Guide to Derivatization Reagents for GC

1

SUPELCO
Bulletin 909

Bulletin 909A

Guide to Derivatization Reagents for GC

A large number of reagents are used to prepare derivatives for
gas chromatography, but most of the derivatization reactions
fit into one of three categories:  acylation, alkylation, or
silylation.  This bulletin describes each category, and presents
information on how to choose the proper reagent based on the
functional group(s) of the compound to be derivatized.

Key Words

●

 acylation ● alkylation ● silylation

●

 derivatization reagents ● derivatives

  Contents

Topic

Page

Considerations When Derivatizing an Analyte

1

Acylation

2

Alkylation

3

Silylation

3

Derivatization Reagent Selection Guide

4

Troubleshooting

8

Ordering Information

9

Glassware for Derivatization
Vials with 0.1-10.0mL capacity accommodate sample plus solvent
and reagent in quantities typically used in gas chromatography.
Vials must be suitable for temperature extremes. Vials supplied
with open-center screw caps can be sealed with rubber septum
stoppers or Teflon®-lined discs. The heavy walls and excellent
sealing properties of SupelcoTM micro-reaction vials allow samples
to be heated safely to moderately high temperatures. Ground
bottoms give these vials added stability on a flat surface, and are
convenient for pencil markings. Thermostatically controlled heat-
ing units with aluminum blocks drilled to fit the vials precisely are
available from several manufacturers, including Supelco.

Note: Although a Teflon lining generally is quite inert, it can be
dissolved by some samples and reagents.

Deactivation of Glassware
Because the surface of laboratory glassware is slightly acidic, it can
adsorb some analytes — particularly amines. In low level analyses,
such losses can be significant. To prevent sample loss through
adsorption, glassware used in low level analyses usually is silanized.
Silanization masks the polar Si-OH groups on the glass surface by
chemically binding a nonadsorptive silicone layer to the surface,
in effect “derivatizing” the glass. In the most common silanization
procedure, the glassware is treated with a solution of 5-10%
dimethyldichlorosilane (DMDCS) in toluene for 30 minutes. The
deactivated glassware is rinsed with toluene, then immediately
thereafter with methanol.

Adsorption also can be reduced by adding a compound that
competes for the adsorptive sites on the glass surface. A small
amount (often less than 1%) of an alcohol, such as butanol, added
to the solvent significantly reduces adsorption losses.

Considerations When
Derivatizing an Analyte
Gas chromatography is used to separate volatile organic com-
pounds. By modifying the functionality of a molecule to increase
– or sometimes decrease – volatility, derivatizing reagents enable
chromatographers to analyze compounds that otherwise are not
readily monitored by GC. Derivatization also reduces analyte
adsorption in the GC system and improves detector response, peak
separations, and peak symmetry.

Derivatives are used for the following reasons:

●

to improve resolution and reduce tailing of polar compounds
(–OH, –COOH, =NH, –NH

2, –SH, and other functional groups)

●

to analyze relatively nonvolatile compounds

●

to improve analytical efficiency and increase detectability

●

to improve stability of compounds

The choice of a derivatizing reagent is based on the functional
group requiring derivatization, the presence of other functional
groups in the molecule, and the reason for performing the
derivatization. The chemical structure and properties of the mol-
ecule influence the reagent choice.

In choosing a suitable derivatization reagent, certain criteria must
be used as guidelines. A good reagent:

●

produces a derivatization reaction that is 95-100% complete

●

will not cause any rearrangements or structural alterations
during formation of the derivative

●

does not contribute to loss of the sample during the reaction

●

produces a derivative that will not interact with the analytical
(GC or HPLC) column

●

produces a derivative that is stable with respect to time

Supelco offers helpful free technical literature for most derivatization
reagents (see page 9).

T196909A

©

1997 Sigma-Aldrich Co.

2

SUPELCO

Bulletin 909

Sample Handling
Most lab personnel transfer samples and reagents with pipettes.
For sensitive reagents, we recommend using a microliter syringe,
which reduces exposure to atmospheric moisture. Syringes with
Teflon-tipped plungers are more convenient than conventional
syringes with all-metal plungers, particularly for transferring vola-
tile reagents. The Teflon plunger tip forms a better seal and
facilitates withdrawal of the reagent from a sealed vial.

Any syringe will retain some reagent in the barrel. A syringe with an
all-metal plunger, if not properly cleaned, is prone to corrosion and
seizing. The best cleaning procedure is to remove and wash the
plunger, and use a vacuum to pull solvent through the syringe. A
seized plunger sometimes can be freed by soaking the syringe in
a container filled with methanol.

Injection Ports
When working with silylating reagents, use a silanized glass injec-
tion port or make injections directly onto a glass column. Use of
a stainless steel injection port frequently yields erratic and
irreproducible results. The problem may not become apparent
until after several weeks of use, when corrective action may include
replacing the injector.

Reaction Time
Reaction time varies greatly among compounds. Many materials
can be derivatized by the reagents described here in a matter of
seconds or minutes at room temperature, while others require
extended periods at elevated temperatures. For a compound with
unknown reactivity, the progress of the derivatization can be
monitored by periodic chromatographic analysis of aliquots of the
reaction mixture. Disappearance of the reagents or appearance of
product peaks can be used to determine the reaction’s progress.

Heating often increases the yield of derivative and/or shortens the
reaction time. Before using heat, consider the thermal stability of
the analytes and reagents involved.

Water
Water in the reaction mixture often can hinder the reaction and/
or hydrolyze the derivative, reducing the yield of derivative for
analysis. Tightly seal opened reagents during storage. If necessary,
add sodium sulfate to the reaction mixture to trap water present
in the sample.

Chromatography
We offer a wide range of general purpose and specially tested
capillary GC columns for evaluating underivatized and derivatized
analytes. For descriptions of our capillary columns, please refer to
the current Supelco catalog.

