CBMS308/825 Chemical Analysis II What is Analytical Derivatisation?

Derivatisation: a technique used in chemistry which transforms

a chemical compound into a product (the reaction's derivate) of
similar chemical structure, called a derivative.
Analytical Derivatisation
Introduction In analytical derivatisation, a specific functional group of an analyte
of interest is targeted in the derivatisation reaction, transforming the
analyte into a derivate of differing reactivity, solubility, boiling point,
Lecture 1
melting point, aggregate state, or chemical composition.
The resulting new chemical properties of the derivate are then used
to advantage for the analysis of the analyte.

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Uses of Analytical Derivatisation Derivatisation for Enabling Analysis

Increasing Volatility:
In a broad sense, analytical derivatisation is employed for one or all
of the following reasons:
To enable analysis of analytes not directly amenable to a
particular mode of analysis due to, for example, volatility,
solubility or stability.
To improve analysis efficiency by improving/adjusting
chromatographic behaviour, for example, improving
resolution or reducing interactions.
To enhance detectability of analytes by increasing
detector response to an analyte.
Used across a wide range of disciplines including the biomolecular
sciences, medical, forensic and environmental sciences.
Low volatility is due:
To size of the molecule e.g. proteins
Intermolecular interactions/attraction in the molecule. e.g.
saccharides.
Derivatisation can not increase volatility of large molecules
In smaller molecules a lack of volatility is generally attributable to
intermolecular forces such as hydrogen bonding. Masking of polar
groups by derivatisation can lead to increased volatility.
Solubility is similarly affected by intermolecular forces.

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Derivatisation for Enabling Analysis Derivatisation for Improving Analysis
Decreasing Volatility: Stability and Interactions:
Derivatisation can be used to form less volatile derivates: Sometimes a sample may be volatile (or soluble) but may
To minimise losses during sample manipulation undergo decomposition or rearrangements under the analytic
conditions used producing ghost peaks etc.
To analyse gaseous samples by HPLC. e.g. aldehydes and e.g. keto-enol rearrangements of polyones
ketones in air (EPA method TO-5).
Separate sample peaks from solvent peak or other peaks in O OH

O
N

GC. O O
N
O

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
time (min)

Chromatogram of 2,3-pentadione and 2,3-pentadione phenyloxime

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Derivatisation for Improving Analysis Derivatisation for Improving Analysis

Stability and Interactions:
Resolution:
Interactions between the analytes themselves or between the compounds that co-elute can frequently be resolved by an
analytes and the chromatographic column may reduce the appropriate derivative.
separation efficiency of many compounds and mixtures.
e.g. closely related compounds are generally poorly separated
e.g. polar samples on polar surfaces of the column
O OH OH OH
O

OH OH
OH

HO Conversion to the chloromethyl-dimethyl-silyl

derivative enables baseline separation of all
1,4-, 1,5- and 1,6-dihydroxynaphthalenes three analytes.
nearly always co-elute on most GC
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 columns.
time (min)
Gas chromatogram of butyl alcohol and butyl acetate

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 Derivatisation for Improving Analysis Derivatisation for Improving Analysis
Group separation: Chiral separation:
Chiral isomers of compounds have identical properties and thus
compounds that have poor resolution from other sample
are not able to be separated on standard chromatography
components during separation can frequently be resolved by an
stationary phases. Resolution can be achieved by forming a
appropriate derivative.
diastereometric derivative with an enantiomerically pure chiral
e.g. in steroid analysis, keto-steroids often co-elute or overlap derivatising agent.
with hydroxy steroids.
e.g. resolution of (R)- and (S)- amphetamines
O
OH

HO HO
O
Androstenediol OH Pregnenolone
The benzyl oxime derivatised steroid will have a
much longer retention time an elute later. (R)- and (S)- amphetamines The N-trifluoroacetyl-L-prolyl derivatised
amphetamines are diastereomers and easily
Identical properties, not resolved.
O O N-trifluoroacetyl-
resolvable
Testosterone L-prolyl chloride
Progesterone

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Derivatisation for Enhancing Detection Derivatisation for Enhancing Detection

Enhancing Detection: Enhancing Detection:
By introducing onto the analyte, atoms or functional groups that By introducing onto the analyte, atoms or functional groups that
interact strongly with the detector and hence improve signal interact strongly with the detector and hence improve signal
identification. identification. O
O
e.g. adding a chromophore Other examples:
or fluorophore adding ECD sensitive OH
O

