6. 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.
NOTE: A modified analyte in this case will be the product,
which is known as the derivative.
NOTE: The derivative may have “similar or closely
related” structure, but not the same as the original non-
modified chemical compound. 6
8. Why ?
To permit analysis of compounds not directly
amenable to analysis due to, for example, inadequate
volatility or stability
Improve chromatographic behaviour or detectability
NOTE: Derivatization is a useful tool allowing the use of
GC and GC/MS to be done on samples that would
otherwise not be possible in various areas of
chemistry such as medical, forensic, and
environmental
8
12. Points to be NOTED
• Volatility
• Volatile or eluted out :
Without thermal decomposition
Or molecular rearrangement
• Functional groups with active Hydrogen
• Derivatization either ↑ or ↓ volatility
12
13. Points to be NOTED
• Generally derivatization is aimed at improving on the
following aspects in GC:
i. Suitability
ii. Efficiency
iii.Detectability
13
15. General Reaction
• The most commonly used derivatization procedures
involve the “substitution of active hydrogens” on the
compound to be derivatized with a variety of functional
groups.
• These functional groups impart the desired
characteristics to the compound, while eliminating the
adverse effects. 15
16. General Reaction
R1—AH + R2—D → R1 —AD + R2—H
Where,
atom “A” = Oxygen, Sulfur, Nitrogen or similar atoms
atom “D” = Functional group on the derivatization
reagent
16
18. Derivatization Reagents
• Definition
• Criteria for selection:
Produce more than 95% derivatives
No structural or molecular alterations
No sample loss
Non – interacting derivatives
Stable derivatives with time
18
22. Alkylation
INTRODUCTION:
•Represents the replacement of active hydrogen by an
aliphatic or aliphatic-aromatic (e.g., benzyl) group in
process referred to as “ESTERIFICATION”.
RCOOH + PhCH2X → RCOOCH2Ph + HX
Where, X = Halogen group
R’ = Alkyl substitution 22
23. Alkylation
NEED:
Conversion “organic acids into esters”, especially methyl
esters that produce of better chromatograms than the free
acids.
To prepare ethers, thioethers and thioesters, N-
alkylamines, amides and sulphonamides.
Alkyl esters formed offer “excellent stability” and can be
isolated and stored for extended periods if necessary.
NOTE: Use of inorganic acids (HCl/SCl) for fats & oils. 23
25. Alkylation
ADV:
Wide range of reagents avail.
Reaction condition can vary
from strongly acidic to strongly
basic.
Some reactions can be done in
aqueous systems.
Derivatives are generally
stable.
DISADV:
Limited to amines and acidic
hydroxyls.
Conditions frequently severe.
Reagents often toxic.
Optimization for particular
compounds usually necessary.
25
27. Acylation
INTRODUCTION:
•An acyl group is introduced to an organic compound.
•In the case of a carboxylic acid, the reaction involves the
introduction of the acyl group and the loss of the hydroxyl
group.
CH3OCOCOCH3 + HOR → CH3OCOR´ + HOCOCH3
Where, R = alkyl grp
R’ = another alkyl substitution 27
28. Acylation
NEED:
•Compounds that contain active hydrogens (e.g., -OH, -SH
and -NH) can be converted into esters, thioesters and
amides, respectively, through acylation.
•Highly polar and volatile derivatives
•Stability from the thermal decomposition
28
29. Acylation
Benefits of Acylation:
•Improve analyte stability by protecting unstable groups.
•Provides volatility on substances such as carbohydrates
or amino acids, which have many polar groups that they
are non-volatile and normally decompose on heating.
•Assists in chromatographic separations which might not
be possible with compounds that are not suitable for GC
analysis.
•Compounds are detectable at very low levels with an
electron capture detector (ECD).
29
31. Acylation
ADV:
Hydrolytically stable.
Perfluro deriv. ↑ volatility.
↑sensitivity by added mol.wt.
↑detectability by ECD by
added halogen atoms.
Reacts with alcohols, thiols and
amines
Can be used to activate -COOH
for esterification.
DISADV:
Derivatives are frequently
difficult to prepare.
Reaction products often must
be removed before analysis.
Reaction must be done in non-
aqueous system.
Reagent are moisture-sensitive
Reagents are hazardous and
odorous. 31
33. Silylation
INTRODUCTION:
•Introduction of a “silyl group” into a molecule, usually in
substitution for active hydrogen such as dimethylsilyl
[SiH(CH3)2], t-butyldimethylsilyl [Si(CH3)2C(CH3)3] and
chloro-methyl-dimethylsilyl [SiCH2Cl(CH3)2].
