
mixture may be resolved efficiently.
 The temperature programming may be carried out in three different modes such
as;
a. Neutral
b. Linear
c. Matrix or Multilinear
 In general, the operation with linear temperature programme is more common.
 The requirements for good temperature programming are discussed below:
i. Dual column system. This compensates for bleeding of liquid phase from
columns during increase of temperature.
ii. Separate heaters for injector, column and oven and detector system.
iii. Thin walled columns.
iv. Low liquid phase loading.
v. Pure dry carrier gas.
vi. Low mass column oven for
rapid heat transfer.
 https://www.slideshare.net/banuman35/derivatization-in-gchplc-by-pravisankar-
vignan-pharmacy-c
 https://www.slideshare.net/suraj_mindgamer/derivatization-in-gc
 https://www.sciencedirect.com/sdfe/pdf/download/eid/1-s2.0-
S0022354915406240/first-page-pdf
 https://www.kopykitab.com/Instrumental-Methods-Of-Analysis-by-Dr-Kavitha-P-N-
Mrs-Vijayanthimala-P