derivatization of polar compounds for gc - sigma-aldrich · pdf filederivatization of polar...
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Agenda
•Introduction
•Basic reactions
• Silylation
• Acylation
• Alkylation
• Esterification and transesterification
•Developing a method
•Troubleshooting
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Basic Information
•What is derivatization?
• Changing the chemical characteristics of an analyte by
reaction with an active agent
•Why do derivatization?
• To make it possible to analyze a compound(s) of interest
using a specific chromatographic technique
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Benefits of Derivatization
•Gas Chromatography
• Increasing volatility or thermal stability
–Ex.: Analysis of fatty acids as their methyl esters
–Ex.: Analysis of sugars by GC
• Decreasing reactivity
–Ex.: Analysis of highly polar compounds by GC
• Increasing sensitivity
–Ex.: Making a molecule detectable by an ECD
• Increasing chiral recognition in GC
–Ex.: Derivatization decreases boiling point and chiral recognition is usually better at lower temperatures
–Ex.: Chiral recognition becomes better with different ligands
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Benefits of Derivatization
•HPLC
• Improve detectability or sensitivity
–Ex.: Use of reagents to increase UV or fluorescence absorption
• Decreasing reactivity
–Ex.: Analysis of highly polar compounds by GC
•TLC
• Make spots visible
•Chiral
• Convert a mixture of enantiomers to diastereomers by reacting
them with one pure enantiomeric reagent and separate the
diastereomers on a non-chiral column
Types of Derivatization for GC
• Silylation
• Acylation
• Alkylation
• Esterification and transesterification
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Silylation: Replaces an active hydrogen on an OH, SH, or NH group:
•Hydrogen replacement - reduces dipole-dipole interactions,
increases volatility
•Reaction mechanism: nucleophilic attack on the silicon atom
in the silylating reagent
•For completion of the reaction, the basicity of the leaving
group on the silyating reagent (X), must be greater than the
group to be replaced on the sample
•Ease of silylation generally follows this trend:
• Alcohol > phenol > carboxylic acid > amine > amide
• Alcohols and Amines: 1° > 2° > 3°
Sample-OH + R3Si-X Sample-O-SiR3 + HX
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Silylation
For:
•Amines, Amides, Alcohols, Thiols, Phenols, Enols, Carboxylic acids
Possible Reagents:
•Based on derivatives of Trimethylsilyl-, t-Butyldimethylsilyl- and other Alkylsilyl or Arylsilyl- functional groups
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Acylation: Replaces an active hydrogen with an acyl group
•Derivative is less polar and more volatile than the parent
compound
•Can be used to add a “protecting” group to heat sensitive
compounds
•Reaction mechanism can involve nucleophilic, electrophilic, or
free radical displacement
O
Sample-OH + R-C-X Sample-O-C-R + HX
O
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Acylation
For:
•Amines, amides, alcohols, thiols, phenols, enols, glycols, unsaturated compounds (C=C), aromatic rings
Possible Reagents:
•Acid anhydrides, Acid halides, Reactive acyl derivatives such as acylatedimidazoles, acylated amides, or acylated phenols
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Alkylation: Replaces an active hydrogen with an alkyl group
•Derivative is less polar and more volatile than the parent
compound
•Can be used to add a “protecting” group to heat sensitive
compounds
•Most common use is the derivatization of organic acids prior
to GC analysis
•Principal reaction involves nucleophilic displacement
•The less acidic H is, the more strongly basic the catalyst
must be
Sample-OH + R-X Sample-OR + HX
Alkylation
•Reagent strength depends on the acidity of the hydrogen to
be replaced
• More acidic => weaker reagent
–Ex: phenols, carboxylic acids
• Less acidic => stronger reagent
–Ex: alcohols, amides 12
For:
•Carboxylic acids, Amines, Amides, Alcohols, Thiols, Phenols and Enols
Possible Reagents:
•Alkyl halides, Nitro substituted chloro or fluorobenzenes, Tetraalkylammoniumhydroxides, Dimethylformamide dialkyl acetals, Diazoalkanes
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Esterification
•Used for the derivatization of organic acids
• Fatty acids to fatty acid methyl esters
•Uses an alcoholic reagent and acid catalyst
• A volatile catalyst (such as HCl) should be used
•Involves the condensation of the carboxyl group in the acid
and the hydroxyl group in an alcoholic reagent, with the
elimination of water
•To help drive the reaction, water should be removed as it is
formed
OH
O
R+
H+
OR'
O
R+ H2OR'OH
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Transesterification:
•Used to displace the alkoxy group in an ester with another
alcohol, producing a new ester and new alcohol
•Occurs when the ester is solvated by the alcoholic reagent
• An excess of the alcohol must be present
• To drive the reaction, the new alcohol should be removed
as it is formed
•Both acidic or basic catalysts are used
• Basic catalysts: transesterification of fats and triglycerides
OR''
O
R+
OR'
O
R+R'OH R''OH
H+ or OH-
Choosing the best derivatization reagent
•Choice of derivatising reagent depends on:
• Functional group to be derivatised
–OH, COOH, NH, etc.