Acylation
Acylation, an alternative to silylation, is the conversion of com-
pounds that contain active hydrogens (-NH, -OH, -SH) into amides,
esters, or thioesters through the action of a carboxylic acid or
carboxylic derivative. Acylation has many benefits:

●

It improves analyte stability by protecting unstable groups.

●

It can confer volatility on substances such as carbohydrates or
amino acids, which have so many polar groups that they are
nonvolatile and normally decompose on heating.

●

It assists in chromatographic separations which might not be
possible with underivatized compounds.

●

Compounds are detectable at very low levels with an electron
capture detector.

In halocarbons, the presence of a carbonyl group adjacent to a
halogenated carbon enhances the electron capture detector (ECD)
response. Acylation also has been used to form fragmentation-
directing derivatives for mass spectrometry and chromogenic
derivatives for HPLC.

Perfluoro Acid Anhydrides
Acylation of amino, hydroxy, and thiol groups to the perfluoroacyl
derivatives reduces polarity. The derivatives also are both stable
and highly volatile. Although fluorinated anhydride derivatives are
used primarily with electron capture detectors (ECD), they can be
used with flame ionization detectors (FID). These reagents react
with alcohols, amines, and phenols to produce stable derivatives.
Fluorinated anhydrides are used in derivatizing samples for drug
of abuse confirmation.

The perfluoro acid anhydrides and acyl halide reagents form acidic
byproducts which must be removed prior to the GC analysis, to
prevent damage to the chromatography column. Acylations with
anhydride reagents normally are performed in pyridine, tetrahy-
drofuran, or other solvent capable of accepting the acid byproduct.
Amine bases also may be used as catalysts/acid acceptors.

Perfluoroacylimidazoles
Perfluoroacylimidazoles offer advantages over perfluoro acid an-
hydrides for preparing perfluoroacyl derivatives. The reactions are
smooth and quantitative, and produce no acid byproducts that
must be removed prior to the injection.

The activated amide reagents also yield no acid byproducts,
producing only imidazole and N-methytrifluoroacetamide, re-
spectively.

The perfluoroacylimidazoles react with hydroxyl groups and both
primary and secondary amines, and quantitatively acylate indole
alkylamines.

General Acylation Reagents
N-Methyl-bis(trifluoroacetamide) (MTBTFA) trifluoroacylates pri-
mary and secondary amine, hydroxyl, and thiol groups under mild
non-acidic conditions. Reactions with amines generally proceed at
room temperature. Hydroxyl derivatizations are slower; heat is
recommended. N-methyltrifluoroacetamide, the principal
byproduct of the derivatization reaction, is stable and volatile, and
does not interfere with the chromatography.

3

SUPELCO
Bulletin 909

Alkylation
Alkylation involves adding an alkyl group (aliphatic or aliphatic-
aromatic) to an active functional (H) group. Replacement of
hydrogen with an alkyl group is important because the derivative
has lower polarity, relative to the parent substance. Alkylation
reagents are used to modify compounds containing acidic hydro-
gens, such as carboxylic acids and phenols. The resulting products
are ethers, esters, thioethers, thioesters, n-alkylamines, and n-
alkylamides. Alkylation of weakly acidic groups (e.g., alcohols)
requires strongly basic catalysts (sodium or potassium methoxide).
More acidic OH groups (phenols, carboxylic acids) require less
basic catalysts (hydrogen chloride, boron trifluoride).

DMF-Dialkylacetals
Dimethylformamide dialkyl acetals are used to esterify acids to
their methyl esters. Hydroxyl groups are not methylated. Carboxy-
lic acids, phenols, and thiols react quickly, to give the correspond-
ing alkyl derivatives. N,N-dimethylformamide dimethylacetals are
moisture sensitive.

Diazoalkales
In the presence of a small amount of methanol as catalyst,
diazomethane (a yellow gas, usually used as an ethereal solution)
reacts rapidly with fatty acids, forming methyl esters. Elimination
of gaseous nitrogen drives the reaction. The yield is high and side
reactions are minimal. However, diazomethane is carcinogenic,
highly toxic, and potentially explosive. Diazomethane is not ideal
for esterifying phenolic acids because the phenolic hydroxyl
groups also are methylated (at a slower rate), which can lead to
mixtures of partially methylated products.

Esterification and Transesterification Reagents
Esterification, the reaction of an acid with an alcohol to form an
ester, is the most popular alkylation method. Alkyl esters offer
excellent stability, and provide quick and quantitative samples for
GC analysis. The process involves the condensation of the carboxyl
group of the acid and the hydroxyl group of the alcohol, with
elimination of water. Results are best in the presence of a catalyst
(e.g., hydrogen chloride), which is removed with the water.

Transesterification is the displacement of the alcohol portion of an
ester by another alcohol. This reaction has been widely used for
making esters of higher alcohols from esters of lower alcohols. In
the presence of an acidic or basic catalyst, methanol can be used
to transesterify fats or oils.

General Alkylation Reagents
Pentafluorobenzylbromide is convenient for making esters and
ethers, and has been used in trace analyses. This strong lachryma-
tor should be used in a hood. Hexacyclooctadecane and
pentafluorobenzylbromide are used to prepare pentafluorobenzyl
- dilute with 50mL 2-propanol. 1mL of this reagent will derivatize
up to 0.3mg phenols.)

Esterate-M is used to prepare methyl and other esters of long chain
fatty acids by reaction with dimethylformamide and dimethylacetal.
Aldehydes and ketones are conveniently derivatized by forming
oximes with o-alkylhydroxylamine HCl reagents. o-
Methylhydroxylamine HCl has been used with ketosteroids, pros-
taglandins, saccharides, aldoacids, and ketoacids. N-butylboronic
acid reacts with 1,2- or 1,3-diols or with 

α- or β-hydroxy acids to

form 5- or 6-member ring nonpolar boronate derivatives. The
derivatives are prepared simply by adding n-butylboronic acid to
a solution of the hydroxy compound in dimethylformamide.

