Advantages and Limitations of Chemical Derivatization for Trace Analysis by Liquid Chromatography

Journal of Chromatographic Science, Vol. 23, November 1985 Advantages and Limitations of Chemical Derivatization for Trace Analysis by Liquid Chromatography James F. Lawrence Downloaded from https://academic.oup.com/chromsci/article/23/11/484/306112 by guest on 27 September 2021 Food Research Division, Food Directorate, Health Protection Branch, Ottawa, Ontario, Canada K1A 0L2 Jim Lawrence is the head of the Food desired concentration level in the sample. There are two ways Additives and Contaminants Section of that this can be done. One is to increase sensitivity by convert the Food Research Division in the Ca ing the compound to a form which produces a greater detector nadian Department of Health & Welfare response, while the other is to alter some of the physical or in Ottawa. Since joining the Division 13 chemical characteristics of the compound to improve the selec years ago, he has been actively involved tivity of the analysis. There are several approaches to modify in the development of analytical meth ods for trace substances in foods by ing analytes, including chemical reactions (1-6), ion-pairing both gas and liquid chromatography. techniques (8), photochemistry (9,10), electrochemistry (11), He has authored or edited a number complexation (12), and metal chelation (13,14). All function to of books and some ninety research publications in this area. Dr. improve sensitivity or selectivity so that determinations at trace Lawrence received his Ph.D. in organic-analytical chemistry from levels can be accomplished. Dalhousie University in Halifax, Nova Scotia, in 1972. A typical example of improved sensitivity is the addition of a highly absorbing chromophoric substituent to a weakly absorb ing analyte to produce a much stronger response using ultraviolet (UV) absorption detection. Not only can this lead to better detec Introduction tion limits, but it can also be used to reduce the actual quantity of sample extract injected into the chromatographic system, Chemical derivatization in association with chromatography has leading to an extension in lifetime of the column if the original become an acceptable and rather widely used means of analysis injections were near overload conditions. of both organic and inorganic analytes. Since the first book ap Many analysts only consider sensitivity enhancement when peared on chemical derivatization in liquid chromatography (1) choosing to derivatize. However, selectivity enhancement should there has been much interest in the technique and a number of also be considered if the best possible means of derivatization books, chapters, and reviews followed on the subject (2-6). These is to be implemented. Selectivity can be achieved in three ways: works included both trace and non-trace analytical applications. the reaction, chromatography, and detection. An example of For trace analysis, particularly in biological matrices such as en selectivity in the reaction is given in Table I, which compares vironmental samples, foods, and animal tissues and fluids, three fluorescent reagents for amines. It can be seen that for chemical derivatization can be extremely useful. However, there primary amines, fluorescamine would be the most selective in are many factors that an analyst must consider before making terms of reactivity, while dansyl chloride would be the least the choice to derivatize and also many choices for the best deriva because it reacts with more types of functional groups to pro tive for his own particular analytical need. This article outlines duce fluorescent products. Thus, although all three reagents these considerations in an attempt to provide a clear understand might produce primary amine derivatives of similar sensitivity, ing to the analyst of how to make the best selection. It incor fluorescamine is superior in terms of reaction selectivity. porates the same general ideas as expressed in an earlier brief Chromatographic selectivity is achieved through the choice publication in this journal (7) but includes more detail on how of reagent for pre-chromatographic derivatization. Table II lists selectivity and sensitivity can be used to the best advantage, and four benzoylation reagents which have found use in the is aimed specifically at trace analysis by liquid chromatography. derivatization of alcohols and phenols. Although the reaction selectivity for many compounds may be similar for the four, the polarity of the derivatives will be different because of the Improved Detectability different nitro- and methoxy-substituents. Thus, under any given set of chromatographic conditions, these derivatives will appear The main purpose of derivatization for trace analysis by liq in different areas of the