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

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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 described in Reference 16. *Approximate absorbance maxima.

485 Journal of Chromatographic Science, Vol. 23, November 1985

ethyl ester, one observes a reduction in the original peak and vantage of post-column reactions is that the chromatographic a new peak appearing precisely where arsenobetaine ethyl ester separation is based on differences in the parent molecules rather should appear, confirming the presence of arsenobetaine. Fur­ than their derivatives. In most pre-chromatographic reactions, ther proof can be obtained by collecting the parent and deriva­ particularly for absorption or fluorescence detection, large bulky tive peaks for mass spectrometric confirmation. chromophoric substituents are added to the parent molecules, which have a tendency to diminish differences between two similar molecules making their separation more difficult. There are several limitations to post-column reaction detection: choice Pre-Column vs. Post-Column Derivatization of reagent and compatibility of the reaction medium with the mobile phase. In pre-column derivatization, the reaction times The choice of using pre-column or post-column derivatiza­ can be varied, the reaction medium fully optimized, and the tion for trace analysis is largely a matter of an individual's situa­ excess reagent can be removed if necessary. In post-column reac­ tion. It is preferable, of course, to do it in the simplest, least tions, time is restricted and one must always deal with the Downloaded from https://academic.oup.com/chromsci/article/23/11/484/306112 by guest on 27 September 2021 time-consuming manner. If the pre-column approach is selected, presence of the mobile phase, which is usually not the optimum then the sample preparation procedure of the derivatization se­ medium for the reaction. Also, reagents must be chosen so that quence will include additional steps. Post-column derivatiza­ any excess will not interfere in the detection of the products. tion is done continuously in an on-line fashion and thus requires Post-column dynamic extraction (8) does offer potential in this no additional sample handling. However, the analyst must ob­ regard, although it has not been extensively evaluated. As a tain the materials to construct the post-column reaction system. result the selection of post-column reagents is rather restricted. In a laboratory where a wide variety of chemicals are to be However, the field is an active one and more and more applica­ analysed by liquid chromatography, pre-column derivatization tions are continually appearing in the literature. may be preferred. It may be simpler to have a bottle of reagent on hand for occasional derivatization when necessary than to have a complete post-column reactor system in storage. This is particularly true if only a few samples at any one time re­ Limitations quire derivatization. However, if a laboratory is involved in con­ tinuously monitoring many samples for the same substances, As has been shown above, chemical derivatization, either pre- then post-column derivatization becomes attractive. or post-column, can be extremely helpful in developing trace A major advantage of post-column reactions over pre-column analytical methodology. However, there are certain limitations ones is that for the former, a reaction need not produce a single and drawbacks which may or may not be important depending well-defined product. In fact, ion-pairing (8) and metal chela­ upon the application, but which should be pointed out. First tion techniques (18) have proven to be very useful for post- of all, derivatization adds an additional step to the analytical column reactions. Also, side reactions or multiple products pose process, whether it is pre- or post-column. In some cases it might no problems in post-column reactions since they elute together be preferable to incorporate an additional purification step in and contribute to the total detector response. The only require­ the procedure so that a purer extract can be analysed directly ment would be that this be done reproducibly. A further ad- rather than carry out a derivatization. Variations in reaction yields due to the sample matrix can lead to error, as can the extra sample handling, particularly for pre-column reactions. In pre-column derivatizations, for exam­ ple, an analyst may optimize a reaction based on a quantity of known pure standard. However, he may find that yields are very poor when the same reaction conditions are used for the derivatization of a sample extract containing μg/g or lower con­ centrations of the analyte. The problem can be due to the fact that other sample co-extractives react with the reagent, thus competing with the analyte, or that the reaction medium is altered, resulting in a polarity or pH change. To overcome this, more reagent and perhaps more vigorous reaction conditions may be required to improve yield. This, then, can lead to inter­ ferences due to excess reagent, reagent impurities, and side reactions. In general, the author has found that for environmental or biological samples, derivatization is limited to concentration levels in the low ng/g range or higher. This, of course, is more than adequate for most applications.

