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Anal. Chem. 1985, 57,224-229
Comparative Study of Postcolumn Reactions for the Detection of Saccharides in Liquid Chromatography Petr Vrltnjr,l U. A. Th. Brinkman, and R. W. Frei* Department of Analytical Chemistry, Free University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
The appllcatlon of a new postcolumn derlvatlzatlon reagent, p -aminobenzoic acid hydrazide (ABH), In the HPLC of saccharides Is described. Comparative studles wlth other available reagents (dlsodlum blclnchanlnate, P-cyanoacetamide, tetrarollum blue, and potassium hexacyanoferrate) indicate that for given Instrumental condltlons and separatlon techniques the ABH reagent offers the best sensltlvtty. By use of a Ca*+-loaded catlon exchanger column and water as an eluent, detectlon limits In the range of IO-' g have been reached. The incluslon of a solid phase catalytlc reactor in the detection system permits detecttion of nonreducing oiigosacchartdes (e.g., sucrose) as well as reducing ollgb- and monosaccharldes. Contrlbutlons of this Catalytic reactor and of other system parts to the total band broadenlng for optlmum experlmental condltlons are glven. Appllcatlons to real samples Illustrate the increased speclficlty and sensltlvlty of this new postcolumn reactlon In comparison wlth other detection methods.
Detection systems based on a specific postcolumn reaction of sugars were the first ones used in the automated liquid chromatography of these important natural compounds. However, these detection reagents based on the use of strong acids (1,2) are difficult to handle, require a specially designed acid-resistant reagent delivery and detection system, cause excessive broadening of peaks, and are not compatible with some solvents such as acetonitrile recently used in the HPLC of carbohydrates. This explains why specialized sugar analyzers (3,4) offering typical analysis times of several hours, were never as widely accepted as, for example, amino acid analyzers for the automated ion exchange chromatography of amino acids. For the analysis of saccharides most investigators preferred the use of conventional HPLC equipment in spite of the very low sensitivity of available detectors (differential refractometer or UV photometer operated at 190 nm) for these compounds and their complete lack of selectivity. In recent times, in conjunction with a renewed interest in detection systems based on sensitive and specific detection modes (5,6)and operating with a substantial reduction in band broadening (7, 8), increased attention is paid to postcolumn reactions for sugars, compatible with HPLC. Unfortunately, most of them (9-16) are only capable of detecting reducing sugars, whereas the recently suggested cuprammonium reagent (17), suitable for detection of all carbohydrates, has only a limited applicability due to the complexity of this reaction system. Another detection reagent far nonreducing sugars has also been reported recently (18) but its detection wavelength of 260 nm makes it more prone to interferences (see Figure 5 below). To adapt these detection reagents also to some nonreducing oligosaccharides, VrltnE et al. (19) suggested use of a solid phase catalytic reactor, which converts them to easily detectable On leave from the Department of Agricultural Systems, University of Agriculture VSZ, Suchdol21, 160 21 Prague, Czechoslovakia.
reducing subunits. This catalytic hydrolysis technique has been optimized recently (20). Most of the postcolumn derivatization techniques suitable for the HPLC of sugars are based on reactions in alkaline media (9-16). Among the most promising ones with respect to mild reaction conditions and a rapid reaction rate is the reaction between hydrazides of organic acids and saccharides, producing yellow anionic chromophores, as described by Lever (10). We have found the p-hydroxybenzoic acid hydrazide (I) to give the best sensitivity of 10 compounds tested in a nonautomated spectrophotometric procedure. However, our results on its use as a HPLC postcolumn derivatization reagent indicate that its response in a detection system using a filter photometer (19) is not linear for absorbancies exceeding 0.35 au. An aim of this study was therefore to find an improved reagent still suitable for detection in the submicrogram range in conjunction with a chromatographic cation exchanger separation column (21). The reagent should also be compatible with the catalytic reaction for the hydrolysis of eluted saccharides. The amino analogue of compound (I), the hydrazide of p-aminobenzoic acid (ABH) (II), was proposed by us as a possible reagent and compared to the available detection systems as listed in this paper. Special attention was paid to the optimization study of ABH and to the influence of various parts of the instrumentation upon its performance.