Silylation
Silylation is the introduction of a silyl group into a molecule, usually
in substitution for active hydrogen. Replacement of active hydro-
gen by a silyl group reduces the polarity of the compound and
reduces hydrogen bonding. The silylated derivative thus is more
volatile, and more stable. Detection is enhanced. Many hydroxy
and amino compounds regarded as nonvolatile or unstable at 200-
300°C have been successfully chromatographed after silylation.
Silyl reagents are compatible with most detection systems but, if
used in excess, can cause difficulties with flame ionization detec-
tors.

The trimethylsilyl (TMS) group, Si(CH

3)3, is the most popular and

versatile silyl group for GC analysis. TMS derivatization enables
better GC separations and application of special detection tech-
niques.  TMS silylating reagents and derivatives react with active
hydrogen atoms.  Consequently, TMS derivatives should not be
analyzed on polyethylene glycol phases or other stationary phases
that have these functional groups. Nonpolar silicone phases, such
as SPB™-1 and SPB-5, combine inertness and stability with
excellent separating characteristics for these derivatives.

Silyl reagents are influenced by both the solvent system and the
addition of a catalyst. A catalyst (e.g., trimethylchlorosilane or
pyridine) increases the reactivity of the reagent. Silyl reagents
generally are moisture sensitive, and should be stored in tightly
sealed containers.

Derivatizing Reagent Selection Guide
The table on pages 4-7 of this bulletin summarizes derivatization
reagent selection, based on sample type. To choose a suitable
reagent, first determine the functional group or compound type
that is of interest to you (far left column in the table). There may
be several options available (second column from left) – consider
what chromatographic tools are available to you, and remember
to consider the criteria for choosing a good reagent (page 1). The
Observations column includes general hints which may assist you
in making your selection.

Troubleshooting Guide
The troubleshooting guide on page 8 can assist you in solving
derivatization problems. The guide lists problem symptoms (far
left column), possible causes of the problem (second column), and
suggested solutions. At the back of this bulletin we have included
a blank page for you to record your own observations when
troubleshooting a derivatization. If you have any helpful hints you
would like to share with others, simply fax them to our Technical
Service group. If you are unable to solve a derivatization problem,
please call our Technical Service group for assistance.

4

SUPELCO

Bulletin 909

O  H

O

H

R–C–NH

2

H

Derivatization Reagent Selection Guide

Functional Group/

Compound Type

P

rocedure

Reagent

Derivative

Observations

Amides

Acylation

TFAA

Trifluoroacetamides

Most 

reactive and volatile of fluorinated anhydrides.  Ideal with FID, ECD, TCD.

Used in identifying methamphetamine.

R–C–NH

2

P

FP

A

Pentafluoropropionamides

Requires lowest analysis temperature of fluorinated anhydrides. Ideal with FID,ECD,TCD.

Primary

Used in identifying opiates, benzoylecgonine.

6

HFBA

Heptafluorobutylamides

Most sensitive fluorinated anhydride for EC detection. Ideal with FID,ECD,TCD.

6

R–C–N–R

Alkylation

TMAH

Methyl 

amides

A favorite reagent for drugs, especially barbiturates. Flash alkylation.

1

Secondary

Exception: meprobamate - analyzed by direct GC analysis as the free base.

DMF-dialkylacetals

N-(N,N-dimethyl)aminomethylenes

Ideal 

for wet samples where excess reagent forms corresponding alcohol.

2

Barbiturates

Silylation

B

S

A

Trimethylsilyl 

amides

Highly reactive, universal reagent. See observations for carbonyls.

Benzodiazepines

BSTFA

Trimethylsilyl 

amides

Highly reactive, universal reagent, more volatile than BSA. See

Imides

observations for carbonyls.

Proteins

BSTFA 

+ 

TMCS

Trimethylsilyl 

amides

TMCS acts as a catalyst - assists in derivatizing amines.

MTBSTFA

TBDMCS 

amides

Strong, yet mild silylating reagent. Derivatives 10,000 times more

stable to hydrolysis than TMS derivatives.

MTBSTFA + TBDMCS

TBDMCS 

amides

TBDMCS acts as a catalyst - assists in derivatizing amines.

Amines

Acylation

Acetic 

anhydride

Acetates

Use with primary and secondary amines.

MBTFA

Trifluoroacetamides

Use with 

primary and secondary amines. Principal byproduct,

N-methyltrifluoroacetamide, is stable, volatile, does not present problems

with GC. Ideal with FID, ECD, TCD. Good for trace analysis with ECD.

Primary

TFAA

Trifluoroacetamides

Most 

reactive & volatile of fluorinated anhydrides. Derivatives

volatile for FID, ECD, TCD.  Good for trace analysis with ECD.

R–C–N–R

TFAI

Trifluoroacetamides

Good for trace analysis with ECD. No

 acid byproducts – byproduct, imidazole, i

s 

inert.

Secondary

P

FP

A

Pentafluoropropionamides

Requires lowest analysis temperature of fluorinated anhydrides.

Ideal with FID, ECD, TCD.  Good for trace analysis with ECD.

Used to identify catecholamines.

Alkaloids

HFBA

Heptafluorobutylamides

Ideal with FID, ECD, TCD. Most sensitive to EC – 

good for trace 

analy

sis 

with ECD.

Amino acids

Used to identify amphetamines, phencyclidine, catecholamines.

6

Amino sugars

Alkylation

PFBBr

Pentafluorobenzyl ethers

Ideal 

with ECD.

Amphetamines

DMF-dialkylacetals

N-(N,N-dimethyl)aminomethylenes

Rapid 

reactions, convenient to use. Use with sterically hindered a

mines.

2

Biogenic

N

B

B

Boronates

Converts 

α

-amino acids, hydroxy acids, hydroxy amines, keto acids,

  catecholamines

diols to more easily chromatographed derivatives.

Carbamates

TMAH

Methyl 

amides

A favorite reagent for drugs, especially barbiturates. Exception:

Hydroxyl amines

meprobamate - analyzed by direct GC analysis as the free base.

