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Derivatizing Reagents for Detection of Organic Compounds By HPLC

Derivatization is the process of chemically modifying a compound to develope a new compound which has properties that are suitable for analysis using HPLC. Derivatization improves the detectability of a target analyte by reaction with suitable derivatizing agent. Derivatization reactions are simple chemical modification of substance that make it compatible with the selected separation method or transforms substance with a low UVabsorption into highly sensitive product. Derivatization reactions in liquid chromatography modify the solutes adding a chomophore for easy UV detection or a fluorophore for sensitive fluorescent detection. The chemical structure of the compound remains same and just modifies the specific functional group for reacting compounds to derivative of deviating chemical and physical properties in order to make them detectable. Introduction of certain elements or groups through chemical derivatization may enhance the detector’s response helpful for the elucidation of structure of analytes. In conclusion, the present review describe various derivatization reagents for pre-column and post column derivatization process in HPLC by UV-visible and fluorescence detection are summarized along with reactions and some practical aspects. The commonly used derivatizing reagents in HPLC are 1-fluoro-2, 4dinitrobenzene, ninhydrine, 4-N-N-dimethylaminoazobenzene-4’-sulfonyl chloride, benzoyl chloride, phenyl isocyanate for UV-visible detection and o-phthalaldehyde, fluorescamine, 1dimethylaminonapthalene-5-sulfonyl chloride (DNS-Cl), 9-fluorenylmethyl chloroformate (Fmoc-Cl), benzofurans for Fluorescence detection. Review Article Chhanikar et al.; AJACR, 9(2): 1-13, 2021; Article no.AJACR.72013 2


INTRODUCTION
Derivatization is chemical reaction which converts an analyte in such a way as to allow its detection. This process whereby a molecule is chemically altered to enhance its detection by a particular analytical method is called derivation. [1][2][3] The chemical structure of the compound remains the same and just modifies the specific functional group of reacting compounds to derivative in order to increase their sensitivity to UV absorption and fluorescence detection. [4,5] Chemical derivatization is used to convert the non-UV absorbing analytes into highly sensitive products that are easily chromatographed or detected.
In pharmaceutical analysis, derivatization is commonly employed if the Active Pharmaceutical Ingredient does not have a chromophore for UV detection or a fluorophore for fluorescence detection [6].
In this paper, the derivatization method for HPLC connecting with UV-VIS detection will be described. The term derivatization means basically the reactions caused by chemical reagents or physical methods to convert a poor detector-responding analyte into a highly detectable product which enhanced chromatographic properties. In other words, derivatization aims to increase the detectability of the analyte.
Derivatization in HPLC can be performed either before the chromatographic analysis (Pre-column derivatization) or after the column separation and before detection (Post-column derivatization).

Pre-Column Derivatization
In pre-column derivatization, the reaction is generally performed manually in vials before HPLC injection i.e. the compounds are derivatized before their injection in the column, but can also be automated. [6,7] This technique allows more flexible working conditions (reaction time, solvent reaction, elimination of excess reagent, etc. [3] Separation of the coloured reagent or UV-absorbing material takes place after their modification by derivatization, the excess of derivatizing agent and the by-products can be easily removed; derivative stability is necessary. [6] This technique is often used to promote improved chromatographic response of the analytes under investigation. [3,7] Important characteristics of this technique involve completion of reaction, stability of the resultant derivatives and ease of preparation. [4]

Post-Column Derivatization
Post-column derivatization has been proposed to overcome the drawbacks associated with precolumn mode. Post-column derivatization technique is typically used for compounds with low or no response to the selected detection system. [4,8,9,10].
In post-column derivatization, the derivatizing reagent is added not only to the mobile phase but also to the target analyte/eluted compound. The reaction is performed in a reactor which is placed between the column and the detector. This is accomplished by inserting a mixing "T" or chamber at the end of the column and pumping system (pump). The derivative is in a reaction coil, the length of which is controlled by the time required for completion of the reaction, and is then measured in the detector. [5,6,11].

Uv-Visible Detection
UV detection is the most commonly used technique in HPLC but it sometimes lacks sensitivity or selectivity for trace analysis of compounds. [4, 11,12] Chemical derivatization modifies substances with a low UV absorption into highly sensitive products. [4,12] The chromatographic detection can be improved by the derivatization of the compounds which do not possess a chromophore.
[4] There have been a variety of chemical reagents that have been used to add a chromophore to a particular type of functional group. [12] The reagents which are used in UV-visible detection are usually highly conjugated aromatic compounds containing a highly reactive group to react with a functional group of the analyte. [4, 10,12].
Derivatization can also improves the resolution of closely eluted compounds and chromatographic retention of polar compounds include aliphatic amines, amino acids, and compounds that have carbonyl and hydroxyl substituents because the derivatives are usually more hydrophobic than the underivatized analyte. [4,13] FDNB is also called as Sanger's reagent. FDNB specifically reacts with N-terminal amino group. Mostly the reaction of FDNB performed in an alkaline medium (borate buffer). FDNB reacts with amino acid or primary amine or secondary amine to form a derivative 2, 4-dinitrophenyl. (Fig. 1.1).

Ninhydrine [1,2,3,6]
Both pre-column and post-column derivatization methods are used for the determination of amino acids in proteins. The former is characterized by an ion-exchange separation and post-column derivatization with ninhydrine. Ninhydrine reacts only with primary amines and giving blue-violet coloured compound (diketohydrindylidenediketohydrindamine) ( Fig. 1.4)

4-N-N-Dimethylaminoazobenzene-4'-Sulfonyl Chloride (DABS-Cl) [1,2,3,6]
DABS-Cl is one of the derivatizing agent for amino acid. DABS-Cl reacts with amine to form sulphonamide derivative ( Fig. 1.5). The resulting sulphonamide show an absorbance maximum at 420 to 450 nm. Lammens and Verzele [21] developed a procedure in which derivatization was performed at 50ᵒ C and at pH 8.9 for short time, and gave a quantitative yield. Reversephase HPLC with isocratic elution and detection at 464 nm was employed.