/p
n
ba
"2
(1)
(11)
EXPERIMENTAL SECTION Apparatus and Materials. The schematic of the apparatus is shown in Figure 1. HPLC was carried out with a Model llOA (Altex, Berkeley, CA) reciprocating pump equipped with a Kontron (Zurich, Switzerland) pulse dampener and a six-port Rheodyne (Berkeley,CA) injection valve. Separation was carried out on a chromatographic column (0.6 X 25 cm), packed with Ostion LG KS 0800 strongly acidic sulfonated polystyrene cation exchanger in the Ca2+form. Effluent from the analytical column may be either led to a catalytic reaction column (ZO),(0.4 X 6 cm) packed with a 4% cross-linked sulfonatedpolystyrene resin (17-pm particles, H+ form; for supplier, see below), for hydrolysis of nonreducingsugars or, in the absence of these, directly to a mixing tee, through which an appropriate detection reagent is added by another pump. For this purpose, a Kratos (Ramsey, NJ) URS 051 postcolumn reaction system was used. The reaction mixture was then led to a reaction capillary coil of 0.012 in. i.d., which was formed either by a stainlesssteel capillary or by knitted PTFE coils of different volumes (Kratos, Ramsey, NJ). Both the chromatographic column and the heterogeneouscatalysis reactor were placed in a water bath (85 "C). The capillary reaction was
0003-2700/85/0357-0224$01.50/00 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985
225
Table I. Reaction Conditions Used for Tests of Various Detection Reagents BCA
composition recommended reaction time (s) actual reaction time (s) reaction temperature ("C) coil volume (ml) eluentlreagent ratio
HCF
TTB
CAA-UV
CAA-F1
ref 15 78"-600* 81 100 1 1.23
ref 13,14
C
78"-1800b 81 100 1 1.23
180b 40 95 0.5 2
ref 12
ref 11
C
60-300" 60 100 1 1.0
3OOb 75 100 1 1.67
60" 63 80 2.5 0.263
ABH
-
Reaction time used in an automated detection system, *Recommendedoptimum reaction time for a batch spectrophotometricprocedures. See Experimental Section. Yn HP
20 0.3
a,
: 0.2 f
w
10
L)
Figure 1. Instrument setup used for the catalytic postcolumn reactor, and the homogeneous postcolumn reaction for sugars: ER, eluent reservoir; HP, high-pressure pump; PD, pulse damper; IV, injection valve; AC, analytical column; CR, catalytic reactor; TW, thermostated water bath; RC, reaction coil; RP, reagent pump: UV, UV absorbance detector, R, recorder.
either immersed in a glycerol bath or placed in the Kratos reactor heating unit and operated at 100 "C. The effluent from the reaction coil was led without cooling to a LC-75 UV-vis photometric detector or a Model 3000 fluorimeter equipped with a LC flow cell adapter (Perkin-Elmer, Norwalk, CT). Two different polystyrene-type cation exchange resins were supplied by ?ne manufacturer (United Chemicaland Metallurgical Works, Usti nad Labem, Czechoslovakia) and were designated as Ostion LGKS 0800 (8% cross-linking,10- or 25-pm particles) and an experimental batch of resin having 4% cross-linkingand 17-pm particles. These two resins are strongly acidic sulfonated styrene-divinylbenzene copolymers with spherical particles. The resins were converted to the H+ form by washing with 2 M HCl on a sintered glass disk and used as packing materials for the reactors. Ostion LGKS 0800 was also used in its Ca2+form as a packing for the analytical column (conversion with 1M CaC12) prior to filling the column. The 8% cross-linkedpolystyrene cation exchanger was packed into the columns as follows: a 50% (v/v) slurry in degased deionized water was added to a slurry reservoir and packed downward under constant pressure (200 bar) using water as a packing liquid. The less cross-linked cation exchanger (4% DVB) was packed similarly using a lower packing pressure (25 bar). Preparation of Reagents and Samples. The following postcolumn derivatization reagents for carbohydrates were tested: disodium bicinchoninate (12) (BCA) (Sigma Chemicals, St. Louis, MO), 2-cyanoacetamide (13-15) (CAA) (zur Synthese, Merck, Darmstadt, F.R.G.),the hydrazide of p-aminobenzoicacid (ABH) (Janssen Pharmaceutica, Beerse, Belgium), tetrazolium blue (9) (TTB) (fur Mikroskopie, Merck, Darmstadt, F.R.G.), and potassium hexacyanoferrate (11) (HCF) (reagent grade, Baker Chemicals, Deventer, The Netherlands). All other chemicals and sugars were of reagent-grade purity. A standard procedure for preparing the working solution of p-aminobenzoylhydrazide was as follows: 1part of its 5% solution in 0.5 M HC1 (stored in a brown bottle in the refrigerator) was mixed with 2 parts of 2.4 M NaOH, ultrafiltered and ultrasonicated under vacuum before use. A fresh working solution is to be prepared daily. For the comparative tests with the tetrazolium blue reagent a 0.34% solution in 0.31 M NaOH was prepared, which was mixed