chromatogram depending upon their uid chromatography is to be able to detect the analyte at the polarity. This enables the analyst to choose a derivative that 484 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission. Journal of Chromatographic Science, Vol. 23, November 1985 will fall in that part of the chromatogram where interference problems during the sample extraction or during the actual from co-extractives may be minimal. quantitation. Formaldehyde, for example, is both volatile and Detection selectivity can also be achieved through the pro reactive. It has been successfully determined in a variety of sam per selection of derivatizing reagent. It may be defined as the ple types after conversion to its 2,4-dinitrophenylhydrazone (15). uniqueness of the product in terms of detectability compared Figure 1 shows the analysis of the compound in a beer sample to other constituents which may be in the sample. For detec using reversed-phase chromatography with UV absorption de tion by UV absorption, for example, the author has found in tection (16). The formaldehyde was distilled directly into a solu many cases that selectivity increases as the wavelength of detec tion of 2,4-dinitrophenylhydrazine for reaction. The resulting tion is increased. This can be put to good advantage when select product is very stable, extractable with hexane and can be stored, ing a derivatization reagent. Table II lists the wavelength max refrigerated, for long periods before analysis. In addition, the ima for several benzoylation reagents. Benzoyl chloride will yield derivative is easily collected from the chromatographic system products whose maxima are near 230 nm, whereas for confirmation later by mass spectrometry. 3,5-dinitrobenzoyl chloride would produce derivatives absorb Downloaded from https://academic.oup.com/chromsci/article/23/11/484/306112 by guest on 27 September 2021 ing maximally around 350 nm. The latter in the first instance would be preferred because of the lower likelihood of detec tion interference from sample co-extractives. Also, if an analyst Confirmation has only the use of a fixed wavelength detector at 254 nm, the reagents of choice might be p-methoxy- or p-nitrobenzoyl Derivatization is also used for confirmatory purposes. The chloride, which provide derivatives with strong absorbance near capacity factor alone, even with the use of a selective detector, that wavelength. Looking at Table I, as mentioned earlier, is often not sufficient to make an unequivocal identification fluorescamine was the most selective reagent for reaction with of the substance in question. Chemical derivatization can add primary amines. However, it may not provide the best derivative valuable information about an unknown. Changes in retention in terms of detection selectivity. The fluorescence excitation and volume or detector response after derivatization can be matched emission maxima are not particularly unique because many com with those of known standards. An example of this is shown pounds in nature fluoresce in the blue region. In terms of in Figure 2, where an extract of sole is analysed for organo- uniqueness, the NBD-derivatives might very well provide the arsenic compounds using off-line atomic absorption detection best derivatives because of their unusually close (and high) ex (17). Only one peak containing arsenic is observed and it cor citation and emission maxima. responds to arsenobetaine, an organoarsenic compound con In developing an analytical method, the analyst should con taining a carboxylic acid moiety. Upon treatment of the extract sider all three types of selectivity when deciding upon the most with ethanol/BF3, which reacts with arsenobetaine to form the appropriate derivatization reagent. In addition, the technical ease and simplicity of the reaction should be considered. Stabilization Derivatization can be useful in helping to stabilize analytes, particularly those that may be volatile or reactive, leading to Table I. Reactivity and Fluorescence Properties of Derivatization Reagents Fluorescence (nm) Reagent Reactivity Ex. Em. Dansyl chloride Primary and secondary 365 500 amines, phenols, thiols NBD chloride Primary and secondary 480 530 amines Fluorescamine Primary amines 390 475 Table II. UV Chromophoric Reagents for Alcohols and Phenols Reagent Derivative Absorbance*(nm) Benzoyl chloride 230 p-Methoxybenzoyl chloride 260 Figure 1. Determination of formaldehyde in beer as the 2,4-dinitrophenylhydra p-Nitrobenzoyl chloride 260 zone derivative. F=formaldehyde derivative; MS=fraction collected for confirma 3,5-Dinitrobenzoyl chloride 350 tion by mass spectrometry. The large peak at 14 min is an unknown carbonyl- containing constituent of the sample. Chromatographic conditions are

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