Nitrosamines

Silylation

B

S

A

Trimethylsilyl 

ethers

Reagent of choice for simultaneous silylation of amino and hydroxyl groups.

Nucleotides

Effective without solvent, but also used with solvents such as pyridine or DMF.

Nucleosides

BSTFA

Trimethylsilyl 

ethers

Reagent and byproducts are volatile. Can act as its own solvent. Can cause

Urea

detector fouling and noise. If DMF is used as a solvent for silylating secondary

amines, n(aminomethylene)-2,2,2-trifluoroacetamides can be formed instead of

the TMS derivatives.

BSTFA 

+ 

TMCS

Trimethylsilyl 

ethers

TMCS acts as a catalyst - enhances reactivity of BSTFA.

HMDS

Trimethylsilyl 

ethers

Used with TMCS to extend practical range of GC. Gaseous byproduc

t 

(N

H

4).

H  H

H

5

SUPELCO
Bulletin 909

1 Flash alkylation: analyte is derivatized in the GC injection port.

2 DMF-dialkylacetals are recommended for sterically hindered aldehydes, amines, carboxylic acids, and phenols. Shorter chain reag

ents produce more volatile derivatives than longer chain

reagents.

These include DMF-DBA, DMF-DEA, DMF-DMA, DMF-DPA, and Esterate-M (DMF-DMA, 2meq/mL in pyridine).

3 In some cases methyl oximes are not resolved from other components of a complex mixture. o-Benzylhydroxylamine HCl forms less v

olatile derivatives which may be separated. If analysis

requires ECD sensitivity, use o-(pentafluorobenzyl)hydroxylamine HCl.

4 With HMDS + TMCS a fine precipitate of NH

4

Cl is produced during derivatization. The precipitate does not affect chromatography.

6 Perfluoro acid anhydrides produce acidic byproducts which must be removed from the reaction mixture before the derivatives are 

injected onto the GC column. With

perfluoroacylimidazole there

are no acid byproducts to remove.

Functional Group/

Compound Type

Procedure

Reagent

Derivative

Observations

Carbohydrates

Acylation

Acetic 

anhydride

Acetates

Generally used with pyridine – 1:1 mixture with pyridine will derivatize alditols.

(CH

2

OH)

n

MBTFA

Trifluoroacetamides

Reagent of 

choice for derivatizing sugars. Forms volatile derivatives of mono-, di-,

Starches

and trisaccharides.

Sugars

TFAI

Trifluoroacetamides

Forms 

volatile derivatives of mono-, di-, and trisaccharides.

Silylation

BSA 

+ 

TMCS

Trimethylsilyl 

ethers

BSA not recommended for carbohydrates – anomerization will occur. Can be used

with some syrups.

BSTFA 

+ 

TMCS

Trimethylsilyl 

ethers

Use with sugar acids, glucuronides.

HMDS

Trimethylsilyl 

ethers

Most popular choice for silylating sugar acids and related substances. TMCS will

increase silylation potential.

HMDS + TMCS

Trimethylsilyl 

ethers

Use 

with 

aldoses.

4

HMDS + TMCS + pyridine

Trimethylsilyl 

ethers

Use 

with 

oligosaccharides.

4

T

FA

Trimethylsilyl ethers

TMSI

Trimethylsilyl 

ethers

Reagent of choice for silylating sugar phosphates in presence of small amounts of

water. Can be used with some syrups. Use neat or with solvent.

TMSI 

+ 

pyridine

Trimethylsilyl 

ethers

Reagent of choice for silylating aldoses, sugar phosphates, disaccharides contain-

ing small amounts of water. Will not derivatize amino groups.

Carbonyls

>C=O

Alkylation

BCl

3

-2-chloroethanol

Chloro 

esters

Use to prepare phenoxy-type acids for ECD.

Acid 

halides

o-Methyloxyamine 

HCl

Oximes

Use with aldehydes, ketones, ketosteroids. Prevents keto groups from forming

Acid anhydrides

enol ethers.

3

Aldehydes

TFAA

Trifluoroacetates

Most 

reactive and volatile of anhydrides. No acid byproduct. Good with ECD.

Enols

Silylation

B

S

A

Trimethylsilyl 

ethers

Under mild reaction conditions forms highly stable products with most organic

Esters

functional groups.  Very volatile. Byproduct, TMS-acetamide, may interfere with early

Ketones

eluting peaks.

Hydrazones

BSA mixtures oxidize to form SiO

2

, which fouls FIDs.

Oximes

BSTFA

Trimethylsilyl 

ethers

Reacts faster and more completely than BSA. BSTFA and its byproducts are highly

Phenoxy acids

volatile and will not interfere with early eluting peaks. Can act as

Steroids (hydroxy/

its own solvent. Combustion product, HF, reacts with SiO

2

, forming volatile

  keto hormones)

products that can cause detector fouling and noise.

BSTFA 

+ 

TMCS

Trimethylsilyl 

ethers

TMCS acts as a catalyst, increasing reactivity of BSTFA.

TMSI 

+ 

pyridine

Trimethylsilyl 

ethers

Use with hindered and unhindered steroids.

6

SUPELCO

Bulletin 909

 –OH

Functional Group/

Compound Type

Procedure

Reagent

Derivative

Observations

Carboxyls

O

Alkylation

PFBBr

Pentafluorobenzyl esters

U

sed with ECD, UV, MS detection. Use with cannabinoids, carboxylic and fatty acids.

R–C–OH

B

C

l 3

-methanol

Chloro 

esters

Used to prepare short chain (C1-C10)  fatty acids for ECD.

Amino acids

B

F

3-butanol

Butyl esters

Used to prepare n-butyl esters of short 

chain (C1-C10) mono- and 

dicarbox

ylic acids.

Cannabinols

B

F

3-

p

ro

p

a

n

o

l

Propyl 

esters

Used to prepare n-propyl esters.

Carboxylic acids

BF

3-methanol

Methyl 

esters

Use with large samples of C8-C24 fatty acids.

