Bulletin 909A

Guide to Derivatization Reagents for GC

A large number of reagents are used to prepare derivatives for Glassware for Derivatization
gas chromatography, but most of the derivatization reactions Vials with 0.1-10.0mL capacity accommodate sample plus solvent
fit into one of three categories: acylation, alkylation, or and reagent in quantities typically used in gas chromatography.
silylation. This bulletin describes each category, and presents Vials must be suitable for temperature extremes. Vials supplied
information on how to choose the proper reagent based on the with open-center screw caps can be sealed with rubber septum
functional group(s) of the compound to be derivatized. 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
Key Words
bottoms give these vials added stability on a flat surface, and are
● acylation ● alkylation ● silylation
convenient for pencil markings. Thermostatically controlled heat-
● derivatization reagents ● derivatives
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
Considerations When dissolved by some samples and reagents.
Derivatizing an Analyte
Gas chromatography is used to separate volatile organic com- Deactivation of Glassware
pounds. By modifying the functionality of a molecule to increase Because the surface of laboratory glassware is slightly acidic, it can
– or sometimes decrease – volatility, derivatizing reagents enable adsorb some analytes — particularly amines. In low level analyses,
chromatographers to analyze compounds that otherwise are not such losses can be significant. To prevent sample loss through
readily monitored by GC. Derivatization also reduces analyte adsorption, glassware used in low level analyses usually is silanized.
adsorption in the GC system and improves detector response, peak Silanization masks the polar Si-OH groups on the glass surface by
separations, and peak symmetry. chemically binding a nonadsorptive silicone layer to the surface,
Derivatives are used for the following reasons: in effect “derivatizing” the glass. In the most common silanization
procedure, the glassware is treated with a solution of 5-10%
● to improve resolution and reduce tailing of polar compounds
dimethyldichlorosilane (DMDCS) in toluene for 30 minutes. The
(–OH, –COOH, =NH, –NH2, –SH, and other functional groups)
deactivated glassware is rinsed with toluene, then immediately
● to analyze relatively nonvolatile compounds thereafter with methanol.
● to improve analytical efficiency and increase detectability Adsorption also can be reduced by adding a compound that
● to improve stability of compounds competes for the adsorptive sites on the glass surface. A small
amount (often less than 1%) of an alcohol, such as butanol, added
The choice of a derivatizing reagent is based on the functional to the solvent significantly reduces adsorption losses.
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: Contents
● produces a derivatization reaction that is 95-100% complete Topic Page

● will not cause any rearrangements or structural alterations Considerations When Derivatizing an Analyte 1
during formation of the derivative Acylation 2
● does not contribute to loss of the sample during the reaction Alkylation 3
● produces a derivative that will not interact with the analytical Silylation 3
(GC or HPLC) column Derivatization Reagent Selection Guide 4
● produces a derivative that is stable with respect to time Troubleshooting 8
Supelco offers helpful free technical literature for most derivatization Ordering Information 9
reagents (see page 9).

T196909A ©1997 Sigma-Aldrich Co.

