[Reader Insight] Introduction to Common Derivatization Methods and Application Examples
This article is submitted by expert chromatographer OU Shuo-Jun. Welch Materials, Inc. is authorized to translate this article to English and publish it on behalf of the author.
Introduction
Derivatization is a frequently employed pretreatment method with objectives such as enhancing detector response, increasing chromatographic retention of target analytes, altering polarity for easier extraction, or separating impurities.
However, selecting the appropriate derivatization method, or even deciding whether derivatization is necessary, depends on factors like the target compound’s structure and properties, the pretreatment method, and the detector type. These considerations make it a complex and worthwhile topic for discussion.
Based on issues encountered during experiments and the principles of derivatization methods applied, the author has summarized several derivatization approaches to broaden their application in sample pretreatment.
1. Methylation of Hydroxyl Groups
Application:
This method targets hydroxyl-containing compounds such as methanol and ethanol. Methylation reduces polarity, facilitating separation from water-soluble impurities.
Common Reagents:
Methyl iodide and dimethyl sulfate under strong alkaline conditions.
Reaction Conditions:
Add 0.5 mL of 4 mol/L sodium hydroxide solution at room temperature, shake vigorously, let stand for 15 minutes, then add 0.5 mL of methyl iodide and vortex for 10 minutes.
Reaction Mechanism:
The hydroxyl group (-OH) reacts with hydroxide ions (OH⁻) to form –ONa and water, followed by a reaction with CH₃I to produce –OCH₃ and NaI.
Limitations:
The target compound must remain stable in a strongly alkaline environment, and the hydroxyl group must exhibit clear reactivity.
2. Amide Formation of Hydroxyl or Amino Groups
Application:
This method is used for hydroxyl- or amino-containing compounds such as alkylamines, biogenic amines, and sterols. Amide formation reduces polarity. Amines reacting with acyl chlorides can introduce specific functional groups like aromatic rings for analytical purposes.
Reaction Conditions:
Add 5 mL each of 0.4% benzyl chloroformate and 1% sodium carbonate solution, then incubate in a water bath at 60°C for 2 hours.
Reaction Mechanism:
Hydroxyl (-OH), amino (-NH₂), or secondary amino groups (-NH-) react with RCOCl to form RCO-NH- and HCl. The reaction is accelerated under weakly alkaline conditions, and specific R groups (e.g., UV- or fluorescence-absorbing groups) can be introduced as needed.
3. Transesterification Reaction
Application:
(This is a complicated reaction and only one example is shown here.)
This method targets different ester forms of acids, such as fatty acids. Transesterification with methanolic potassium methoxide (KOCH₃) or alcohols (ROH) converts acids into a single ester form (e.g., methyl ester). The R group introduced by ROH can be tailored for analytical needs.
Reaction Conditions:
Add 5 mL of 10% acetyl chloride-methanol solution and incubate in a water bath at 60°C for 2 hours.
Reaction Mechanism:
R₁CO-OR₂ reacts with CH₃O⁻ to form R₁CO-OCH₃ and R₂OH.
4. Halogen Introduction
Application:
Rarely used in practical determinations, this method applies to compounds such as saccharin and alkylamines. Introducing halogen groups can impart UV absorption to compounds lacking conjugated structures. Various halogen substitution reactions are available, but only practical examples from analytical testing are provided.
Reaction Conditions:
Add 5 mL of 50% sulfuric acid and 5 mL of sodium hypochlorite (effective chlorine: 15%-20%), and react at room temperature for 5 minutes.
Reaction Mechanism:
In a strongly acidic and oxidative environment, amino groups (-NH-R or -NR₂) react with hypochlorite to form N-Cl bonds, which exhibit UV absorption.
5. Hydrolysis of Anhydrides or Lactones
Application:
This method is suitable for compounds like benzoic anhydride and maleic anhydride. Compounds without easily ionizable groups often exhibit low sensitivity in mass spectrometry due to lack of ionizable sites. Hydrolysis introduces carboxyl groups with higher electrophilicity, enhancing response.
Reaction Conditions:
Add 3 mL of 10% nitric acid and incubate in a water bath at 60°C for 3 hours.
Reaction Mechanism:
See image below.
6. Derivatization of α-Amino Acids
Application:
Since amino acids are highly water-soluble and lack UV absorption, derivatization introduces conjugated structures such as aromatic rings. This reduces polarity, increases chromatographic retention, and allows maximum UV absorption at 260 nm.
Reaction Conditions:
Dissolve amino acids in an aqueous solution containing 60% pyridine and an appropriate amount of phenyl isothiocyanate (PITC), then incubate in a water bath at 40°C for 1 hour.
Reaction Mechanism:
PITC reacts with amino acids to form phenylthiocarbamoyl derivatives (PTH-amino acids).
Conclusion
In addition to the derivatization methods described above, reactions such as silylation and hydrazone formation between aldehydes and phenylhydrazine are also commonly used.
Before employing derivatization, it is crucial to clarify the purpose, considering compound stability (under reaction conditions), required functional groups, polarity modifications, and chromatographic behavior optimization. The reactivity of functional groups and reaction conditions should be carefully selected and optimized to achieve the best response.
Derivatization should be purposeful and aimed at achieving the best results with minimal effort, avoiding unnecessary derivatization that detracts from the analytical process.