Glycerides

Trimethylsilyldiazomethane

Methyl 

esters

Used with carboxylic acids. Excess reagent in the presence of methanol reacts

Hydroxy acids

instantly and quantitatively. Reaction easily monitored by disappearance of yellow color of reagent.

5

Lipids/phospholipids

DMF-dialkylacetals

Methyl 

esters

Alkylates carboxyl groups; use with sterically hindered carboxylic acids. Also reacts

Prostaglandins

with amines, amino acids, phenols.

2

Steroids (bile,

Methanolic base

Methyl esters

Use with mono-, di-, triglycerides, glycolipids, sphingolipids.

  hydroxy/keto

Methanolic 

HCl

Methyl 

esters

Use with fatty acids C9 and longer. Useful for esterifying difficult carboxylic acids 

(bile acids).

  hormones)

Methanolic 

H

2

SO

4

Methyl 

esters

Use with carboxylic acids and esters (transesterification).

N

B

B

Cyclic 

boronates

Use with carbohydrates, catecholamines, ceramides, sphingosines, corticosteroids,

hop resin acids, 

α

- and 

β-hydroxy acids, monoglycerides, monoglyceryl ethers, prostaglandins.

Reaction achieved by mixing equimolar amounts of sample and reagent in appropriate

solvent (several minutes, room temp.). Polar groups on analyte must be on adjacent

carbons, or separated by only 1 carbon.

TMAH

N-Methyl 

esters

Use with reactive amino, carboxyl, or hydroxyl groups. Flash alkylation.

1

Silylation

B

S

A

Trimethylsilyl ethers

Derivatives easily formed but generally not stable – analyze quickly.

BSTFA

Trimethylsilyl ethers

Reacts faster and more completely than BSA. See observations for carbonyls.

BSTFA + TMCS

Trimethylsilyl ethers

TMCS acts as a catalyst, increasing reactivity of BSTFA.

TMSI

Trimethylsilyl ethers

Use with fatty acids, cannabinols, steroids. Will derivatize most hindered and

unhindered steroid hydroxyls. Can be used with some salts.

Ethers

≡C–O–C

≡

Silylation

HMDS + TMCS + pyridine

Trimethylsilyl ethers

Use 

with epoxides that do not react rapidly with TMCS.

4

Epoxides

TMCS

Trimethylsilyl ethers

Use 

with chlorohydrins.

Hydroxyls

Acylation

Acetic anhydride

Acetates

Use with alcohols, phenols.

R

O

H

M

B

T

FA

Trifluoroacetates

Good for trace analysis with ECD.

Alcohols

TFAA

Trifluoroacetates

Good for trace analysis with ECD.

Alkaloids

TFAI

Trifluoroacetates

Good for trace analysis with ECD.

Cannabinoids

PFPA

Pentafluoropropionates

Use with alcohols, phenols. Derivatives volatile for FID, ECD. Good for trace analysis with ECD.

6

Glycols

HFBA

Heptafluorobutyrates

Use with alcohols, phenols. Derivatives volatile for FID, ECD. Good for trace analysis with ECD.

6

Alkylation

PFBBr

Pentafluorobenzyl 

ethers

Use with alkoxides 

only. Use with ECD.

Trimethylsilyldiazomethane

5

Methyl 

esters

Not ideal for esterifying phenolic acids – phenolic hydroxyl groups also are

methylated (at a slower rate) – can 

lead to mixtures of 

partially methylated 

products.

TMAH

N-Methyl 

esters

Flash alkylation of phenolic alkaloids.

1

Phenols

DMF-dialkylacetals

Methyl 

esters

Use with sterically hindered phenols.

2

Hexaoxacyclooctane

Pentafluorobenzyl 

phenols

Use with p

henols for US EPA Method 604 (see General Alkylation Reagents – page 3).

Silylation

B

S

A

Trimethylsilyl ethers

Most often used. Good choice for silylating phenols when used in DMF.

BSTFA

Trimethylsilyl ethers

Good thermal stability.

BSTFA + TMCS

Trimethylsilyl ethers

Poor hydrolytic stability.

HMDS

Trimethylsilyl ethers

Use 

with unhindered alcohols and phenols. Weak donor, usually used with TMCS.

Appropriate solvent (pyridine, DMF, DMSO) may increase reaction rate.

MTBSTFA

Trimethylsilyl ethers

TBDMSIM

Trimethylsilyl ethers

Use with alcohols. Derivatives 10,000 times more stable to hydrolysis than TMS ethers.

TMCS

Trimethylsilyl ethers

Weak donor, usually used with HMDS. Can be used with salts. Excellent catalyst for

forming TMS ethers.

TMSI

Trimethylsilyl ethers

Strongest silylation reagent for hydroxyls. No reaction with amines or amides.

Derivatizes sugars in presence of water. Can be used with syrups.

7

SUPELCO
Bulletin 909

Functional Group/

Compound Type

P

ro

cedure

Reagent

Derivative

Observations

Nitriles

R–C

≡N

Undergo many of the same reactions as carboxylic acids – see

 Carbonyls.

Thiols

Acylation

MBTFA

Trimethylsilyl ethers

Reaction occurs under mild, non-acidic conditions.

PFBBr

Trimethylsilyl 

ethers

Use 

with ECD.

6

R–SH

Alkylation

Trimethylsilyldiazomethane

5

Methyl esters

Mercaptans

DMF-dialkylacetals

2

Methyl esters

Silylation

TMSI

Trimethylsilyl ethers

Sulfides

Silylation

TMSI

Trimethylsilyl ethers

R–S

Sulfonic Acids

Alkylation

TMAH

N-Methyl esters

R–SO

2

O

H

PFBBr

6

Trimethylsilyl ethers

Silylation

TMSI

Trimethylsilyl ethers

Reaction is with hydroxyl group.

Sulfonamides

Acylation

TFAA

Trifluoroacetates

Stable derivatives.

PFBBr

Trimethylsilyl ethers

Stable derivatives. Enhances ECD.