Cl O
groups to steroids O
O
Chromatogram of various carbonyl azine derivatives (CF2)7CF3
separated on Zorbax ODS column with Fluorescence
detection: 1, Azine reagent; 2, Formaldehyde-azine; 3, O O
Acetaldehyde-azine; 4, Acetone-azine; 5, Acrolein-
Cl (CF2)7CF3
azine; 6, Propionaldehye-azine; 7, Methylethyl ketone- O
azine; 8, Crotonaldehyde-azine; 9, Butylaldehyde-
azine; 10, Isobutylaldehyde-azine; 11, Benzaldehyde-
azine; 12, Isovaleraldehyde-azine; 13, Valeraldehyde- Forming pyrollidone derivatives to locate double bonds in
azine; 14, p-Tolualdehyde-azine; 15, Hexanaldehyde-
azine; 16, Unknown-from reagent; 17, Heptanaldehyde- fatty acids by MS.
azine; 18, Unknown-from reagent.
Redox sensitive derivatives to enhance electrochemical
detection
2-diphenylacetyl-1,3-
indandion-1-hydrazone (DAIH)

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Goals of Analytical Derivatisation Goals of Analytical Derivatisation
Analytical derivatisation reactions are a form of synthetic organic
chemistry in which the goal is a 100% yield of a single pure The most common issues include:
product. Occurrence of side products.
Achieved through a combination of high purity reagents and Contamination, interferences, co-elution
large relative excesses. Incomplete reaction
The formation of multiple products
General Requirements:
The reaction should:
be quantitative
Be fast and simple
Should work well with small amounts of material (mg to fg)

Ideally, both reagent and the products should be stable.

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Derivative Type and Reagents Alkylation

The replacement of acid hydrogen in polar groups (e.g. OH, SH, Derivatisation by alkylation involves the replacement of active
NH) account for the large proportion of analytical derivatisation hydrogens with an alkyl group.
reactions. Nucleophilic and electrophilic addition reactions account
Typically this is achieved by nucleophilic displacement.
for the rest. The principle reaction involved fall into 4 main classes of
reaction: For alcohol and phenols the products are ethers:
1. Alkylation
2. Acylation
3. Silylation For carboxylic acids the products are esters:
4. Condensation

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Alkylation Alkylation
For thiols the products are thioethers: Alkylation of compounds with weakly acid protons (e.g. OH, NH)
require the use of a strongly basic catalyst to generate the
nucleophile from the analyte. e.g.
methyl sulfinyl carbanion (CH3-SO-CH2-)
For amines and amides the products are N-alkyl amines and N-alkyl potassium in ammonia (NH2-)
amides:

More acidic protons such as those on phenols and carboxylic acids

require less strongly basic catalysts (i.e. milder reaction conditions),
e.g.
hydroxide ion (OH-)
carbonate ion (CO32-)

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Alkylation Alkylation
The alkylating reagent are typically alkyl or aryl bromides or iodides.
e.g. pentafluorobenzyl bromide, 2,4 dinitrobenzyl bromide
F F
Br Br
F O 2N

F F NO 2

Getting base, reagent and analyte together can be problematic

requiring the use of a phase transfer reagent (e.g. crown ethers or
quaternary ammonium salts)

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Alkylation Alkylation
For analytes with strongly acidic protons a variety of reagents are For analytes with strongly acidic protons a variety of reagents are
available to overcome the need for aqueous bases and/or phase available to overcome the need for aqueous bases and/or phase
transfer reagents. transfer reagents.
Flash Alkylation reagents: Dimethyl formamide dialkyl acetals :
e.g. trimethyl-anilinium hydroxide e.g. Dimethyl formamide dimethyl acetal (Methyl-8)

O R'
O O
+ R' R' OH
R OH N O R' +
R O
DMF

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Alkylation Silylation
For analytes with strongly acidic protons a variety of reagents are Silyl derivatives are formed by the replacement of active hydrogen on
available to overcome the need for aqueous bases and/or phase OH, -SH and NH groups. The general reaction for silylation is:
transfer reagents.
Diazoalkyles :
e.g. Diazomethane (CH3N2)

The trimethylsilyl group is the most commonly employed

functionality. Other variants include t-butyl-dimethyl-silyl groups.

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 Silylation Silylation
The mechanism involves nucleophilic attack on the Si: Common TMS reagents
listed in order of
R'
increasing donor strength:
X R' R'
R OH + R' Si R' R R Si
O Si X O R' + HX
R'
H
R' R'

Thus the properties of X are very important:

Low basicity
Ability to stabilise the ve charge in the transition state.