• Molecular structur
–Acidity of hydrogen
–Sterical hindrance
• Consideration of final analytical method
–GC column (e.g. Wax phases are not compatible with silylderivatives)
–Detection (FID, ECD, MSD?)
• Literature
–Publications
–Manufacturer informations15
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What is the best derivatization reagent?
•Reagent sampler kits
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Choosing a Derivatization Reagent:
Common Silylation Reagents
Reagent Abbreviation Applications
N,O-bis(trimethylsilyl)acetamide BSA OH, COOH, amides, amines
N,O-bis(trimethylsilyl)trifluoroacetamide BSTFA OH, Ar-OH, COOH, carbohydrates, amides,
amines, acid anhydrides, sulfonamides
Dimethyldichlorosilane DMDCS Deactivating glass
Hexamethyldisilazane HMDS OH, Ar-OH, COOH, amines
N-t-butyldimethylsilylimidazole TBDMSIM Unhindered OH and Ar-OH
Trimethylchlorosilane TMCS Silylation catalyst; used w/other reagents
N-trimethylsilylimidazole TMSI OH, COOH, carbohydrates, fatty acids, sulfonic
acids, Ar-OH, R-SH
BSA + TMCS OH, alkaloids, amines, biogenic amines,
carbohydrates, COOH, Ar-OH, steroids
BSA + TMCS + TMSI OH, amines, amides, amino acids, COOH, Ar-OH,
steroids
BSTFA + TMCS OH, alkaloids, amides, amines, biogenic amines,
COOH, Ar-OH, steroids
HMDS + TMCS Amino acids, amipicillin, carbohydrates
HMDS + TMCS + pyridine OH, bile acids, carbohydrates, Ar-OH, steroids,
sterols, sugards
TMSI + pyridine C=O, steroids
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Choosing a Derivatization Reagent:
Common Acylation Reagents
Reagent Abbreviation Applications
Acetic Anhydride OH, Ar-OH, carbohydrates, amines
Trifluoroacetic acid TFA Amides, amines, C=O, OH, sulfonamides,
silyl catalyst
Trifluoroacetic acid TFAA OH, amino acids, amides, amines, Ar-OH, steroids
Pentafluoropropionic acid anhydride PFPA
Heptafluorobutyric acid anhydride HFPA
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Choosing a Derivatization Reagent:
Common Alkylation/Esterification Reagents
Reagent Abbreviation Applications
Boron trichloride-2-chloroethanol Esterifying/halogenation for ECD work
Phenoxy acid herbicides
Boron trichloride-Methanol BCl3-MeOH COOH, transesterification
Boron trifluoride-Butanol BF3-BuOH Short chain carboxylic acids, transesterification
Boron trifluoride-Methanol BF3-MeOH Long chain carboxylic acids, transesterification
Methanolic Sulfuric acid MeOH-H2SO4 COOH, transesterification
Methanolic base (metallic sodium in
methanol)
Na in MeOH Transesterification of triglycerides, cholesteryl
esters, phospholipids
Methanolic HCl MeOH-HCl Fatty acids
Pentafluorobenzyl bromide PFBBr Halogenated derivatives of COOH, mercaptans, Ar-
OH, sufonamides
Trimethylanilinium hydroxide TMAH Carbamates, hydroxyl amines, barbituates
Reaction Solvent
•Must not be capable of reacting with the derivatization
reagent
• Non-protic solvents
•Can be used to facilitate the derivatization reaction
• Common solvents: ACN, pyridine, DMF, DMSO, THF
•Use the highest purity possible
• Be aware of any added preservatives
•In some cases, the derivatization reagent can act as the
solvent
• Excess may have to be removed prior to chromatographic
analysis
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Reaction Temperature and Time
•Start with what is recommended in the literature
• Used by others; application specific
• Recommended by reagent manufacturer
•Increase or decrease if necessary
• Increase if yield is not sufficient
• Decrease if interfering side reactions are evident
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Confirming the Identity of the Derivative
•A Mass selective detector (MSD) is an invaluable tool
•If derivatizing a mixture, do each analyte separately if
possible
• This will help in identifying multiple derivatives
•ALWAYS do a derivative blank along with the sample
• Blank should contain the reagent and any solvents used
• It will help ensure that “artifact” peaks are not miss-
identified as analyte derivatives in the final sample
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Using an MSD to Identify Derivatives
• There will be an increase in molecular weight
• Calculate the total increase based on the derivative type
and number of active H+’s replaced
• Look for logical losses in the mass spectrum based on the
MW of the derivatized functional groups 23
Functional Group Derivative Increase in MW
per group
OH, COOH, NH, NH2 TMS 72
NH2 TMS 144 (if 2 H replaced)
OH, COOH, NH, NH2 TBDMS 114
OH, NH, NH2 Acetyl 42
COOH Methyl ester 14
NH, NH2 TFA 96
Using an MSD to Identify Derivatives
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100 150 200 250 300 350 400 450 500 5500
200000
400000
600000
800000
1000000
1200000
1400000
1600000
m/z-->
Abundance
Scan 153 (3.569 min): 1624_55_INST04_20070105006.D186
73
260
288147
103330216126 239 355 415 475387 503 549443
M-15
M-57
M-85
M-159
CH3
CH3
CH3
CH3
CH3
Si
O
NH
OCH3
CH3
CH3CH3
CH3
CH3
CH3
Si
MW=345
15 = CH3
57 = C(CH3)3
85 = C(CH3)3 + CO
159 = C(O) –O-C(CH3)3
The molecular weight of underivatized valine is 117. With
the addition of two TBDMS groups, it is now 345.