6

HFBA

Trimethylsilyl ethers

Stable derivatives. Enhances ECD.

6

R–SO

2

NH

2

Alkylation

DMF-dialkylacetals

2

Methyl esters

Silylation

BSTFA

Trimethylsilyl ethers

1 Flash alkylation: analyte is derivatized in the GC injection port.

2 DMF-dialkylacetals are recommended for sterically hindered aldehydes, amines, carboxylic acids, and phenols. Shorter chain reag

ents produce more volatile derivatives than longer chain

reagents. These include DMF-DBA, DMF-DEA,DMF-DMA, DMF-DPA, and Esterate-M (DMF-DMA, 2meq/mL in pyridine).

5 Safer substitute for diazomethane.

6 Perfluoro acid anhydrides produce acidic byproducts which must be removed from the reaction mixture before the derivatives are 

injected onto the GC column. With

perfluoroacylimidazole there are no acid byproducts to remove.

Useful Literature

Handbook of Analytical Derivatization Reactions

D.R. Knapp

23561

For additional information and prices refer to the current Supelco catalog.

Supelco Technical Literature

Product Specification sheets containing detailed information about re-

agent physical properties, typical derivatization procedures, reaction

mechanisms, storage information, etc. are available, free, for most of the

acylation, alkylation, and silylation reagents described in this bulletin. To

request this free literature see the table on page 9.

Supelco also produces a wide variety of technical literature, all of which

is free. You may request this literature by using the business reply card

located at the back of our catalog or by calling our Ordering and Customer

Service Department (800-247-6628 or 814-359-3441).

8

SUPELCO

Bulletin 909

Troubleshooting the Derivatization Reaction and Analysis
With few exceptions, possible causes and remedies listed here specifically address the derivatization process. It is assumed that an
appropriate column and analytical conditions, and other general considerations, are used.

Symptom

Possible Cause

Remedy

Missing peaks or

1. Impurities in solvent, starting material,

1. Use only highest purity materials at all steps in sample prepartion

solvent peak

catalysts, or extract may interfere with

process.

only

derivatization (e.g., plasticizers from vial,
inorganics used in sample synthesis,
preservatives or antioxidants in solvents).

2. Reagent  deteriorated.

2. Store reagent properly to prevent oxygen/water contamination,

temperature damage (see product specification sheet).

3. Reagent:sample ratio too low.

3. Use more reagent for same amount of sample.

4. Rate of reaction too slow.

4. Reevaluate reagent concentration, time, temperature. Consider

heating the reaction mix (consider thermal stability of the
analytes and reagents).
A catalyst will increase the reactivity of some reagents.

5. Water in reaction mix.

5. Remove water by adding sodium sulfate to sample.

Store reagent properly to prevent oxygen/water contamination.

6. Wrong reagent.

6. Reevaluate reagent selection.

7. Sample adsorbed to glassware.

7. Deactivate glassware, inlet sleeve, and column by silanization.

Extra peak(s)

1. Reagent interacting with column.

1. Verify that reagent is compatible with analytical column.

2. Impurities from sample, solvent,

2. Inject solvent and reagent blanks, solvent rinse from unused vial,

reagents, sample vial, other labware.

etc. to isolate source of impurities.

3. Derivative undergoing hydrolysis.

3. Remove water by adding sodium sulfate to sample.

Store reagent properly to prevent oxygen/water contamination.

4. Derivative reacting with solvent.

4. Use a solvent that does not have an active hydrogen, alcohol,

or enolizable ketone group (e.g., hexane, toluene, etc.).

Detector

1. Low yield of derivative – reaction

1. Add more reagent, increase temperature or heating time, or add

response low

did not go to completion.

catalyst. Water may be present; add sodium sulfate to sample.

2. Detector (FID) dirty.

2. Clean FID per instrument manual.

3. Sample components absorbed by

3. Inject standard on column known to be performing well. If

inlet liner or column.

results are good, remove inlet liner and check cleanliness. Use
new, deactivated liner or replace glass wool and packing.
Rinse bonded phase column or remove 1-2 coils from inlet end of
nonbonded column. If performance is not restored, replace
column.

No sample separation 1. Septum in reaction vial not sealed.

1. Prepare a new sample and derivatize. Be sure vial is sealed.

after adding reagent
and heating

Low Yield

1. Improper handling technique: extra

1. Reevaluate technique, if possible eliminate steps in which analyte

steps allow more room for error (e.g.,

could be adsorbed or otherwise lost (unnecessary transfers, etc.).

low boiling components could be lost
during sample concentration); sample
too dilute; wrong solvent.

2. Impurities in solvent, starting material,

2. Use only highest purity materials at all steps in the sample

catalysts, or extract interfering with

preparation process.

derivatization (e.g., plasticizers from vial,
inorganics used in sample synthesis,
preservatives or antioxidants in solvents).

3. Reagent  deteriorated.

3. Store reagent properly to prevent oxygen/water contamination,

temperature damage (see product specification sheet).

4. Reagent:sample ratio too low.

4. Use more reagent for same amount of sample.

5. Rate of reaction too slow.

5. Reevaluate reagent concentration, time, temperature. Consider

heating the reaction mix (consider thermal stability of the
analytes and reagents).
A catalyst will increase the reactivity of some reagents.

6. Water in reaction mix.

6. Remove water by adding sodium sulfate to sample.

Store reagent properly to prevent oxygen/water contamination.

7. Wrong reagent.

7. Reevaluate reagent selection.

8. Sample adsorbed to glassware.

8. Deactivate glassware, inlet sleeve, and column by silanizing.

9. Carrier, air, detector (FID) hydrogen,

9. Measure flows and set according to instrument manufacturer’s

or make-up gas flow set incorrectly.

recommendations.

9

SUPELCO
Bulletin 909

Product specification sheets for most Supelco reagents are available free of charge. These publications contain information about the
reagent: physical properties, use, benefits, mechanism of action and typical derivatization procedures, toxicity, hazards, and storage.
To obtain free copies, contact our Order Processing department.