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Silylation Acylation
Easy of reactivity towards silylation is as follows: Acylation is another commonly used derivatisation method for polar
OH, -SH and NH groups.
Typical reagents for acylation include acyl anhydrides, acyl or sulfonyl
Alcohol OH > Phenol OH > COOH > amine > amide
halides or activated acyl amides.
Reaction with anhydrides and acyl/sulfonyl halides forms acid by-
Reactivity is also affected by steric hindrance. Thus: products:
O O
O O
primary > secondary > tertiary R' O R' R +
O R' HO R'
O
O
Silyl derivatives are sensitive to moisture and are generally prepared R OH + R' Cl R
O R' + HCl
in sealed vessels. Alternative silyl reagents can impart greater
O
moisture stability. e.g. t-butyl-dimethyl-silyl O
R' S Cl
CH3 O H 3C CH3 O O S R' + HCl
O R O
H3 C Si O C R H3 C Si O C R
CH3 H 3C CH3

Readily hydrolysed Stable in water

by water
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Acylation Acylation
Why are acid by-products bad? The solution is to use an acid scavenger e.g. pyridine or triethylamine
O O
R NH2 + R O O
R' Cl N R' + HCl
R NH 2
H + R +
HCl R' Cl N R' NH Cl
N H
O
R NH3 + No Reaction
R' Cl
Acylation of basic groups (such as amines) using acyl anhydrides,
acyl or sulfonyl halides must be performed in the presence of an acid
CH 3 CH 3 CH3 CH 3 scavenger.
H2 O
O Si O Si O Si OH HO Si
CH 3 CH 3 CH3 CH 3
n

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Acylation Acylation
Activated acyl amide reagents are advantageous in that they do not The acetyl (or trifluoroacetyl) group is the most commonly employed
yield acid byproducts. acyl derivative in GC.
e.g. Commonly used functions for HPLC detection enhancement include:
TFAI (trifluoroacetylimidazole) and O Cl
Cl O
MBTFA (N-methyl bis(trifluoroacetamide) O
O S O H
N O
N
Give imidazole and N-methyl trifluoroacetamide respectively
O
N O
N
AQC
FMOC-Cl DANS-Cl 6-aminoquinolyl-N-
9-fluorenylmethoxycarbonyl chloride Dimethylaminonaphthalene hydroxysuccinimidyl carbamate
(9-fluorenyl methyl chloroformate) sulfonyl chloride (specific for NH, long Stokes)
UV and fluroescent Strong UV and fluorescence (long
Stokes shift, but quenched in
aqueous mobile phases)

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 Condensation Condensation
Condensation, in this context, refers to the joining of two molecules Condensation of carbonyl groups the most common
with the loss of water. derivatisation reaction for carbonyls are conversion to oximes,
hydrazaone or schiff bases.
Some common condensation reactions employed for derivatisation:
Esterification of carboxylic acids can be performed on an
R N R'
analytical scale using either Bronstead acid (e.g. HCl) or Lewis acid H 2N O R' O Oxime

(e.g. BF3) catalysts. O R

H R N R' Hydrazone
+ H 2N N R' N
R R H
R
O O R N
+ H2 O H 2 N R' R'
+ R' OH R' imine (Schiff Base)
R OH R O R
BF3 or HCl

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Condensation General Considerations

Condensation of carbonyl groups derivatisation of carbonyl groups It is important, when at all possible, to use an internal standard with
is primarily for protection (e.g. to prevent enol-keto tautomerism) or similar reactivity to the compounds you are analysing.
detection enhancement. Care must be taken to ensure purity of the reagents used. If
e.g. pentafluorobenzyl oximes are among the most sensitive ECD possible run a blank
derivatives known. Highest quality solvents should be used for analytical derivatisations
F F F F
O R
R R + H2 N O F N O F Often it is important to be able to remove any excess reagent prior
R to analysis to avoid swamping the detection system, or reacting
F F F F
with stationary phases.
2,4 dinitrophenyl hydrazine derivatives of aldehydes and ketones allow F F O F F
O O
20 fold lower detection limits in HPLC using electrochemical detection + F R F
than is possible by UV detection. R OH Br
F F F F
H2 N OH
DANS-hydrazine gives strong HN NH2
R
HN N
UV chromophore and fluorescence. O S O
O S O R F F
+
H3 N O H+ F F
O
F H 2N O
R R + F
N F F
N F F

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