Amino acid chromatographic analysis methods are pivotal in unraveling the intricate composition of amino acids, essential components of proteins. These methods, broadly categorized into the direct and derivative approaches, offer distinct strategies for analysis. The direct method employs sophisticated ion exchange chromatography amino acid analyzers, exemplified by the AAA-Direct system, enabling immediate analysis without the need for derivatization. In contrast, the derivative method utilizes various reagents like Fluorescamine, FMOC-Cl, OPA, DNFB, Dansyl Chloride, PITC, and AQC to enhance the detectability of amino acids through post-column derivatization.
Amino Acid Chromatographic Analysis Methods
Direct Method
The direct method employs an ion exchange chromatography amino acid analyzer for direct analysis without the need for derivatization. This approach utilizes high-efficiency anion exchange chromatography with integrated pulse amperometric detection, eliminating the need for intricate derivatization steps. The process is straightforward, offers high sensitivity, and achieves excellent separation results.
Representative of this approach is the AAA-Direct system introduced by Dionex decades ago. This system utilizes high-performance anion exchange chromatography with integrated pulse amperometric detection (HPAEC-IPAD). While effective, this method has limitations such as high cost, extended analysis time, strong specificity, significant investment, and limited versatility, hindering its widespread adoption and applicability.
Derivative Method
Due to the simple structure of most amino acids and their similarities, they often lack intrinsic fluorescence or chromophores, making them challenging to detect or differentiate. Derivatization, which can be carried out either before or after chromatographic separation, becomes essential. Post-column derivatization involves separating the sample through chromatography, followed by derivatization and subsequent detection.
In comparison to pre-column derivatization, post-column derivatization offers advantages such as excellent reaction reproducibility and the simultaneous separation and detection of non-derivatized compounds with absorbance. Post-column derivatization allows for versatility in using detectors based on different principles, but it requires a small dead volume, rapid reaction rates, and may be susceptible to interference from excess reagents or hydrolysis products. Specific equipment components are often necessary. Despite these challenges, pre-column derivatization is gaining increasing attention due to its advantages.
Amino Acid Analysis Derivatization Reagents
There is a wide variety of derivatization reagents for amino acids, and an ideal reagent should rapidly form a stable, singular product with amino acids that reflects their original structures. The derivatization reaction should be simple, highly reproducible, and any excess reagent should not interfere with subsequent chromatographic separation and detection or should be easily removable if necessary. Several commonly used derivatization reagents include:
Fluorescamine
Fluorescamine, abbreviated as FA, is a derivative formed rapidly when benzene acetaldehyde reacts with amino acids in the presence of excess phthalhydrazide. This reagent exhibits some selectivity, reacting only with primary amino acids to generate fluorescent derivatives. Secondary amino acids also undergo a reaction with Fluorescamine, but they do not form fluorescent derivatives. Fluorescamine is an ideal reagent for detecting primary amino acids due to its selectivity, and the reaction conditions are not stringent. The derivatization process is rapid, and the reagent itself has minimal interference with the reaction. The fluorescence excitation wavelength is 390 nm, and the emission wavelength is 475 nm.
Reaction of fluorescamine with an amino acid (Skelley et al., 2003)
9-Fluorenyl Methyl Chloroformate (FMOC-Cl)
9-Fluorenyl methyl chloroformate, abbreviated as FMOC-Cl, reacts with amino acids and can be used for both fluorescence and UV detection. The fluorescence excitation wavelength is 265 nm, and the emission wavelength is 310 nm. The derivatization reaction is rapid, and the resulting product is stable, remaining unaffected when stored at 4°C in the dark for up to 13 days. However, the selectivity of this derivatization reagent is relatively low, and the hydrolysis product of FMOC-Cl, FMOC-OH, exhibits fluorescence interference similar to FMOC-amino acids.
Ortho-Phthaldialdehyde (OPA)
Ortho-phthaldialdehyde, abbreviated as OPA, reacts with primary amino acids in the presence of the reducing agent mercapto reagent to produce isoindole derivatives rapidly, completing the reaction in just 1 minute at room temperature. OPA serves as a derivatization reagent with the advantages of simplicity in the derivatization steps, fast reaction kinetics, and the absence of fluorescence interference from excess derivatization reagent during detection. However, OPA does not react with secondary amino acids, and the stability of the derivatization product is relatively low, limiting its application scope.