Example: TBDMS derivative of valine
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Equipment for derivatisation
•Vials/Reaction vessels
•Syringes
•Concentrator/Evaporator
•Block Heater
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Micro reaction vessels
•Useful for small samples sizes
•Can withstand heating
•Contain volume graduations
• These graduations are not accurate! Do not use for
measuring exact volumes.
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Syringes
•Clean & dry
• Removable needle
• Plunger guides
• Chaney adapter
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Concentrator/Evaporator
•To dry sample prior to derivatization
•To remove unwanted solvents prior to GC analysis
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Block Heater
•Should have variable temperature control
•During use, always monitor temperature with a thermometer
Moisture
•For many derivatization reactions, water is the enemy
• Interferes by competing with the sample for the
derivatization reaction
• Both degradation of reagents and derivatives themselves
•Sufficiently dry samples prior to derivatization
• This can be done with gentle heating and/or under a
stream of dry nitrogen
•If there is high humidity in the room, it may be helpful to
store syringes, vials, etc. in a dry box
•Silyl reagents are used in excess and can tolerate very
small amounts of moisture – but still try to keep things dry!
•Chemical removal of water by 2,2-dimethoxypropane30
Handling of Reagents
•Once opened, store reagents in tightly closed containers in
a dry environment
•Allow refrigerated reagents to come to room temperature
prior to use
•Gently mix reagents prior to use (including those in sealed
ampules)
•BSTFA will darken when exposed to moisture
• Colorless/light yellow =>yellow/amber
•Silyl reagents can withstand small amounts of moisture
• Water reacts with reagent and is removed chemically
• To be applied in excess
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Chromatographic Issues
•Byproducts
• Does the procedure produce byproducts that may be
detrimental to the chromatographic column or interfere
with the analysis? For example, inorganic acids
•Detector fouling
• Use of silylating reagents consistently may cause buildup
on a flame ionization detector (FID)
•Column compatibility
• If excess derivatization reagent is present, is it compatible
with your GC column? For example silyl derivatives
should not be analyzed on a polyethylene glycol (PEG)
based phase such as SUPELCOWAX or Carbowax32
What if, the results are not as expected?
•Missing peaks and low response (poor yield)
• Reagent is bad/not fresh any more
• Insufficient reagent amount
• Wrong reagent used (not reactive enough for sample)
• Reaction temperature and / or time too low
• Interferences in the reaction mixture (such as water)
•Additional peaks
• Impurities from solvents, reaction vessel, and/or reagents
used
• Side products from derivatization reaction
• Decomposition products of the derivative itself
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Troubleshooting, Example 1:Interference from a contaminant
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0 10 20
Time (min)
contaminant
1,3-diolein derivative; poor response
0 10 20
Time (min)
1,3-diolein derivative
Sample prepared with contaminated pipette
Sample prepared with clean pipette
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1: 1-Decanol
2: Tridecane
3: 1-Decanol-TMS ether
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1 23
2
3
No BSTFA
50 µl BSTFA
1000 µL BSTFA
Troubleshooting, Example 2:Insufficient volume of derivatization reagent
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Brochure „Derivatization Reagents“ (KDI)
• Products sorted by technique (GC, HPLC, TLC and Chiral)
• Reagents also listed by application
• Vials, syringes and other useful items for derivatization reactions
• Up-to-date application information and references
• Some tips and tricks for derivatization
• Articles on special derivatization procedures
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Web Page
•www.sigma-aldrich.com/derivatization
Technical Literature
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Dziękuję za uwagę!
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