A Quick Guide to Derivatization Reagents

Derivatization reagents are designed to simplify chromatography by increasing sample volatility, allowing for simple GC-FID detection.  Your choice of derivatization reagent will depend on your compound of interest.  The following is a quick guide to selecting the right reagent for your application.  Please contact our Technical Team for more information.
 

 


 

Alkylation of Organic Acids
 

BF3 in Methanol (Boron Trifluoride, 14% in Methanol)
BF3 in Methanol is the most commonly used method of forming methyl esters of organic acids.

3.0 N HCl in n-Butanol
3N HCl in n-Butanol is required for newborn screening for metabolic disorders. Neonatal screening, which has become a standard procedure in many countries, measures amino acids and acylcarnitines from a single drop of blood. Blood concentration of one or several of these compounds is either abnormally high or low in a variety of metabolic disorders in newborns. Derivatization with 3N HCl in n-Butanol ensures butylation of the carboxyl acid group of the analyte and formation of butyl ester, which forces ionization or makes charging of the analytes more efficient. Although direct analysis of extracted acycarnitine without derivatization is possible, according to different reports, butylesterification is superior with regard to sensitivity and specificity. 3 N HCl in n-Butanol from Regis Technologies is manufactured under cGMP protocols to assure the highest quality and lot-to-lot consistency for this reagent. Each lot is tested by tandem mass spectrometry to ensure absence of contaminants which may interfere with analysis.

 


 

Silylation of Alcohols, Thiols, Secondary Amines, Tertiary Amines, Carboxylic Acids
 

BSA (Bis(trimethylsilyl)acetamide)
BSA forms highly stable TMS derivatives with most organic functional groups under mild reaction conditions.

BSTFA- + TMCS-Regisil® (1%, 10%) (Bis(trimethylsilyl)trifluoroacetamide Trimethylchlorosilane)
BSTFA reacts faster and more completely than BSA, due to the trifluoroacetamide leaving group. The high volatility of BSTFA and its byproducts results in non-co-elution of early eluting peaks. Additionally, the highly volatile and stable products result in low detector noise and fouling. Addition of TMCS to BSTFA catalyzes reactions of hindered functional groups and other difficult functionalities, such as secondary hydroxyls and amines.

Deriva-Sil (BSTFA+TMCS+TMSI in Pyridine)
Derivatizes sterically-hindered compounds. Reacts with carbohydrates,hydroxy- and keto-steroids, fatty acids and some amines and amides. Derivatizations are complete in minutes. Also available in concentrate form without pyridine for applications where pyridine is not desirable (e.g. 3-ketosteroids).

MSTFA (N-Methyltrimethylsilyltrifluoroacetamide)
MSTFA is the most volatile of the TMS-acetamides and useful in the analysis of volatile trace materials. MSTFA is more volatile than BSA or BSTFA but has similar silylation strength.

MtBSTFA (N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide)
Derivatization is usually complete upon dissolution with this exceptionally strong, yet mild silylating reagent. MtBSTFA derivatives are 104 times more stable to hydrolysis than their corresponding TMS derivatives and produce easily interpreted mass spectra for GC/MS.

MtBSTFA + t-BDMCS (N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide + 1% tert-Butylchlorodimethylsilane)
Addition of t-BDMCS to MtBSTFA catalyzes reactions of hindered alcohols and amines.

 


 

Silylation of Hydroxyl Groups (not amines or amides)
 

TMSI (Trimethylsilylimidazole)
TMSI is a potent, selective TMS donor that reacts with alcohols and phenols but not amines or amides. TMSI derivatizes wet sugar samples, hindered hydroxyl groups in steroids, and amino acids in fluorinated acylation reagents. TMSI is used in the preparation of dual perfluoroacyl and TMS derivatives.

 


 

Silylation of Carbohydrates, Organic Acids, Unhindered Alcohols and Phenols, Some Amines
 

HMDS, Hydox-Sil (1,1,1,3,3,3-Hexamethyldisilazane + Chlorotrimethylsilane + Pyridine)
HMDS is a weak TMS donor, used for silylation of carbohydrates. Used as a mixture with pyridine and trifluoroacetic acid (Hydox-sil) for alcohols, acids and amines. Hydrox-sil concentrate without pyridine is also available.

 


 

Silylation for Reaction Catalyzation of Alcohols, Thiols, Secondary Amines, Tertiary Amines
 

TMCS (Trimethylchlorosilane)
TMCS is used as a catalyst to increase reactivity of other silylation reagents.

 


 

Acylation of Hydroxyls, Thiols, Secondary Amines, Tertiary Amines
 

MBTFA (N-Methyl-bis(trifluoroacetamide)
MBTFA reacts rapidly under mild conditions with primary and secondary amines, while it reacts more slowly with alcohols, phenols, and thiols. MBTFA works well in the analysis of sugars.

 


 

Acylation of Alcohols, Phenols, Amines
 

HFBI (Heptafluorobutyrylimidazole)
HFBI readily forms derivatives with phenols, alcohols, and amines suitable for ECD. The reactions are fast and mild, and imidazole is not acidic; so no decomposition or corrosion occurs on columns.

HFIP (1,1,1,3,3,3-Hexafluoro-2-Propanol)
Esterification reagent for the determination of aromatic acids in tissue by GC and electron capture detection (ECD).

HFBA (Heptafluorobutyric Anhydride)
HFBA is frequently used for the confirmation of drugs of abuse and reacts with alcohols, amines, and phenols. Bases such as triethylamine and trimethylamine can be added to promote reactivity to HFBA. HFBA is most commonly used for ECD because its derivatives are most sensitive to ECD.

PFPA (Pentafluoropropionic Anhydride)
PFPA is most commonly used for ECD and reacts with alcohols, amines, and phenols. Bases such as triethylamine and trimethylamine can be added to promote reactivity. PFPA is frequently used for the confirmation of drugs of abuse. PFPA derivatives require the lowest analysis temperatures.

PFPOH (Pentafluoropropanol)
Use in combination with PFPA to make derivatives of the most common functional groups, especially polyfunctional bio-organic compounds. The formed derivatives are highly suitable for ECD.

TFAA (Trifluoroacetic Anhydride)
TFAA is most commonly used for ECD and reacts with alcohols, amines, and phenols. Bases such as triethylamine and trimethylamine can be added to promote reactivity to TFAA. TFAA is frequently used for the confirmation of drugs of abuse and is the most reactive and volatile of the anhydrides.

 


 

Acylation of Chiral Analytes
 

(R)-(-)-MTPA-Cl ((R)-(-)-a-Methoxy-a-(trifluoromethyl)phenylacetyl chloride)
(R)-(-)-MTPA-Cl, or Mosher's Acid Chloride, is a GC Chiral and Specialty Derivatization Reagent that is used for the determination of enantiomeric purities of alcohols and amines.

MCF ((-)-(1R)-Menthylchloroformate 0.1 M in Chloroform)
MCF is a companion reagent to TPC. MCF is most generally used for the resolution of optically active alcohols (MCF reacts with amines also, but the resulting diastereomers are harder to separate than are the TPC derivatives).

TPC (N-Trifluoroacetyl-L-prolyl chloride 0.1 M in Chloroform)
TPC is the reagent of choice for the resolution of optically active amines by gas chromatography. TPC provides sample volatility and couples with amines to form diastereomers which can be separated on GC columns. Its use in the determination of amphetamines and other drugs of abuse testing has attracted considerable interest.