0.1
h(nrn)
Figure 2. Selection of Xop, for the HPLC detection system based on the reaction of sugars with ABH: HPLC, column 0.6 X 25 cm, packed with 10-Fm Ostion LG KS 0800 (Ca2+ form), eluent was water at 30 mL/h, t = 85 OC; detection, catalytic hydrolysis on solid phase reaction column, t = 85 O C , followed by ABH reagent solution addition at 14.5 mL/hr, reaction time was 40.4 s, t = 95 OC, pump for reagent delivery was a Kratos URS 051; ( 0 )sucrose, (0)fructose, (0)glucose, ( 0 ) raffinose; (soli lines) signal-to-noise ratio for 0.018 p g of saccharides, (broken lines) peak heights for 0.6 p g of saccharides.
with a 3.75-fold excess of 95% ethanol after ultrasonication. The resulting solution was then ultrafiltered and ultrasonicated in vacuo. Other reagents were prepared according to the literature (9, 11-15). A standard solution of sugars was prepared by appropriate dilution from a 2% stock solution, containing raffinose, sucrose, glucose, and fructose in 10% 2-propanol. Samples of ethanolic plant extracts and wine were purified by passing a 2-mL aliquot through a 1.5-mLbed of Dowex 50W-X8 (50-100 mesh) in the H+ form and elution of the column with water to a 25-mL volumetric flask, containing 2.5-mL of 2propanol.
RESULTS Optimization of Detection Conditions with g-Aminobenzoic Acid Hydrazide. Although the reaction of ABH with reducing sugars was not examined until now, the main attention in this section will be given to the tests of its practical applicability as a detection reagent and not to the studies of the reaction itself as regards its spectral and kinetic characteristics. However, it has been demonstrated by preliminary batch experiments with glucose that the reaction is faster than for most other reagents (10-16), maximum absorbance being reached within 3 min. Optimum Wavelength. The spectral characteristics of the reaction of sugars with ABH were determined by running a series of chromatograms (for conditions, see Table I) using
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985
Table 11. Comparison of Results Obtained with Selected Photometric Reagents CAA-UV"
TTB
ABH"
response (au) for 1 pg of raffinose
sucrose glucose fructose measurement wavelength (nm) noise level relative to ABH detection limit (2x noise) for sucrose (moles) "Cf.Figure 4. different detection wavelengths in the range 370-420 nm. It is apparent from Figure 2, that the peak heights of all tested saccharides increased, some of them almost linearly, with a decrease in A. However, plotting the signal-to-noise (peakheight-to-noise) ratio against the detection wavelength in this figure clearly indicates that a low wavelength is not suitable for high-sensitivity measurements and that the optimum detection wavelength is approximately 410 nm. This A, was used in all subsequent experiments. Basicity. Like other hydrazides of organic acids (IO),ABH reacts with sugars only in an alkaline medium. The photometric response with ABH for a given reaction time and temperature increases with increasing concentrationof sodium hydroxide in the reaction mixture. A maximum of 2.4 M NaOH for the reagent preparation (see Experimental Section), corresponding to a 0.55 M concentration in the final reaction mixture was chosen as an optimum assuming an acceptable stability of the alkaline working solution of ABH during a day. Temperature. A study of the dependence of the chromophore-producing reaction upon the reaction temperature in a postcolumn derivatization system reveals that no optimum was reached over the whole tested range (80-100 "C), since the reaction time (40 s) is significantly below plateau condition as determined by the manual procedure. The standard temperature was 95 "C because of occasional problems with base line stability encountered at 100 "C, probably due to bubble formation. Flow Rates. Figure 3 shows the dependence of the height of peaks produced by the reaction of different