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
________________________________________________________
________________________________________________________
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________________________________________________________
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________________________________________________________
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________________________________________________________
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________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________
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]
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SUPELCO ● 595 North Harrison Road, Bellefonte, PA 16823-0048 ● 814-359-3441
T196909
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