Fluorescence detector derivatizing reagents

The aromatic nucleus adsorbs in the UV and thus, the derivative can be detected by a UV detector. This is the most common type of chemical derivatization but the derivative may be chosen to be appropriate for different types of detector. For example, the solute can be reacted with a fluorescing reagent, producing a fluorescent derivative and thus be detectable by the fluorescence detector. Alternatively, a derivative can be made that is easily oxidized and, consequently, would be detectable by an electrochemical detector. 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

Fluorescence detection, alone or with the aid of derivatizing reagents to enhance detector responses and improve the chromatographic resolution, has also been used for the determination of biogenic amines. Lakshmana and Trichur (1997) used native fluorescence to analyze NE, DA, and 5HT in rat brain utilizing an isocratic separation on an ODS CIS column. The detection limits reported were 100-250 pg on column. 

In their study on the automation of the derivatization reaction of carboxylic acids, Wolf and Korf (34) chose bromomethylmethoxycoumarin (Br-MMC) as a label because of its commercial availability. The automation of this reaction is problematic, because elevated temperatures are required in the manual procedures and the reaction had to take place in an aprotic environment. The addition of a solid salt also complicates the procedure. They describe a solution to this problem using a suspension of potassium carbonate and appropriate reagent concentrations. The samples were detected by a fluorescence detector equipped with a 5-ju.l cell, using an excitation wavelength of 325 nm and a cutoff filter of 398 nm. 

Derivatives for LC Detection. The lack of a universal detector for LC and the popularity of the UV detector have caused chromatographers to seek derivatization reactions that introduce UV chromophores into sample analytes. In those instances where the derivative also fluoresces, additional sensitivity can be obtained by fluorometric detection. Table 6 contains a list of the most common derivatizing reagents for this purpose. 

The inability to detect precludes the ability to develop a separation. The selected technique is defined by the required limit of detection. If low-pg/mL levels are needed, it is fruitless to use a UV/visible absorbance detector. Laser-induced fluorescence (LIF) is usually appropriate, provided derivatization reagents are available if the solute does not have significant native fluorescence [2], Limits of detection of 10 10 M are easily achieved using LIF, provided the solute absorbs at a laser emission wavelength and has a reasonable fluorescence quantum yield. 

In some cases, the filter is used as both particulate trapping medium and as a substrate for holding a derivatizing reagent that reacts with and stabilizes the particulate matter. For example, l-(2-pyridyl)pip-erazine-coated glass fiber filters are used to trap and react with particulate methylene bisphenyl isocyanate to form a derivative that is then analyzed using HPLC coupled with a fluorescence detector. 

If a fluorescent derivatizing reagent that is non-fluorescent when unreacted (and therefore detector-transparent) but fluoresces when it becomes the tag (i.e., the part of the derivatizing reagent that is chemically bonded to the analyte) cannot be found. 

The UV detector has been the most widely used in the LC determination of pesticides [59,63,69,71,77,106,107,109,111,122,147,148]. However, at present, the diode array detector is usually preferred to obtain the UV spectrum for each individual compound and confirm e presence of target analytes [35 8,45,48,50,71,74,76,108,149]. Carbamate pesticides are usually determined with fluorescence detector, following LC-postcolumn derivatization of methylamine (formed in the previous hydrolysis of pesticide) with OPA reagent [68,74]. Recently, a postcolumn detection system was used for the... 

Chemical derivatization has become a very popular technique for increasing the sensitivity of a specific type of detector to compounds for which it normally exhibits little or no response. An examples of this procedure would be the reaction of an aliphatic alcohol, which contains no UV chromophore, with benzoyl chloride to form the benzyl ester which could then be detected by the UV detector. Derivatization can also be used to permit the use of an alternative type of detector to increase sensitivity. For example an amino acid may exhibit only weak absorption in the UV, but when reacted with a suitable fluorescing reagent, could be detected by means of the fluorescence detector at concentration levels one or two orders of magnitude lower than with the UV detector. It is clear that derivatization procedures can increase significantly the versatility of many detectors. 

For the liquid chromatographic separation of amino acids and amino compounds, indirect separation techniques after precolumn labeling of the amino group are also widely used. By the precolumn derivatization approach, amino compounds are converted into structures that are suitable for separation and suitable for detection by various sensitive detectors. For the separation of the labeled amino compoimds, a wide variety of separation columns, including reversed-phase, can be used. Concerning detection, UV—Vis absorbance, fluorescence, and also MS (MS/MS) detectors are widely used, depending on the properties of the derivatization reagents. 

An excellent discussion on derivatization techniques has been given by Lawrence (17) including a detailed discussion on pre-column derivatization (18) and post-column derivatization (19). Probably, the more popular procedures are those that produce fluorescing derivatives to improve detector sensitivity. One of the more commonly used reagents is dansyl chloride (20), 5-dimethylamino-naphthalene-1-sulphonyl chloride (sometimes called DNS-chloride or DNS-C1). The reagent reacts with phenols and primary and secondary amines under slightly basic conditions forming sulphonate esters or sulphonamides. 

Quite often, neither of the three aforementioned detection systems will provide adequate sensitivity and selectivity to complete the analysis with specified lower limits of detection. In these cases, an increasingly popular trend has been to either pre- or postcolumn derivatize the analyte or analytes to convert them to species that can be adequately detected with reliable and commercially available detectors. Tables VII and VIII list a variety of reagents for the derivatization of specific organic moieties that provide increased UV absorption and fluorescence, respectively (39). 

Fluorometric detectors are sensitive to compounds that are inherently fluorescent or that can be converted to fluorescent derivatives either by chemical transformation of the compound or by coupling with fluorescent reagents at specific functional groups. If derivatization is required, it can be done before chromatographic separation or, alternatively, the reagent can be introduced into the mobile phase just before its entering the detector. 

Likewise, the luminescence properties of many analytes can be altered in the presenoe of surfactant aggregates (4,7.,8.). Consequently, addition of micelle-forming surfactants (present either in the LC mobile phase or added post-column) can improve the sensitivity of fluorimetric LC detectors (49,482). Micellar spray reagents have been utilized to enhance the fluorescence densitometric detection of dansylamino acids or polycyclic aromatic hydrocarbons (483). The effect was observed for TLC performed on cellulose or polyamide stationary phases with the micellar spray reagent being either CTAC, SB-12, or NaC (483). More recently, use of nonionic Triton X-100 has been found to improve the HPLC detection of morphine by fluorescence determination after post-column derivatization (486) as well as improve the N-chlorination procedure for the detection of amines, amides, and related compounds on thin-layer chromatograms (488). 

---