SUPELCO
Bulletin 909 1
Sample Handling Acylation
Most lab personnel transfer samples and reagents with pipettes. Acylation, an alternative to silylation, is the conversion of com-
For sensitive reagents, we recommend using a microliter syringe, pounds that contain active hydrogens (-NH, -OH, -SH) into amides,
which reduces exposure to atmospheric moisture. Syringes with esters, or thioesters through the action of a carboxylic acid or
Teflon-tipped plungers are more convenient than conventional carboxylic derivative. Acylation has many benefits:
syringes with all-metal plungers, particularly for transferring vola- ● It improves analyte stability by protecting unstable groups.
tile reagents. The Teflon plunger tip forms a better seal and
facilitates withdrawal of the reagent from a sealed vial. ● It can confer volatility on substances such as carbohydrates or
amino acids, which have so many polar groups that they are
Any syringe will retain some reagent in the barrel. A syringe with an
nonvolatile and normally decompose on heating.
all-metal plunger, if not properly cleaned, is prone to corrosion and
seizing. The best cleaning procedure is to remove and wash the ● It assists in chromatographic separations which might not be
plunger, and use a vacuum to pull solvent through the syringe. A possible with underivatized compounds.
seized plunger sometimes can be freed by soaking the syringe in ● Compounds are detectable at very low levels with an electron
a container filled with methanol. capture detector.
In halocarbons, the presence of a carbonyl group adjacent to a
Injection Ports
halogenated carbon enhances the electron capture detector (ECD)
When working with silylating reagents, use a silanized glass injec- response. Acylation also has been used to form fragmentation-
tion port or make injections directly onto a glass column. Use of directing derivatives for mass spectrometry and chromogenic
a stainless steel injection port frequently yields erratic and derivatives for HPLC.
irreproducible results. The problem may not become apparent
until after several weeks of use, when corrective action may include
Perfluoro Acid Anhydrides
replacing the injector.
Acylation of amino, hydroxy, and thiol groups to the perfluoroacyl
derivatives reduces polarity. The derivatives also are both stable
Reaction Time
and highly volatile. Although fluorinated anhydride derivatives are
Reaction time varies greatly among compounds. Many materials used primarily with electron capture detectors (ECD), they can be
can be derivatized by the reagents described here in a matter of used with flame ionization detectors (FID). These reagents react
seconds or minutes at room temperature, while others require with alcohols, amines, and phenols to produce stable derivatives.
extended periods at elevated temperatures. For a compound with Fluorinated anhydrides are used in derivatizing samples for drug
unknown reactivity, the progress of the derivatization can be of abuse confirmation.
monitored by periodic chromatographic analysis of aliquots of the
reaction mixture. Disappearance of the reagents or appearance of The perfluoro acid anhydrides and acyl halide reagents form acidic
product peaks can be used to determine the reaction’s progress. byproducts which must be removed prior to the GC analysis, to
prevent damage to the chromatography column. Acylations with
Heating often increases the yield of derivative and/or shortens the anhydride reagents normally are performed in pyridine, tetrahy-
reaction time. Before using heat, consider the thermal stability of drofuran, or other solvent capable of accepting the acid byproduct.
the analytes and reagents involved. Amine bases also may be used as catalysts/acid acceptors.

Water Perfluoroacylimidazoles
Water in the reaction mixture often can hinder the reaction and/ Perfluoroacylimidazoles offer advantages over perfluoro acid an-
or hydrolyze the derivative, reducing the yield of derivative for hydrides for preparing perfluoroacyl derivatives. The reactions are
analysis. Tightly seal opened reagents during storage. If necessary, smooth and quantitative, and produce no acid byproducts that
add sodium sulfate to the reaction mixture to trap water present must be removed prior to the injection.
in the sample.
The activated amide reagents also yield no acid byproducts,
Chromatography producing only imidazole and N-methytrifluoroacetamide, re-
spectively.
We offer a wide range of general purpose and specially tested
capillary GC columns for evaluating underivatized and derivatized The perfluoroacylimidazoles react with hydroxyl groups and both
analytes. For descriptions of our capillary columns, please refer to primary and secondary amines, and quantitatively acylate indole
the current Supelco catalog. 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.

