Why Use GC Derivatization Reagents
Gas Chromatography is used to separate volatile organic compounds. Derivatizing reagents are used to modify an analyte’s functional groups to enable chromatographic separation and/or detection. Benefits of derivatization include:
- Increased sample volatility
- Improved selectivity and chromatographic efficiency
- Enhanced detectability
How To Choose Which Derivatizing Reagent To Use for Gas Chromatography:
A good derivatizing reagent and procedure should produce the desired chemical modification of the compound(s) of interest and be reproducible, efficient, and nonhazardous.
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 column
- Produces a derivative that is stable with respect to time
Chrom Tech is proud to distribute Regis GC Derivatization Reagents. For more than 60 years, Regis has been manufacturing high-purity reagents under highly controlled manufacturing procedures with strict quality control specifications. For GC, there are three basic types of derivatization reactions: silylation, acylation, and alkylation. Silylating reagents react with compounds containing active hydrogens; these reagents are the most common type used in GC. Acylating reagents react with highly polar functional groups, such as amino acids or carbohydrates. Alkylating reagents target active hydrogens on amines and acidic hydroxyl groups.
Silylation is the most widely used derivatization procedure for sample analysis by GC. The popularity of silylation reagents is enhanced by their ease of use and formation of derivatives. In silylation, an active hydrogen is replaced by an alkylsilyl group, such as trimethylsilyl (TMS), or t-butyldimethylsilyl (t-BDMS). Compared to their parent compounds, silyl derivatives are more volatile, less polar, and more thermally stable.
Silylation reagents are generally more moisture sensitive, requiring them to be sealed to prevent deactivation. The derivatives of TMS reagents are also more sensitive. In response to this challenge, t-BDMS reagents were introduced, which enables the formation of derivatives 10,000 times more stable against hydrolysis than the TMS ethers.
Both TMS and t-BDMS reagents are suitable for a wide variety of compounds, offer excellent thermal stability, and can be used under a variety of GC conditions and applications. Analysis by GC/MS often requires special sample derivatization and MtBSTFA is very effective for these applications.
Acylation reagents offer the same advantages as silylation reagents: creating less polar, more volatile derivatives. However, in comparison to silylation reagents, acylation reagents can more readily target highly polar, multi-functional compounds, such as carbohydrates and amino acids. In addition, acylation reagents offer the distinct advantage of introducing electron-capturing groups, which enhance detectability during analysis.
Generally, these reagents are available as acid anhydrides, acyl derivatives, or acyl halides. The acyl halides and acyl derivatives are highly reactive and may be suited for use where issues of steric hindrance may be a factor. Acid anhydrides are available in a number of fluorinated configurations, which improve detection. These fluorinated anhydride derivatives are used primarily for Electron Capture Detection (ECD) but can also be used for Flame Ionization Detection (FID). Fluorinated anhydrides are often used in derivatizing samples to confirm drugs of abuse. Despite the special utility of these reagents, their acidic nature requires that any excess or byproducts be removed prior to analysis to prevent deterioration of the column.
As with other derivatization reagents, alkylation reagents reduce molecular polarity by replacing active hydrogens with an alkyl group. These reagents are used to modify compounds with acidic hydrogens, such as carboxylic acids and phenols. Akylation reagents can be used alone to form esters, ethers, and amides or they can be used in conjunction with acylation or silylation reagents. A two-step approach is commonly used to derivatize amino acids, where multiple functional groups on these compounds may necessitate protection during derivatization.
Esterification, which is the reaction of an acid with an alcohol in the presence of a catalyst to form an ester, is the most popular method of alkylation. Alkyl esters are stable, can be formed quickly, and, by altering the length of the substituted alkyl group, retention of the derivative can be varied. In addition to the formation of esters, alkylation reagents can be used in extraction procedures where biological matrices are present.
Other: Chiral Derivatization Reagents
Gas chromatographic chiral analysis of enantiomeric compounds on achiral stationary phases requires the use of enantiopure derivatization reagents. These reagents generally target one specific functional group to produce diastereomers of each of the enantiomeric analytes. From the resulting chromatograms, calculations are conducted to determine the enantiomeric concentration of the analyte.
Other Considerations: Chromatography Vials Used for Derivatization
Autosampler 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. Chrom Tech’s NextGen™V Vials® from DWK are suitable small scale durable reaction vials with screw caps for micro scale derivatization reactions.
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.
Reduced Surface Activity (RSA™) Vials
Instead of coating the vials to mask the polar silanol groups, the RSA™ Vials are NOT coated, but they’re made from a proprietary manufacturing process that does not create silanols (OH groups) on the Type 33, borosilicate glass. Since the RSA™ vial surface has virtually no silanols it makes them mostly inert and a low adsorption vial, and therefore, can be used for GC derivatization reactions.
Derivatizing compounds for GC often is necessary to obtain reproducible chromatographic results. Eliminating this step to save time can be costly and produce inaccurate and unreliable results. A well-chosen derivatization procedure, based on the chemical composition of the target compounds, can significantly improve your chemical separations. Also, careful consideration is essential when selecting your glassware for derivatization and Chrom Tech HPLC vials are a good option. Please contact Chrom Tech to discuss your chromatography needs today.