Figure 2. Constructed chromatograms of organoarsenic compounds. Top: arsenobe- Conclusion taine and arsenobetaine ethyl ester standards; middle: extract of sole; bottom: esterified sole extract. HPLC conditions: Waters C18 μ Bondapak column with a mobile phase of 10% v/v methanol in water adjusted to pH 3.5 with glacial This article has provided practical ideas on how to best apply acetic acid. Flow rate, 1.5 ml/min. chemical derivatization to trace analysis by liquid chromatog­ raphy. It should be made clear that the approach has a valuable

486 Journal of Chromatographic Science, Vol. 23, November 1985

place in trace analysis, but in order to take advantage of it, the 8. J.F. Lawrence, U.A.Th. Brinkman, and R.W. Frei. Ion-pairing as analyst should be aware of all of the factors involved in success­ a means of detection in liquid chromatography. In Post-Column fully developing a method employing derivatization. Analysts Reaction Detection in HPLC. I. Krull, ed., Marcel Dekker, New should also consider derivatization for gas chromatography since York, 1986. 9. M.S. Gandelman, J.W. Birks, U.A.Th. Brinkman, and R.W. Frei. this type of analysis can be advantageous and there exists an Liquid chromatographic detection of cardiac glycosides and sac­ abundance of reagents for such derivatization. In developing charides based on the photoreduction of anthraquinone-2,6- analytical methodology, the simplest method should be tried disulfonate. J. Chromatogr. 282: 193-209 (1983). first. This means that direct analysis would be preferred to 10. J.W. Birks and R.W. Frei. Photochemical reaction detectors in derivatization. If derivatization is required, the analyst should HPLC. Trends Anal. Chem. 1: 361-64 (1982). seek the simplest solution to his problem, be it by gas or liquid 11. P.T. Kissinger, K. Bratin, G.C. Davis, and L.A. Pachla. The poten­ chromatography, or pre- or post-column reaction. tial utility of pre- and post-column chemical reactions with electro­ It is likely that derivatization techniques in liquid chromatog­ chemical detection in liquid chromatography. J. Chromatogr. Sci. raphy will continue to be developed. New reagents and prac­ 17: 137-46 (1979). Downloaded from https://academic.oup.com/chromsci/article/23/11/484/306112 by guest on 27 September 2021 tical applications demonstrating improved sensitivity and selec­ 12. T. Gnanasambandan and H. Freiser. Paired-ion chromatographic separation of neutral species. Anal. Chem. 53: 909-11 (1981). tivity are regularly appearing in the literature. Such develop­ 13. R.M. Cassidy. The separation and determination of metal species ments will make chemical derivatization even more attractive by modern liquid chromatography. In Trace Analysis, Vol. 1. J.F. in the future. Lawrence, ed. Academic Press, New York, 1981. 14. I.S. Krull. Trace metal analysis by high performance liquid chro­ matography. In Liquid Chromatography in Environmental Anal­ ysis. J.F. Lawrence, ed. Humana, Clifton, ΝJ, 1984. References 15. J.F. Lawrence. Prechromatographic chemical derivatization in liq­ uid chromatography. In Chemical Derivatization and Modifica­ 1. J.F. Lawrence and R.W. Frei. Chemical Derivatization in Liquid tion Techniques in Analytical Chemistry, Vol. 2. R.W. Frei and Chromatography. Elsevier, Amsterdam, 1976. J.F. Lawrence, eds. Plenum Publishers, New York, 1982. 2. K. Blau and G.S. King. Handbook of Derivatives for Chromatog­ 16. J.F. Lawrence and J.R. Iyengar. The determination of formaldehyde raphy. Heyden, London, 1977. in beer and soft drinks by HPLC of the 2,4-dinitrophenylhydrazone 3. R.W. Frei and J.F. Lawrence, eds. Chemical Derivatization and derivative. Int. J. Environ. Anal. Chem. 15: 47-52 (1983). Modification Techniques in Liquid Chromatography, Vols. 1 17. P. Michalik, G. Tam, H.B.S. Conacher, and J.F. Lawrence. Iden­ and 2. Plenum Publishers, New York, 1981 and 1982. tification of arsenobetaine and arsenocholine in some Canadian 4. J.F. Lawrence. Organic Trace Analysis by Liquid Chromatog­ fish and shellfish. J. Agric. Food Chem. Submitted for publication. raphy. Academic Press, New York, 1981. 18. R.W. Frei. Reaction detectors in liquid chromatography. In Chemi­ 5. J.F. Lawrence (guest editor). Derivatization in chromatography, cal Derivatization and Modification Techniques in Analytical part I. J. Chromatogr. Sci. 17: 113-76 (1979). Chemistry, Vol. 1. R.W. Frei and J.F. Lawrence, eds. Plenum 6. J.F. Lawrence (guest editor). Derivatization in chromatography, Publishers, New York, 1981. part II. J. Chromatogr. Sci. 17: 177-95 (1979). 7. J.F. Lawrence. Practical aspects of chemical derivatization in Manuscript received June 28, 1985; chromatography. J. Chromatogr. Sci. 17: 113-14 (1979). revision received September 23, 1985.

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