SUPELCO
2 Bulletin 909
Alkylation Silylation
Alkylation involves adding an alkyl group (aliphatic or aliphatic- Silylation is the introduction of a silyl group into a molecule, usually
aromatic) to an active functional (H) group. Replacement of in substitution for active hydrogen. Replacement of active hydro-
hydrogen with an alkyl group is important because the derivative gen by a silyl group reduces the polarity of the compound and
has lower polarity, relative to the parent substance. Alkylation reduces hydrogen bonding. The silylated derivative thus is more
reagents are used to modify compounds containing acidic hydro- volatile, and more stable. Detection is enhanced. Many hydroxy
gens, such as carboxylic acids and phenols. The resulting products and amino compounds regarded as nonvolatile or unstable at 200-
are ethers, esters, thioethers, thioesters, n-alkylamines, and n- 300°C have been successfully chromatographed after silylation.
alkylamides. Alkylation of weakly acidic groups (e.g., alcohols) Silyl reagents are compatible with most detection systems but, if
requires strongly basic catalysts (sodium or potassium methoxide). used in excess, can cause difficulties with flame ionization detec-
More acidic OH groups (phenols, carboxylic acids) require less tors.
basic catalysts (hydrogen chloride, boron trifluoride). The trimethylsilyl (TMS) group, Si(CH3)3, is the most popular and
versatile silyl group for GC analysis. TMS derivatization enables
DMF-Dialkylacetals better GC separations and application of special detection tech-
Dimethylformamide dialkyl acetals are used to esterify acids to niques. TMS silylating reagents and derivatives react with active
their methyl esters. Hydroxyl groups are not methylated. Carboxy- hydrogen atoms. Consequently, TMS derivatives should not be
lic acids, phenols, and thiols react quickly, to give the correspond- analyzed on polyethylene glycol phases or other stationary phases
ing alkyl derivatives. N,N-dimethylformamide dimethylacetals are that have these functional groups. Nonpolar silicone phases, such
moisture sensitive. as SPB™-1 and SPB-5, combine inertness and stability with
excellent separating characteristics for these derivatives.
Diazoalkales Silyl reagents are influenced by both the solvent system and the
In the presence of a small amount of methanol as catalyst, addition of a catalyst. A catalyst (e.g., trimethylchlorosilane or
diazomethane (a yellow gas, usually used as an ethereal solution) pyridine) increases the reactivity of the reagent. Silyl reagents
reacts rapidly with fatty acids, forming methyl esters. Elimination generally are moisture sensitive, and should be stored in tightly
of gaseous nitrogen drives the reaction. The yield is high and side sealed containers.
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
Derivatizing Reagent Selection Guide
mixtures of partially methylated products. The table on pages 4-7 of this bulletin summarizes derivatization
reagent selection, based on sample type. To choose a suitable
Esterification and Transesterification Reagents reagent, first determine the functional group or compound type
that is of interest to you (far left column in the table). There may
Esterification, the reaction of an acid with an alcohol to form an
be several options available (second column from left) – consider
ester, is the most popular alkylation method. Alkyl esters offer
what chromatographic tools are available to you, and remember
excellent stability, and provide quick and quantitative samples for
to consider the criteria for choosing a good reagent (page 1). The
GC analysis. The process involves the condensation of the carboxyl
Observations column includes general hints which may assist you
group of the acid and the hydroxyl group of the alcohol, with
in making your selection.
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 Troubleshooting Guide
ester by another alcohol. This reaction has been widely used for
making esters of higher alcohols from esters of lower alcohols. In The troubleshooting guide on page 8 can assist you in solving
the presence of an acidic or basic catalyst, methanol can be used derivatization problems. The guide lists problem symptoms (far
to transesterify fats or oils. 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
General Alkylation Reagents
troubleshooting a derivatization. If you have any helpful hints you
Pentafluorobenzylbromide is convenient for making esters and would like to share with others, simply fax them to our Technical
ethers, and has been used in trace analyses. This strong lachryma- Service group. If you are unable to solve a derivatization problem,
tor should be used in a hood. Hexacyclooctadecane and please call our Technical Service group for assistance.
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.
SUPELCO
Bulletin 909 3
 Derivatization Reagent Selection Guide

4
Functional Group/
Compound Type Procedure Reagent Derivative Observations
Amides Acylation TFAA Trifluoroacetamides Most reactive and volatile of fluorinated anhydrides. Ideal with FID, ECD, TCD.
O Used in identifying methamphetamine.
R–C–NH2 PFPA Pentafluoropropionamides Requires lowest analysis temperature of fluorinated anhydrides. Ideal with FID,ECD,TCD.
Primary Used in identifying opiates, benzoylecgonine.6
O H 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 BSA 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.
H MBTFA Trifluoroacetamides Use with primary and secondary amines. Principal byproduct,
R–C–NH 2 N-methyltrifluoroacetamide, is stable, volatile, does not present problems
H with GC. Ideal with FID, ECD, TCD. Good for trace analysis with ECD.
Primary TFAA Trifluoroacetamides Most reactive & volatile of fluorinated anhydrides. Derivatives
H H volatile for FID, ECD, TCD. Good for trace analysis with ECD.
R–C–N–R
H TFAI Trifluoroacetamides Good for trace analysis with ECD. No acid byproducts – byproduct, imidazole, is inert.
Secondary PFPA 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 analysis 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 amines.2
Biogenic NBB 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 BSA 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 byproduct (NH4).