sugars with ABH upon the flow rate of ABH reagent. Other experimental conditions including the mobile phase flow rate were kept constant. Curves for all saccharides show a maximum at about 15 mL/h. From the point of view of quantitative analysis the results in Figure 3 are encouraging since in the range 12-24 mL/h the opposing effects of the reagent concentration and of decreasing the reaction time lead to a fair stabilization (plateau) of the photometric response, making thus the reproducibility of the quantitative analysis quite insensitive to small variations in the reagent flow rate in the mentioned range. In addition, fairly constant signal-to-noise ratios are obtained in this range as well (Figure 3). A study of the evaluation of peak height dependence upon the flow rate of the mobile phase has shown that peak height was inversely related to the analytical column flow rate and a 50% gain in sensitivity was realized when this flow rate was reduced from 58 to 23 mL/h. This in turn increased the reaction time from 23 to 45 s and produced a %fold increase in the reagent-to-eluent ratio. On the other hand, eluent flow rates lower than 20 mL/h lead in our system to a less stable base line. Plotting the resolution (Rs)between sucrose-glucose and glucose-fructose against the eluent flow rate indicates no significant change in resolution between 20 and 30 mL/h, whereas for higher flow velocities a gradual decrease in resolution can be observed; 30 mL/h therefore was chosen as an optimum eluent flow rate, allowing for reasonable analysis times of about 12 min and giving a maximum back pressure
0.115 0.224 0.152 0.185 520
5 1.2
x
10-10
0.019
0.116
0.027 0.040 0.001 276 0.5 2.1 x 10-10
0.214 0.150 0.166 410 1 4.3 x 10-1'
0.E
0.E
;
51 P
8
6
f
F
0.4
G
5( n
R
0.2
C
20
10 V( mI/h )
Figure 3. Dependence of the sensitivity of detection upon the flow rate of ABH reagent. HPLC and detection are as in Figure 2, but Altex Model 110A reciprocating pump was used for the reagent delivery. Detection was at 410 nm. Injected amounts of saccharides were 2 pg. Dotted line ( A - - - A ) is 20X noise level.
of the whole chromatographic system of about 40 bar. On the basis of the above mentioned tests the following conditions, considered as an optimum for our chromatographic system, were chosen (see also Table I for the comparison with other reagents): reagent composition: prepared as described in Experimental Section, 1.67% solution of ABH in 1.43 M NaOH + 0.167 M NaC1; detection conditions, reagent flow rate about 15 mL/h, reagent to effluent ratio 1:2, reaction time 40 s, reaction temperature 95 "C, detection wavelength 410 nm. Application of the ABH Reagent. With the above conditions, the linearity of the detection system response including the solid phase catalytic reaction (second column) was investigated. Straight lines going through zero were obtained for all four saccharides tested up to 1-pg quantities (for absorbance values, see Table 11). Under these experimental conditions detection limits were as follows: raffinose, 28 ng; sucrose, 15 ng; glucose, 21 ng; and fructose, 19 ng at a noise level of 1.6 X absorbance units and a 3:l signal-to-noise ratio. A chromatogram obtained for the above mentioned mixture of saccharides in 1-pg quantities is shown in Figure 4. The second trace shown in this figure was obtained by using the same chromatographic (column I) and hydrolytic (column 11) conditionsbut with a different detection reagent (CAA) which will be discussed later.
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985
227
W
0.3-
8 3" u wl P YE:
la
0.2a,
5
; n
0.1a -
b
I 0
,
5
tR(min) 10
Flgure 4. HPLC of saccharides (1 pg) employing different detection reagents; HPLC and solid phase catalytic reactor as in Figure 2: (a) ABH reagent, conditions as In Figure 2, but flow rate 16.4 mL/h, detection at 410 nm; (b) CAA reagent, for detector conditions see Table I, detection at 276 nm.
0
5
tR(rnin) 10
Flgure 6. HPLC analysis of a catlonsxchange-cleaned ethanolic extract of sugar beet leaves (cf. Figure 5, but diluted 10-fold) monitored by reaction with ABH. Chromatographic conditions are given in Figure 2. Detection conditions were as follows: 6a, as in Figure 2; 6b, the solid phase catalytic reactor was excluded from the detection system. Detection wavelength was 410 nm.