Refer to These Publications for Descriptions
of Reagents and Step-by-Step Procedures
for Derivatization

Reagent

Publication No.

Acetic Anhydride

T497121

BSA (N,O-bis(trimethylsilyl)acetamide)

T496017

BSA + TMCS

T496018

BSA + TMCS + TMSI

T496019

BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide)

T496020

BSTFA + TMCS

T496021

DMDCS (dimethyldichlorosilane)

T496022

DMDCS in toluene

T496023

HMDS (hexamethyldisilazane)

T496024

HMDS + TMCS

T496025

HMDS + TMCS + pyridine

T496026

Methanolic Base, 0.5N

T497007

Methanolic HCI, 0.5N, 3N

T497099

Methanolic H

2SO4

T497018

Perfluoro Acid Anhydrides

T497104

Pentafluorobenzyl Bromide, Hexaoxacyclooctadecane

T497103

Rejuv-8TM

T496066

TBDMSIM (N-t-butyldimethylsilylimidazole)

T496065

TMAH

T496180

TMCS (trimethylchlorosilane)

T496028

TMSI (N-trimethylsilylimidazole)

T496029

TMSI + pyridine

T496030

Trifluoroacetic acid

T496027

BCl

3-2-chloroethanol

T496122

BCl

3-methanol

T496123

BF

3-butanol

T496124

BF

3-methanol

T496125

Ordering Information:

Acylation Reagents

Description

Cat. No.

Acetic Anhydride

10 x 2mL

33085

HFBA

10 x 1mL

33170-U

MBTFA

10 x 1mL

39,4939-10X1ML

5mL

39,4939-5ML

PFPA

10 x 1mL

33167

25mL

33168

TFAA

10 x 1mL

33165-U

25mL

33164

TFAI

10 x 1mL

39,4920-10X1ML

5mL

39,4920-5ML

Acronyms

Acronym

Chemical Name [CAS No.]

BSA

N,O-Bis(trimethylsilyl)acetamide [10416-59-8]

BSTFA

Bis(trimethylsilyl)trifluoroacetamide [25561-30-2]

Diazald-

N-Methyl-13C-N-nitroso-

p-

N-methyl-13C

toluenesulfonamide [60858-95-9]

Diazald-

N-methyl-13C- N-Methyl-13C-d

3-N-nitroso-p-

N-methyl-d

3

toluenesulfonamide [102832-11-1]

DMDCS

Dimethyldichlorosilane [75-78-5]

DMF-DBA

N,N-Dimethylformamide / Di-

tert-butyl acetal [36805-97-7]

DMF-DEA

N,N-Dimethylformamide / Diethyl acetal [1188-33-6]

DMF-DMA

N,N-Dimethylformamide / Dimethyl acetal [4637-24-5]

DMF-DPA

N ,N-Dimethylformamide / Dipropyl acetal [6006-65-1]

DMP

2,2 Dimethoxypropane [77-76-9]

HFBA

Heptafluorobutyric anhydride [336-59-4]

HMDS

1,1,1,3,3,3-Hexamethyldisilazane [999-97-3]

MBTFA

N-Methylbis(trifluoroacetamide) [685-27-8]

MNNG

1-Methyl-3-nitro-1-nitrosoguanidine [70-25-7]

MTBSTFA

N-(

tert-Butyldimethylsilyl)-N-methyl-

trifluoroacetamide [77377-52-7]

NBB

n-Butylboronic acid [4426-47-5]

PFBBr

Pentafluorobenzylbromide [1765-40-8]

PFPA

Pentafluoropropionic anhydride [356-42-3]

TBDMCS

t-Butyldimethylchlorosilane [18162-48-6]

TBDMSIM

N-(

tert-Butyldimethylsilyl)imidazole

TFAI

1-(Trifluoroacetyl)imidazole [1546-79-8]

TMCS

Trimethylchlorosilane [75-77-4]

TMSDEA

Trimethylsilyldiethylamine

(N,N-Diethyl-1,1,1-trimethylsilylamine) [996-50-9]

TMSI

Trimethylsilylimidazole [18156-74-6]

Alkylation Reagents

Description

Cat. No.

DMF-Dialkylacetals
DMF-DBA

10 x 1mL

39,5005-10X1ML

5mL

39,5005-5ML

25mL

39,5005-25ML

DMF-DEA (1,1-Diethoxytrimethylamine)

10 x 1mL

39,4971-10X1ML

5mL

39,4971-5ML

25mL

39,4971-25ML

DMF-DMA

10 x 1mL

39,4963-10X1ML

5mL

39,4963-5ML

25mL

39,4963-25ML

DMF-DPA

10 x 1mL

39,4998-10X1ML

5mL

39,4998-5ML

25mL

39,4998-25ML

Diazoalkales

■

Diazald

25g

D28000-25G

100g

D28000-100G

500g

D28000-500G

1kg

D28000-1KG

Diazald-N-methyl-13C (99 atom % 13C)

250mg

27,7614-250MG

1g

27,7614-1G

Diazald-N-methyl-13C-N-methyl-d

3

(99 atom % 13C, 99 atom % d

3)

250mg

29,5981-250MG

1g

29,5981-1G

MNNG

10g

12,9941-10G

25g

12,9941-25G

(Trimethylsilyl)diazomethane
(2.0M solution in hexanes)

5mL

36,2832-5ML

25mL

36,2832-25ML

■ For more information, request Aldrich publication AL-180.

Trademarks
Omegawax, REACTA-SIL, Rejuv-8, SP, SPB, Supelco, Sylon — Sigma-Aldrich Co.
Teflon — E.I. du Pont de Nemours & Co., Inc.

10

SUPELCO

Bulletin 909

Alkylation Reagents (contd.)

Description

Cat. No.