Bulletin 909
SUPELCO
 Functional Group/
Compound Type Procedure Reagent Derivative Observations

Bulletin 909
Carbohydrates Acylation Acetic anhydride Acetates Generally used with pyridine – 1:1 mixture with pyridine will derivatize alditols.

SUPELCO
(CH2OH)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
TFA 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 BCl3-2-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 BSA 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 SiO2, 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 SiO2, 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.
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 reagents 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 volatile derivatives which may be separated. If analysis
requires ECD sensitivity, use o-(pentafluorobenzyl)hydroxylamine HCl.
4
With HMDS + TMCS a fine precipitate of NH4Cl 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.

5
 Functional Group/

6
Compound Type Procedure Reagent Derivative Observations
Carboxyls
O Alkylation PFBBr Pentafluorobenzyl esters Used with ECD, UV, MS detection. Use with cannabinoids, carboxylic and fatty acids.
R–C–OH BCl3-methanol Chloro esters Used to prepare short chain (C1-C10) fatty acids for ECD.
Amino acids BF3-butanol Butyl esters Used to prepare n-butyl esters of short chain (C1-C10) mono- and dicarboxylic acids.
Cannabinols BF3-propanol Propyl esters Used to prepare n-propyl esters.
Carboxylic acids BF3-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 H2SO4 Methyl esters Use with carboxylic acids and esters (transesterification).
NBB 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 BSA 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.
ROH MBTFA 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.
Trimethylsilyldiazomethane5 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 phenols for US EPA Method 604 (see General Alkylation Reagents – page 3).
Silylation BSA 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.
–OH 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.

Bulletin 909
SUPELCO
 Functional Group/
Compound Type Procedure Reagent Derivative Observations

Bulletin 909
Nitriles
R–C≡N Undergo many of the same reactions as carboxylic acids – see Carbonyls.

SUPELCO
Thiols Acylation MBTFA Trimethylsilyl ethers Reaction occurs under mild, non-acidic conditions.
PFBBr Trimethylsilyl ethers Use with ECD.6
R–SH Alkylation Trimethylsilyldiazomethane5 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 OH PFBBr6 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 2NH2 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 reagents 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).

7
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.

SUPELCO
8 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 Ordering Information:

of Reagents and Step-by-Step Procedures
for Derivatization Acylation Reagents
Reagent Publication No. Description Cat. No.
Acetic Anhydride T497121 Acetic Anhydride
BSA (N,O-bis(trimethylsilyl)acetamide) T496017 10 x 2mL 33085
BSA + TMCS T496018 HFBA
BSA + TMCS + TMSI T496019 10 x 1mL 33170-U
BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) T496020 MBTFA
BSTFA + TMCS T496021 10 x 1mL 39,4939-10X1ML
DMDCS (dimethyldichlorosilane) T496022 5mL 39,4939-5ML
DMDCS in toluene T496023 PFPA
HMDS (hexamethyldisilazane) T496024 10 x 1mL 33167
HMDS + TMCS T496025 25mL 33168
HMDS + TMCS + pyridine T496026 TFAA
Methanolic Base, 0.5N T497007 10 x 1mL 33165-U
Methanolic HCI, 0.5N, 3N T497099 25mL 33164
Methanolic H2SO4 T497018 TFAI
Perfluoro Acid Anhydrides T497104 10 x 1mL 39,4920-10X1ML
Pentafluorobenzyl Bromide, Hexaoxacyclooctadecane T497103 5mL 39,4920-5ML
Rejuv-8TM T496066
TBDMSIM (N-t-butyldimethylsilylimidazole) T496065
TMAH T496180
TMCS (trimethylchlorosilane) T496028 Alkylation Reagents
TMSI (N-trimethylsilylimidazole) T496029
TMSI + pyridine T496030 Description Cat. No.
Trifluoroacetic acid T496027
DMF-Dialkylacetals
BCl3-2-chloroethanol T496122
DMF-DBA
BCl3-methanol T496123
10 x 1mL 39,5005-10X1ML
BF3-butanol T496124
5mL 39,5005-5ML
BF3-methanol T496125
25mL 39,5005-25ML
DMF-DEA (1,1-Diethoxytrimethylamine)
10 x 1mL 39,4971-10X1ML
Acronyms 5mL 39,4971-5ML
25mL 39,4971-25ML
Acronym Chemical Name [CAS No.] DMF-DMA
BSA N,O-Bis(trimethylsilyl)acetamide [10416-59-8] 10 x 1mL 39,4963-10X1ML
BSTFA Bis(trimethylsilyl)trifluoroacetamide [25561-30-2] 5mL 39,4963-5ML
Diazald- N-Methyl-13C-N-nitroso-p- 25mL 39,4963-25ML
N-methyl-13C toluenesulfonamide [60858-95-9] DMF-DPA
Diazald- 10 x 1mL 39,4998-10X1ML
N-methyl-13C- N-Methyl-13C-d3-N-nitroso-p- 5mL 39,4998-5ML
N-methyl-d3 toluenesulfonamide [102832-11-1] 25mL 39,4998-25ML
■
DMDCS Dimethyldichlorosilane [75-78-5] Diazoalkales
DMF-DBA N,N-Dimethylformamide / Di-tert-butyl acetal [36805-97-7] Diazald
DMF-DEA N,N-Dimethylformamide / Diethyl acetal [1188-33-6] 25g D28000-25G
DMF-DMA N,N-Dimethylformamide / Dimethyl acetal [4637-24-5] 100g D28000-100G
DMF-DPA N ,N-Dimethylformamide / Dipropyl acetal [6006-65-1] 500g D28000-500G
DMP 2,2 Dimethoxypropane [77-76-9] 1kg D28000-1KG
HFBA Heptafluorobutyric anhydride [336-59-4] Diazald-N-methyl-13C (99 atom % 13C)
HMDS 1,1,1,3,3,3-Hexamethyldisilazane [999-97-3] 250mg 27,7614-250MG
MBTFA N-Methylbis(trifluoroacetamide) [685-27-8] 1g 27,7614-1G
MNNG 1-Methyl-3-nitro-1-nitrosoguanidine [70-25-7] Diazald-N-methyl-13C-N-methyl-d3
MTBSTFA N-(tert-Butyldimethylsilyl)-N-methyl- (99 atom % 13C, 99 atom % d3)
trifluoroacetamide [77377-52-7] 250mg 29,5981-250MG
NBB n-Butylboronic acid [4426-47-5] 1g 29,5981-1G
PFBBr Pentafluorobenzylbromide [1765-40-8] MNNG
PFPA Pentafluoropropionic anhydride [356-42-3] 10g 12,9941-10G
TBDMCS t-Butyldimethylchlorosilane [18162-48-6] 25g 12,9941-25G
TBDMSIM N-(tert-Butyldimethylsilyl)imidazole (Trimethylsilyl)diazomethane
TFAI 1-(Trifluoroacetyl)imidazole [1546-79-8] (2.0M solution in hexanes)
TMCS Trimethylchlorosilane [75-77-4] 5mL 36,2832-5ML
TMSDEA Trimethylsilyldiethylamine 25mL 36,2832-25ML
(N,N-Diethyl-1,1,1-trimethylsilylamine) [996-50-9] ■
For more information, request Aldrich publication AL-180.
TMSI Trimethylsilylimidazole [18156-74-6]

