Transcript Lecture 12
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, CN
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