DERIVATIZATIONS IN
SEPARATIONS
WHAT
IS
DERIVATIZATION?
Reaction
 Reagents
 Change chemical nature analyte
 Improve analysis

WHAT
IS
DERIVATIZATION?
Typically focuses on analyte
 Matrix ideally should remain unaffected



Occasionally get some components in matrix reacting –
not good
Why?
Not usually first step in sample preparation
 Clean-up
 Concentration
 Change property of analyte for separation
 Change property of analyte for better detection
 Can be pre- or post
DERIVATIZATION

Chemical reactions

Typically replace active hydrogens (OH, COOH, NH, CONH)
Desired property
Y–H+R–X
Analyte
H-donor
Reagent
X is a replacing group
R carries the property
Y – R + HX
DERIVATIZATION

Chemical reactions
Increased nucleophile properties
DERIVATIZATION

Chemical reactions

Efficiency probably most important criteria
Must be complete
 Time must be reasonable
 Little to no loss of analyte
 Stable

DERIVATIZATION

Chemical reactions





Forming alkyl or aryl derivatives
Silylation
Acyl derivatives
Carbon-hetero multiple bonds
Cyclic formation
DERIVATIZATION

Chemical reactions

Alkylation and Arylation
Analyte acts as a nucleophile (Y:, Y:H, Y:-)
 SN substitution with alkylating reagent (R – X); X is usually a leaving
group and R alkyl group
 Alkyl iodides and bromides very common
 Catalysts may be required

Y:H + R – X
Y – R + X:H
DERIVATIZATION

Chemical reactions

Alkylation and Arylation
Y:H + R – X

Y – R + X:H
2 mechanisms
slow
R–X
Y: - H + R+
Y: - H + C
X
fast
Y:
R+ + XY – R + H+
C
X
(SN1)
Y
C
+ HX
(SN2)
DERIVATIZATION

Chemical reactions

Alkylation and Arylation

Strong acid present then OH group is very reactive because
DERIVATIZATION

Chemical reactions

Alkylation and Arylation

Acid may also act as a catalyst
DERIVATIZATION

Chemical reactions

Alkylation and Arylation

Salts of heavy metals act as catalysts
DERIVATIZATION

Chemical reactions
DERIVATIZATION

Chemical reactions

Alkylation and Arylation

For SN2 reactions, rate depends on nature of nucleophile
1.
Nucleophile negative charge better than its conjugated acid
2.
Nucleophiles whose attacking atom in same row of periodic
table – nucleophilicity parallels bascity
3.
Nucleophiles whose attacking atom is in a higher period –
nucleophilicity increases
4.
Freer the nucleophile, the higher rate – both atoms have
unshared pair of electrons
DERIVATIZATION

Chemical reactions

Alkylation and Arylation



Order of nucleophilicity – General order
NH2- > RO- >OH- > R2NH > ArO- > NH3 > Pyridine > F- > H2O
Arylation

Similar to SN2
DERIVATIZATION

Chemical reactions

Silylation
Replaces active H (OH, COOH, SH, NH, CONH, POH, SOH) with
a silyl group (trimethylsilyl)
 Purpose
 Reduce polarity of analyte
 Increase analyte stability
 Improve analyte behavior for GC
 Can be used with solvent to aid in extraction and detection,
especially useful in analyzing crude matrix

DERIVATIZATION

Chemical reactions

Silylation
DERIVATIZATION

Chemical reactions

Silylation

Similar to SN2

Efficiency
 Nature of X (leaving group); more stable as free entity better
leaving property;
 Higher acidy better silyl donor ability
DERIVATIZATION

Chemical reactions

Silylation

Similar to SN2
OCOR leaving group better donor than
OR leaving group – more stable b/c
two resonance structures formed
DERIVATIZATION

Chemical reactions

Silylation
DERIVATIZATION

Chemical reactions

Silylation

Nature of Y:H determines silylation efficiency
DERIVATIZATION

Chemical reactions

Silylation

Solvent affect silylation - bad
 H2O, H2O2, HCl, HNO3, H2SO4, H2SO3, H3BO4, H3PO4, H4SiO4

H2O very important, try to eliminate or minimize to as low as possible
Ammonium salts
 Solvent affect silylation – good
 Dimethylformamide, pyridine, acetonitrile

DERIVATIZATION

Chemical reactions

Acylation
Replace active hydrogens
 Reduces polarity
 Improves behavior of analyte in chromatographic column
 Detectability – very useful here

