DERIVATIZATION IN

ANALYTICAL
TECHNIQUES
 DERIVATIZATION
 Derivatization is a technique used in chemistry which
converts a chemical compound into a product (the
reaction's derivate) of similar chemical structure, called a
derivative.
 Generally, a specific functional group of the compound
participates in the derivatization reaction and transforms
the part of compound to deviate reactivity, solubility,
boiling point, melting point, aggregate state, or chemical
composition.
 DERIVATIZATION IN
CHROMATOGRAPHY
 In gas chromatography, this technique increases the
volatility and temperature stability of compound.
 Polar solutes interact strongly; hence more thermal
energy is needed to separate polar molecules.
 Derivatization reactions will be used to decrease solute
polarity, the associated decrease of boiling point will
make GC separation possible.
 DERIVATIZATION IN
CHROMATOGRAPHY
 It block certain functional groups (hydroxyl and
carboxylic acid) that cause excessive tailing through
hydrogen bonding.
 In liquid chromatography derivatives are prepared to
block hydrogen bonding sites and to reduce compound
polarity.
 DERIVATIZATION IN SPECTROSCOPY
 Most drugs absorb weakly in UV- region in spectroscopy.
 A reagent species is added to the weakly absorbing drug
solution, which on reaction, modifies the absorption
spectrum of the reagent.
 For enhancing the electron affinity in MS, the
derivatization with reagents containing fluorinated
moieties is practiced which increases the sensitivity of
separation.
 DERIVATIZATION IN SPECTROSCOPY
 This technique is useful for compounds which show
spectral overlapping in MS
 Derivatization with the purpose of obtaining a compound
that forms more informative fragments in the mass
spectrum can be very useful for compounds having mass
spectrum that matches more then one compound.
 TYPES OF DERIVATIZATION
 There are three basic types of derivatization reactions:

1. Silylation
2. Acylation
3. Alkylation
1. SILYLATION
Mechanism
 Silylation is the introduction of a silyl group into a molecule,
usually in substitution for active hydrogen (-OH, -SH, and –
NH) groups.
 Compounds containing active hydrogen atoms amendable to
silylation are acids, alcohols, thiols, amines, amides, and
enolizable ketones and aldehydes.
 Silylation occurs the nucelophilic attack (SN2). The better the
leaving group, the better the silylation.
 Reaction
Sample-OH + R3Si – X Sample-O-Si-R3 + HX
IMPORTANCE OF SILYLATION
 Replacement of active hydrogen by a silyl group reduces
the polarity of the compound and reduces hydrogen
bonding.
 Silyl derivatives generally are more volatile, less polar,
and thermally more stable.
 Many hydroxy and amino compounds regarded as
nonvolatile or unstable at 200-300°C have been
successfully chromatographed after silylation.
 Silyl reagents are compatible with most detection systems
but if used in excess, can cause difficulties with flame
ionization detectors.
SOLVENTS AND PRECAUTIONS
 Silylation reagents will react with H2O & alcohols first
care must betaken to ensure that both sample & solvent
are dry.
 Solvent should be as pure as possible, this will eliminate
excessive peaks.
 Try using as little solvent as possible as this will prevent a
large solvent peak.
 In many cases, the need for a solvent is eliminated with
silylatingreagents (if a sample readily dissolves in the
reagent, it usually is asign that the derivatization is
complete.
SILYLATION-ADVANTAGES AND
DISADVANTAGES
Advantages:
 Ability to silylate a wide variety of compounds.

 Large number of silylating reagents available.

 Easily prepared.

Disadvantages:
 Silylation reagents are moisture sensitive.

 Must use aprotic (no proton available) organic solvents.

2. ACYLATION
Mechanism
 Acylation, an alternative to silylation, is the conversion of
compounds with active hydrogen (–OH, -SH, and –NH) into
esters, thioesters and amides.
 Acylation enhances GC performance as the analyte volatility is
increased and peak shape improved because of reduced surface
adsorption.
 Acylation can reduce the polarity of a molecule, making it more
retentive in reversed-phase HPLC applications.
 Reaction
IMPORTANCE OF ACYLATION
 It improves analyte stability by protecting unstable
groups.
 It can confer volatility on substances such as
carbohydrates or amino acids, which have so many polar
groups that they are nonvolatile and normally decompose
on heating.
 It assists in chromatographic separations which might not
be possible with underivatized compounds.
ACYLATION – ADVANTAGES AND
DISADVANTAGES
Advantages
 Addition of halogenated carbons increased detectability by ECD.

 Derivatives are hydrolytically stable.

 Increased sensitivity by adding molecular weight.

 Acylation can be used as a first step to activate carboxylic acids

prior to esterfication (alkylation).
Disadvantages
 Acylation derivatives can be difficult to prepare.

 Reaction products (acid by-products) often need to be removed

before analysis.
 Acylation reagents are moisture sensitive.

 Reagents are hazardous and odorous.

 3. ALKYLATION
 Alkylation involves adding an alkyl group (aliphatic or aliphatic
aromatic) to an active functional (H) group.
 Replacement of such a hydrogen with an alkyl group is important
in chromatographic analysis because of the decreased polarity of
the derivative as compared with the parent substance, facilitating
analysis by chromatographic techniques.
 The principal reaction employed for preparation of these
derivatives is nucleophilic displacement
 Reaction:
IMPORTANCE OF ALKYLATION
 Alkylation reagents are used to modify compounds
containing acidic hydrogen, such as carboxylic acids and
phenols.
 Alkylation of weakly acidic groups (e.g.,alcohols)
requires strongly basic catalysts (sodium or potassium
methoxide).
 More acidic OH groups (phenols, carboxylic acids)
require less basic catalysts (hydrogen chloride, boron
trifluoride).
ALKYLATION-ADVANTAGES AND
DISADVANTAGES
Advantages
 Wide range of alkylation reagents available.

 Reaction conditions can vary from strongly acidic to strongly

basic.
 Some reactions can be done in aqueous solutions.

 Alkylation derivatives are generally stable.

Disadvantages
 Limited to amines and acidic hydroxyls.

 Reaction conditions are frequently severe.

 Reagents are often toxic.

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REFERENCES
 https://academic.oup.com/chromsci/article-abstract/17/3/1
13/282710?redirectedFrom=PDF
 https://www.intechopen.com/books/gas-chromatography-
derivatization-sample-preparation-application/derivatizati
on-methods-in-gc-and-gc-ms
 https://www.researchgate.net/publication/316375667_Sa
mple_Derivatization_in_Separation_Science
 https://www.sigmaaldrich.com/content/dam/sigma-aldrich
/migrationresource4/Derivatization%20Rgts%20brochure
.pdf
 https://
www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/
Supelco/Application_Notes/4537.pdf