Benzoyl chloride [3,6]
Benzoyl chloride is the preferred derivatizing agent polyamines, and it reacts with most of the naturally occurring di-amines and polyamines

Ninhydrine
Amino acid (tosyl-, dansyl or benzoyl chloride). Benzoyl chloride is particularly advantageous because it implies short derivatization procedure and has a long elution time. A rapid and simple method using benzoyl chloride was originally described by Redmond and Tseng [22]. The derivatization method was improved by dissolving benzoyl chloride in methanol thereby enhancing the reaction with polyamines. The benzoylated polyamines are eluted by RP-HPLC using methanol-water (60:40) as the mobile phase. The sensitivity of this method is 100 p/mol. The benzoylated polyamines can be stored upto 3 weeks at -20ᵒC.

Acetic anhydride [2,3]
Tertiary aliphatic amine can be separated on HPLC column packed with Nucleosil 5N (CH3)2 resin, and detected by post-column derivatization after reaction with a color reagent consisting of an acetic anhydride solution of citric acid. The reaction temperature was 120ᵒC. UV detection was performed at a wavelength of 550 nm.

Phenyl isocyanate (PIC) [1,2,3,6]
Phenyl isocyanate (PIC) reacts with primary and secondary amines to form N-N'-disubstituted urea ( Fig. 1.7). UV detection measured at 255 nm. Bjorkqvist proposed a method in which derivatization was carried out in N-N'dimethylformamide, and aliphatic alcohol was added to destroy the excess of reagent. A polymeric benzotriazole reagent containing the FMOC group (Fig. 1.8) for UV and fluorescence detection, was applied in the determination of cadaverine and putrescine, normally occurring polyamines in human urine [24]. Polymeric FMOC reagent was also used for the determination of volatile amines in air by on-line solid phase derivatization and HPLC with UV and fluorescence detection.

FLUORESCENCE DETECTION
Fluorescence detection is a very sensitive procedure used in HPLC. Fluorescence takes place when a molecule absorbs a photon, typically in the UV range, which causes the emission of another photon at a higher wavelength in the visible range. [4,5] Generally many compounds have natural fluorescence, but in case if any compound or analyte is not fluorescent then it can be induced by creating a derivative. Derivatization is used for producing fluorescent compound. In addition, the unique property of fluorescent can allow for the selective identification of a molecule in a complex mixture. [6,27] Fluorescence usually has a wider linear range of response vs. concentration compared to optical (UV-VIS) absorbance and is quantifiable at lower concentrations.

O-pthalaldehyde (OPA) [1,2,3,6,28]
OPA is non-fluorescent itself but it is widely used for derivatization of primary amines and amino acids. OPA reacts with primary amino group in an aqueous basic medium in the presence of alkylthiol (such as 2-mercaptoethanol) to form a highly fluorescent isoindolederivative (Fig. 2.1.).
The derivatization reaction was performed in a mixture of borate buffer (pH 6-8 for amines, pH 9.7-10.0 for amino acids) and methanol at room temperature in 2 minutes. Excitation of fluorescence can be achieved at 340 nm. Fluorescence emission is measured at 455 nm. The OPA reaction can be applied to post column derivatization because of its short reaction time and fluorogenic property (non-fluorescent).

Fluorescamine [1,2,3,4,6,28]
Fluorescamine reacts with primary amines and amino acids in borate buffer (pH 9.5-10) at room temperature. The reaction is complete in few minutes and forms a fluorescent Pyrrolidone  Fig. 2.2). Fluorescamine is a non-fluorescent compound, but it offer enhanced detectability of primary amino compounds with fluorescence detection. Fluorescamine reacts with compounds with nucleophilic functional groups such as primary amines, secondary amines, alcohol, water, etc. but only primary amines form a fluorescent derivative. Therefore, fluorescamine is specific reagent for compounds of primary amino group.

1-dimethylaminonapthalene-5-sulfonyl chloride (DNS-Cl) [1,2,3,6]
DNS-Cl reacts with primary and secondary amino compound under slightly alkaline condition (alkaline pH) and forms a fluorescent derivative. The reaction time (30-120 minutes) depends on the type of amino acid compound. The reagent is hydrolyzed in the derivatization procedure to produce a highly fluorescent 1dimethylaminonaphthalene-5-sulfonic acid ( Fig.  2.3). Hence this reaction is mainly used for precolumn derivatization. The derivative of amino acids are separated on reverse-phase column with UV-visible or fluorimetric detection.

Phenyl isocyanate [2,3,28]
Phenyl isocyanate reacts with primary and secondary amine to form urea (Fig. 2.5). It also reacts with alcohols, phenols, water, carboxylic acids. The reaction completes within few minutes at room temperature and for this reaction requires excess reagent.
Isocyanates react quite rapidly with water and alcohols to give urethanes. For this reason they are usually replaced by the less reactive isothiocyanates.

Phenyl Isothiocyanate [2,3,28]
It is also known as Edman's reagent. It is not only used in the pre-column determination of amino acids, but also in the microanalysis of peptide sequence. The reaction is performed in an acidic environment and the derivative was determined by fluorimetric detection (Fig.  2.6).
The reaction with NBD-F is ten times faster than with NBD-Cl, and is complete in 1 minute. Both the reagents NBD-Cl and NBD-F are used for post-column derivatization. NBD-Cl also reacts with aliphatic amine, amino acids, peptides and proteins.