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

SUPELCO
Bulletin 909 9
Alkylation Reagents (contd.) Silyl Reagents (contd.)
Description Cat. No. Description Cat. No.
Esterification Reagents BSTFA, derivatization grade
BCl3-2-Chloroethanol (11% w/w) 144 x 0.1mL 33084
10 x 1mL 33056-U 20 x 1mL 33024
BCl3-Methanol (12% w/w) 25mL 33027
20 x 1mL 33353 BSTFA + TMCS, 99:1 (Sylon BFT)
20 x 2mL 33089-U 144 x 0.1mL 33154-U
400mL 33033 20 x 1mL 33148
BF3-Butanol (10% w/w) 25mL 33155-U
10 x 5mL 33126-U 50mL 33149-U
100mL 33125-U
BF3-Methanol (10% w/w) HMDS
20 x 1mL 33356 30mL 33350-U
19 x 2mL 33020-U 100mL 33011
10 x 5mL 33040-U HMDS + TMCS, 3:1 (Sylon HT)
5mL 26,4121-5ML 20 x 1mL 33046
®
250mL 26,4121-250ML REACTA-SIL Concentrate (HMDS:TMCS, 2:1)
400mL 33021 25mL 39,4610-25ML
BF3-Propanol (14% w/w) HMDS + TMCS + Pyridine, 3:1:9 (Sylon HTP)
5g 15,6825-5G 20 x 1mL 33038
100g 15,6825-100G 25mL 33039
500g 15,6825-500G
Methanolic Base (0.5N) N-Methyl-N-(trimethylsilyl)trifluoroacetamide
2N, 30mL 33352 10 x 1mL 39,4866-10X1ML
2N, 100mL 33080 5mL 39,4866-5ML
Methanolic HCl 25mL 39,4866-25ML
0.5N, 20 x 1mL 33354 MTBSTFA, derivatization grade
0.5N, 10 x 5mL 33095 10 x 1mL 39,4882-10X1ML
3N, 20 x 1mL 33355 5mL 39,4882-5ML
3N, 10 x 3mL 33051 25mL 39,4882-25ML
3N, 400mL 33050-U MTBSTFA + TBDMCS, 99:1
Methanolic H2SO4 (10% H2SO4 v/v in methanol) 10 x 1mL 37,5934-10X1ML
6 x 5mL 506516 5mL 37,5934-5ML
TMAH, 0.2M in methanol 25mL 37,5934-25ML
10 x 1mL 33358-U
TFA
10mL 33097-U
10 x 1mL 33077
General Alkylation Reagents 25mL 33075
DMP (2,2-Dimethoxypropane), 25g 33053 100mL 33076
Esterate M, 25mL 33140 TMCS, derivatization grade
Hexaoxacyclooctadecane (18 crown 6), 25g 33003-U 100mL 33014
O-Methoxyamine HCl, 5g 33045-U
Pentafluorobenzyl bromide, 5g 33001 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
Silyl Reagents for Deactivation of Glassware
20 x 1mL 33036 and Chromatographic Supports
25mL 33037-U Note: All SupelcoTM glass GC columns are silane treated.
TM
BSA + TMCS, 5:1 (Sylon BT)
20 x 1mL 33018 Qty. Cat. No.
25mL 33019-U DMDCS
BSA + TMCS + TMSI, 3:2:3 (Sylon BTZ) 100mL 33009
144 x 0.1mL 33151 5% DMDCS in Toluene (Sylon CT)
20 x 1mL 33030 400mL 33065-U
25mL 33031-U
Rejuv-8 Silylating Agent
25mL 33059-U

SUPELCO
10 Bulletin 909
 Description Cat. No. Description Cat. No.
Acylation Sampler Kit 505862 FID Alkylation Sampler Kit 505854
3 x 1mL of each of the following (except as noted): 3 x 1mL of each of the following:
Acetic anhydride (3 x 2mL) BF3-Methanol
Heptafluorobutyric anhydride Methanolic Base
Pentafluoropropionic anhydride Methanolic HCl (0.5N)
Trifluoroacetic anhydride Methanolic HCl (3N)
Silylation Sampler Kit 505846 TMAH, 0.2M in methanol
3 x 1mL of each of the following: ECD Alkylation Sampler Kit 505870
BSA 3 x 1mL of each of the following (except as noted):
BSTFA BCl3-2-Chloroethanol, 11% w/w
BSTFA + TMCS, 99:1 (Sylon BFT) BCl3-Methanol, 12% w/w
HMDS + TMCS, 3:1 (Sylon HT) Hexaoxacyclooctadecane, 18 crown 6 (1 gram)
TMSI Pentafluorobenzylbromide

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.
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SUPELCO
Bulletin 909 11
 sigma-aldrich.com/supelco
Order/Customer Service 800-247-6628, 800-325-3010 ● Fax 800-325-5052 ● E-mail supelco@sial.com
Technical Service 800-359-3041, 814-359-3041 ● Fax 800-359-3044, 814-359-5468 ● E-mail techservice@sial.com

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

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The SIGMA-ALDRICH Family
© 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
T196909 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
BGL conditions may apply. Please see reverse side of the invoice or packing slip.
SUPELCO
12 Bulletin 909