Esterification Reagents
BCl

3-2-Chloroethanol (11% w/w)

10 x 1mL

33056-U

BCl

3-Methanol (12% w/w)

20 x 1mL

33353

20 x 2mL

33089-U

400mL

33033

BF

3-Butanol (10% w/w)

10 x 5mL

33126-U

100mL

33125-U

BF

3-Methanol (10% w/w)

20 x 1mL

33356

19 x 2mL

33020-U

10 x 5mL

33040-U

5mL

26,4121-5ML

250mL

26,4121-250ML

400mL

33021

BF

3-Propanol (14% w/w)

5g

15,6825-5G

100g

15,6825-100G

500g

15,6825-500G

Methanolic Base (0.5N)

2N, 30mL

33352

2N, 100mL

33080

Methanolic HCl

0.5N, 20 x 1mL

33354

0.5N, 10 x 5mL

33095

3N, 20 x 1mL

33355

3N, 10 x 3mL

33051

3N, 400mL

33050-U

Methanolic H

2SO4 (10% H2SO4 v/v in methanol)

6 x 5mL

506516

TMAH, 0.2M in methanol

10 x 1mL

33358-U

10mL

33097-U

General Alkylation Reagents
DMP (2,2-Dimethoxypropane),  25g

33053

Esterate M, 25mL

33140

Hexaoxacyclooctadecane (18 crown 6),  25g

33003-U

O-Methoxyamine HCl, 5g

33045-U

Pentafluorobenzyl bromide,  5g

33001

Silyl Reagents (contd.)

Description

Cat. No.

BSTFA, derivatization grade

144 x 0.1mL

33084

20 x 1mL

33024

25mL

33027

BSTFA + TMCS, 99:1 (Sylon BFT)

144 x 0.1mL

33154-U

20 x 1mL

33148

25mL

33155-U

50mL

33149-U

HMDS

30mL

33350-U

100mL

33011

HMDS + TMCS, 3:1 (Sylon HT)

20 x 1mL

33046

REACTA-SIL

® Concentrate (HMDS:TMCS, 2:1)

25mL

39,4610-25ML

HMDS + TMCS + Pyridine, 3:1:9 (Sylon HTP)

20 x 1mL

33038

25mL

33039

N-Methyl-N-(trimethylsilyl)trifluoroacetamide

10 x 1mL

39,4866-10X1ML

5mL

39,4866-5ML

25mL

39,4866-25ML

MTBSTFA, derivatization grade

10 x 1mL

39,4882-10X1ML

5mL

39,4882-5ML

25mL

39,4882-25ML

MTBSTFA + TBDMCS, 99:1

10 x 1mL

37,5934-10X1ML

5mL

37,5934-5ML

25mL

37,5934-25ML

TFA

10 x 1mL

33077

25mL

33075

100mL

33076

TMCS, derivatization grade

100mL

33014

TMSI, derivatization grade

25mL

33068-U

TMSI + Pyridine, 1:4 (Sylon TP)

20 x 1mL

33159-U

25mL

33156-U

t-Butyldimethylsilylimidazole-dimethylformamide

10 x 1mL

33092-U

Silyl Reagents

Description

Cat. No.

BSA, derivatization grade

144 x 0.1mL

33035-U

20 x 1mL

33036

25mL

33037-U

BSA + TMCS, 5:1 (Sylon

TM BT)

20 x 1mL

33018

25mL

33019-U

BSA + TMCS + TMSI, 3:2:3 (Sylon BTZ)

144 x 0.1mL

33151

20 x 1mL

33030

25mL

33031-U

Silyl Reagents for Deactivation of Glassware
and Chromatographic Supports
Note:  All SupelcoTM glass GC columns are silane treated.

Qty.

Cat. No.

DMDCS

100mL

33009

5% DMDCS in Toluene (Sylon CT)

400mL

33065-U

Rejuv-8 Silylating Agent

25mL

33059-U

11

SUPELCO
Bulletin 909

Notes on Derivatization Procedures

________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________

We recommend using this space to record helpful tips and troublesome problems you encounter in working with
derivatization reagents.  You may want to photocopy this form and send us tips we can share with others, or let
us know your problems and we will try to help.  Please fax this information to 800-359-3044 or 814-359-5468.

Description

Cat. No.

Acylation Sampler Kit

505862

3 x 1mL of each of the following (except as noted):

Acetic anhydride (3 x 2mL)
Heptafluorobutyric anhydride
Pentafluoropropionic anhydride
Trifluoroacetic anhydride

Silylation Sampler Kit

505846

3 x 1mL of each of the following:

BSA
BSTFA
BSTFA + TMCS, 99:1 (Sylon BFT)
HMDS + TMCS, 3:1 (Sylon HT)
TMSI

Description

Cat. No.

FID Alkylation Sampler Kit

505854

3 x 1mL of each of the following:

BF

3-Methanol

Methanolic Base
Methanolic HCl (0.5N)
Methanolic HCl (3N)
TMAH, 0.2M in methanol

ECD Alkylation Sampler Kit

505870

3 x 1mL of each of the following (except as noted):

BCl

3-2-Chloroethanol, 11% w/w

BCl

3-Methanol, 12% w/w

Hexaoxacyclooctadecane, 18 crown 6 (1 gram)
Pentafluorobenzylbromide

12

SUPELCO

Bulletin 909

© 2003 Sigma-Aldrich Co.  Printed in USA. Supelco brand products are sold through Sigma-Aldrich, Inc. Sigma-Aldrich, Inc. warrants that its products conform to the
information contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and
conditions may apply. Please see reverse side of the invoice or packing slip.

The SIGMA-ALDRICH Family

We are committed to the success of our Customers, Employees and Shareholders through leadership in Life Science, High Technology and Service.

Order/Customer Service 800-247-6628, 800-325-3010 ● Fax 800-325-5052 ● E-mail [email protected]

Technical Service 800-359-3041, 814-359-3041 ● Fax 800-359-3044, 814-359-5468 ● E-mail [email protected]

SUPELCO ● 595 North Harrison Road, Bellefonte, PA  16823-0048 ● 814-359-3441

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T196909

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