To overcome these limitations, OPA is often used in combination with FMOC-Cl. The addition of indole with the mercapto reagent, mercaptoacetic acid, reduces the hydrophobicity of amino acids, resulting in earlier elution of OPA derivatives compared to FMOC derivatives. This allows for excellent separation of primary and secondary amino acids. In efforts to improve the stability of derivatives, some researchers have replaced the mercapto reagent with sodium sulfite and potassium cyanide, enhancing the stability of the reaction product. Automation of derivatization can be achieved using an auto-sampler, expediting peak elution. The advantages of OPA include rapid reaction with mercapto reagents, easy implementation of online automation, simplicity in instrument setup suitable for common laboratories, and minimal column wear for the derivatization product.
Precolumn derivatization analysis of amino acids with HPLC (Salmanizadeh et al., 2020).
2,4-Dinitrofluorobenzene (DNFB)
2,4-Dinitrofluorobenzene, abbreviated as DNFB, is a derivatization reagent with easily controllable reaction conditions. The derivatization process is straightforward, and the resulting derivative exhibits excellent stability. Under light-avoiding conditions at 60°C, DNFB can react with both primary and secondary amino acids, forming a stable derivative. Its advantages include the simultaneous reaction with both primary and secondary amino acids, stability of the derivatization product (with no significant changes in response values under light-avoiding conditions at 40°C for a month), high reaction sensitivity reaching picomoles, but it comes with the drawback of high toxicity.
Dansyl Chloride
Dansyl chloride serves as a derivatization reagent for liquid-phase chromatography, offering advantages such as simple derivatization procedures, quantitative completion of sulfonation reactions, and strong fluorescence and UV absorption. It finds widespread applications in the liquid-phase chromatographic separation and analysis of amino acids, particularly suitable for HPLC-based quantification of free cysteine in biological materials. However, its derivatization reaction has poor reactivity and slow kinetics, requiring at least 35 minutes to achieve maximum yield. Despite its drawbacks, including poor reproducibility and the generation of multi-derivatives, it remains a valuable tool in amino acid analysis.
Phenyl Isothiocyanate (PITC)
Phenyl isothiocyanate, abbreviated as PITC, is a column-pre-derivatization reagent that reacts rapidly and produces stable derivatives when used with primary and secondary amino acids. However, sample preparation with PITC is intricate, and the derivatized sample needs rapid evaporation drying or treatment with hexane to remove interfering factors and filtration to eliminate impurities. PITC, as a derivatization reagent, not only poses toxicity concerns but also requires specialized derivatization equipment for anhydrous conditions. Additionally, trace amounts of PITC reagent in the sample can shorten the lifespan of the analytical column.
6 - Aminoquinolyl-N-hydroxysuccinimidyl Carbamate (AQC)
Aminoquinolyl-N-hydroxysuccinimidyl carbamate, commonly known as AQC, has become a widely used derivatization reagent in recent years for amino acid precolumn derivatization. The AQC derivatization method was introduced in 1993 and has gained extensive application due to the stability of its derivatives and the minimal interference from derivatization by-products compared to other precolumn derivatization methods. AQC reacts rapidly with amino acids and is suitable for both fluorescence and UV detection.
However, one limitation of AQC is the potential interference from hydrolysis by-products during the separation process. When hydroxyproline and hydroxylysine are present in the sample, by-products of the reaction may significantly interfere with quantification.
Amino acid precolumn derivatization techniques in HPLC analysis have been widely adopted in the industry for their high sensitivity, short detection cycles, and high reproducibility. The choice of derivatization method depends on various factors, including the derivatization speed, analytical sensitivity, ability to detect secondary amino acids, and susceptibility to interference, tailored to specific analytical requirements.
References
- Skelley, Alison M., et al. "Mars Organic Detector III: a versatile instrument for detection of bio-organic signatures on Mars." First Jet Propulsion Laboratory In Situ Instruments Workshop. Vol. 4878. SPIE, 2003.
- Salmanizadeh, Hossein, and Neda Sahi. "Determination of amino acid profile for argininosuccinic aciduria disorder using High-Performance Liquid Chromatography with fluorescence detection." Acta Biochimica Polonica 67.3 (2020): 347-351.