•Replacement of “active hydrogen” by a silyl group
reduces the polarity of the compound and reduces
33
34. Silylation
INTRODUCTION………..Contd..:
•Many hydroxyl and amino compounds regarded as non-
volatile or unstable at 200 – 300 °C have been successfully
analyzed in GC after silylation.
•The silylated derivatives are more volatile and more
stable and thus yielding narrow and symmetrical peaks
(Kataoka, 2005).
34
35. Silylation
MECHANISM:
•Replacement of the active hydrogen (in -OH, -COOH,
-NH, -NH2, and –SH groups) with a trimethylsilyl group.
•Silylation then occurs through nucleophilic attack (SN2
),
where the better the leaving group, the better the
siliylation.
•This results to the production of a bimolecular transition
state in the intermediate step of reaction mechanism.
35
37. Silylation
MECHANISM ………. Contd….:
NOTE: Moisture sensitive, thereby should be tightly
stored.
NOTE: Solvents used should be as pure and as little as
possible as it will eliminate excessive peaks and prevent a
large solvent peak.
NOTE: Ease of reactivity of functional grps:
Alcohol > Phenol > Carboxyl > Amine > Amide /hydroxyl
NOTE: For alcohols, the order will be as follows:
Primary > Secondary > Tertiary 37
43. Silylation
INTRODUCTION
•Involves reaction of an enatiomeric molecule with an
enantiomerically pure Chiral Derivatizing Agent (CDA) to
form two “diastereomeric” derivatives that can be
separated in this case using GC.
•Any molecule having asymmetric carbon is called as
“CHIRAL” molecule.
NOTE: Chirality of analyte molecules requires special
consideration in their analysis and separation techniques.
43
44. Silylation
METHODS OF SEPARATION
Separation on an optically active stationary phase.
Preparation of diastereomeric derivatives that can be
separated on a non chiral stationary phase.
REAGENTS
TPC :- N-trifluoroacetyl-L-prolyl chloride
ITPC :- (S)-(–)-N-(Trifluoroacetyl)-prolylchloride
MTPA:- (–)-α-Methoxy-rifluoromethyl-phenylacetic acid
44
46. Summary
INTRODUCTION
•Choice of derivatization technique depends
upon:
Available reagent
Sample
•Derivatives must be suitable, detectable and
efficient for GC analysis.
•For acid analytes, the first choice for
derivatization is esterification.
46
47. Summary
INTRODUCTION
•Nearly all functional groups which present a
problem in gas chromatographic separation can
be derivatized by silylation reagents.
•Chiral GC complex due to different reaction
rates, but could be reduced by proper selection
of reagents.
47
The main reason for derivatizing is to impart volatility to otherwise nonvolatilecompounds. The low volatility may result from the size of the molecule and the resultant large dispersion forces holding the molecule together. Smaller molecules may have a low volatility due to the strong intermolecular attractions between polar groups. In the latter case, masking the polar groups by derivatization can yield dramatic increases in volatility.
Derivatization can also be used to decrease volatility to allow analysis of very low molecular weight compounds, to minimize losses in manipulation and to help separate sample peaks from solvent peak.
Polar samples tend to adsorb on the active surfaces of the column walls and the solid support. Reduction of this adsorption can be accomplished by derivatization.
In general, the halogenated substituents increase electron affinity in the following order I > Br> Cl > F (Though they show little increase in volatility).
Derivatization serves to accentuate the differences in the sample compounds to facilitate the chromatographic separation.
1. For GC analysis, compounds containing functional groups with active hydrogens such as -SH, -OH, -NH and -COOH are of primary concern. AS the tendency of these functional groups to form intermolecular hydrogen bonds. These intermolecular hydrogen bonds affect the inherent volatility of compounds containing them, their tendency to interact with column packing materials and their thermal stability.
1.
2. to produce good peak resolution and symmetry for easy identification and practicability in GC analysis & reduce Interactions
3. achieved either by increasing the bulk of the compound or by introducing onto the analyte compound, atoms or functional groups that interact strongly with the detector and hence improve signal identification. (Ex: halogen add in ECD & TMS ether for identifying the well fragmented peaks)
4.
the substance that is used to chemically modify a compound to produce a new compound which has properties that are suitable for analysis in GC or LC.