Derivatization in GC

An introductory review on "Derivatization Techniques" in Gas Chromatography.

1. D
erivatization
of G
C
S
u
raj C
.
S
u
raj C
.A
A
C
P
A
A
C
P

2. P
P
T
. P
ack
age
2

3. Overview
3

4. Overview
4

5. Introduction
5

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

7. Why ?
7

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

9. Outcome/Accomplish
9

10. Why ?
Impart Volatility
Detect Volatility
Reduction in column absorption
Improve detectability
Accentuate differences among the compounds
Analysis of non-volatile products
Stabilization of compounds for GC 10

11. Points to be
NOTED
11

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

14. General
Reaction
14

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
R
1
—AH + R
2
—D → R
1
—AD + R
2
—H
Where,
atom “A” = Oxygen, Sulfur, Nitrogen or similar atoms
atom “D”= Functional group on the derivatization
reagent
16

17. Derivatization
Reagents
17

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

19. Methods
19

20. Types
Alkylation
Silylation
Acylation
Chiral Derivatization
20

21. Alkylation
21

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 + PhCH
2
X → RCOOCH
2
Ph + 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

24. R
E
A
G
E
N
T
S
24

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

26. Acylation
26

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.
CH
3OCOCOCH
3 + HOR → CH
3OCOR´ + HOCOCH
3
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

30. R
E
A
G
E
N
T
S
30

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

32. Silylation
32

33. Silylation
INTRODUCTION :
•Introduction of a “silyl group” into a molecule, usually in
substitution for active hydrogen such as dimethylsilyl
[SiH(CH
3
)
2
], t-butyldimethylsilyl [Si(CH
3
)
2
C(CH
3
)
3
] and
chloro-methyl-dimethylsilyl [SiCH
2
Cl(CH
3
)
2
].
•Replacement of “active hydrogen” by a silyl group
reduces the polarity of the compound and reduces
hydrogen bonding.
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 (SN
2
),
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

36. Silylation
MECHANISM ………. Contd….:
36

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

38. R
E
A
G
E
N
T
S
38

39. Silylation
ADV:
Wide range of applications
Variety of reagents available
Easily prepared
Excellent thermal stability
Excellent chromatographic
characteristics
DISADV:
Moisture-sensitive
TMS & TBD-MCS derivatives
are easily hydrolyzed
No aqueous solutions.
Must use aprotic org. solvents
Reacts with column materials
Silicone residues build up in
GC detectors
39

40. Derivatization
Solvents
40

41. Derivatization Solvents
•Definition
41

42. GC Chiral
Derivatization
42

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

45. Summary
45

46. S
um
m
ary
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. S
um
m
ary
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

48. 48