DERIVATIZATION

Chemical reactions

Acylation
Most are nucleophilic substitutions, analyte the nucleophile (Y:, Y:H,
Y:-)
 React with acylating group that contains a leaving group X
 Acid generation from the reaction is a hindrance and should be
removed
 Halogen most commonly used are fluorinated acyl groups
 Good reaction for analytes with weakly reactive H

DERIVATIZATION

Chemical reactions

Acylation

SN2 substitution
DERIVATIZATION

Chemical reactions

Acylation
Carbonyl cyanides – similar to acyl
chlorides
HCN weaker acid compared to HX
DERIVATIZATION

Chemical reactions

Acylation
Anhydrides can be used as an
alternative b/c it produces a weak
organic acid – reduces undesired
modifications that can result with
strong acids
Volatility may be lower
DERIVATIZATION

Chemical reactions

Carbon-hetero multiple bonds
Representative groups – C=O, C=S, C=N, CN
 Two modes for derivatization
 Derivatize active H with a hetero-multiple bond derivatizing
agent
 Derivatize analytes with hetero-multiple bond
 Catalyzed by both acid and base conditions

DERIVATIZATION

Chemical reactions

Carbon-hetero multiple bonds
Elimination rxn
Nucleophilic or
Electrophilic - proton
R’s – H, R, Ar in Aldehydes and Ketones; OH in acids, OR in esters, NH or NHR in
amides
DERIVATIZATION

Chemical reactions

Carbon-hetero multiple bonds
Very polar (C=O), b/c displacement of  electron toward oxygen
 Thus, it is about 20% ionic

Nucleophilic attack
Elimination rxn not likely to
occur when substituents
are H, alkyl, aryl
Electrophilic attack usually
predominates under acid
In both attacks – nucleophilic
attack is rate limiting
DERIVATIZATION

Chemical reactions

Carbon-hetero multiple bonds

However, if forms
cyclic acetals or
ketals very stable
for derivatization
Aldehydes and ketones
 b/c of active methylene characteristic they can undergo
condensation reactions – presence of an OH group in the
condensation product is not desired in derivatization
 Formation of hemiacetals and acetals – unstable
DERIVATIZATION

Chemical reactions

Carbon-hetero multiple bonds

N=C in isocyanates (N=C=O) and isothiocyanates
 Very good dervatization for attaching chromophores or
fluorescent groups
 Unstable thermal properties make it uncommon for GC
applications
DERIVATIZATION

Chemical reactions

Cyclic formation

Formation of new cyclics or replacement of old cyclics
 Nonaromatic cyclics containing O
 Aromatic cyclics containing one N
 Azoles or related compounds
 Azines or related compounds
 Cyclic siliconides
 Cyclic phosphothioates
 Cyclic boronates
DERIVATIZATION

Chemical reactions

Peroxyacid – peracetic,
perfomic, perbenzoic
Cyclic formation

Formation of new cyclics or replacement of old cyclics
 Nonaromatic cyclics containing O
Epoxides
DERIVATIZATION

Chemical reactions

Cyclic formation

Formation of new cyclics or replacement of old cyclics
 Aromatic cyclics containing one N

Involve bifunctional molecules
DERIVATIZATION

Chemical reactions

Cyclic formation

Formation of new cyclics or replacement of old cyclics
 Azoles or related compounds

Aromatic five membered heterocycles with a nitrogen and some
other hetero atom (N, O, S)
DERIVATIZATION

Chemical reactions

Cyclic formation

Formation of new cyclics or replacement of old cyclics
 Azines or related compounds

Six membered heterocyclics with more than 1 N or 1 N and some
other heteroatom
DERIVATIZATION

Chemical reactions

Cyclic formation

Formation of new cyclics or replacement of old cyclics
 Cyclic siliconides
DERIVATIZATION

Chemical reactions

Cyclic formation

Formation of new cyclics or replacement of old cyclics
 Cyclic phosphothioates
DERIVATIZATION

Chemical reactions

Cyclic formation

Formation of new cyclics or replacement of old cyclics
 Cyclic boronates
DERIVATIZATION

Chemical reactions

Other derivatizations

Addition across = bond

Oxidation/Reduction
 Ninhydrin

Hydrolysis
DERIVATIZATION

Chemical reactions

Other derivatizations

Aromatic substitutions


Electrophilic substitution – useful for aromatic cmpds with activating
groups O-, OH, OR, OCOR, NH2, NHR, NR2
Complexation