UV photometry at 195 nm reveals the presence of numerous
0.3
0.2
8 m
: 2 0 .I
5 t,(min)
'0
Figure 5. HPLC of sugars monitored by direct UV photometry: HPLC as In Figure 2: (a) sample, catlon-exchangscleanedethanolic extract of sugar beet leaves, corresponding to 1.60 mg of fresh leaves, detection wavelength 195 nm; (b) HPLC of the same sample as in 5a monitored at 276 nm; (c) sample, 100 pg of pure sugar standards; detection wavelength 195 nm. Sensitivity for all chromatograms was 0.32 au.
A main purpose of the present study was to work out a rapid, sensitive, and specific chromatographic technique for the determination of sugars in complex biological matrices, where, for selectivity reasons, they cannot be monitored using conventional HPLC detectors (RI or UV). Figure 5 shows in the lowest trace a chromatogram of the underivatized sugars (same mixture as in Figure 4), but in 1OO-bg quantities, as monitored by direct UV photometry a t 195 nm. The upper trace shows the chromatogram of an ethanolic extract of sugar beet leaves, monitored at the same wavelength. This extract was prepurified by using the cation-exchange column in the H+ form, which in addition to cations, which might cause a partial elution of the Ca2+loaded on the chromatographic column, removes also some plant pigments by adsorption (see Experimental Section). Despite this prepurification, direct
contaminants, whereas the amounts of saccharides in the sample are still too low to be detected. The middle trace shows the analysis of the same amount of sample using a higher detection wavelength (276 nm, also used for detection of 2cyanoacetamide derivatives). A substantial number of interfering peaks can be observed even at this detection wavelength, where the direct photometry of sugars without derivatization is impossible. The two chromatogramspresented in Figure 6 demonstrate the advantage of using the solid phase catalytic reactor in combination with the ABH reaction detection system for a given sample and separation. The sugar beet extract, whose chromatograms are shown in Figure 5 was diluted 10-fold, applied to the same analyticaI column as in Figure 5, and run under identical chromatographic conditions. The upper trace shows the chromatogram obtained with the solid phase catalytic reactor included in the system (note the detection of sucrose) whereas the lower trace, obtained without the reaction column reveals only peaks of reducing sugars, i.e., glucose and fructose. Only the largest interference peak observable in Figure 5a can be seen as a slight increase of the base line just before the sucrose peak, not affecting the separation of the sugars. Similar elution patterns as shown for the sugar beet leaf extracts in Figure 6 have been obtained for samples of natural and sweetened (Vermouth) white wines with peaks for the plant saccharides, sucrose, glucose, and fructose. Evaluation of Previously Described Derivatization Reagents. The newly developed detection technique using AJ3H reagent was compared with other commercially available detection reagents under conditions summarized in Table I. The same chromatographic conditions (column I) and hydrolytic conditions (column 11)were used as in previous studies with ABH. A quantitative comparison of reagents which have shown the best performance is given in Table 11. The most important observations regarding the use of those other reagents are reported below. Disodium Bicinchoninate (BCA). In spite of the positive experience (12)with the routine use of this reagent in conjunction with the anion-exchange chromatography of borate complexes of sugars, BCA was found to be incompatible with
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985
the present chromatographic system. Mixing of this reagent characterized by a high carbonate content with the effluent from the analytical column caused a significant distortion of peak shapes. Moreover, its use in connection with Ca2+-loaded columns is rather hazardous since the elution of some calcium from the column by, e.g., other cations in the sample can result in clogging of the reaction coil. The noise level obtained with this reagent was about 8 X au at 560 nm. Potassium Hexacyanoferrate (HCF). Attempts to use this reagent were not successful because of the exceptionally high noise levels (1.4 X au). Moreover, the detection wavelength (237 nm) to be used with this reagent is not very suitable for the monitoring of sugars in natural samples (see Figure 5), because of possible interferences of many other sample constituents. Tetrazolium Blue (TTB). The problems encountered with the use of tetrazolium blue are different from those with previous reagents. The main shortcoming is the poor solubility of the resulting dye in the aqueous mobile phase, so that a high ethanol content in the resulting reaction mixture should be maintained. To assure this and simultaneously to keep the tetrazolium blue concentration in the reaction mixture at the level recommended by Mopper (9) it was necessary to prepare its solution in ethanolic NaOH as described under the Experimental Section. This solution appears to be unstable at room temperature and changes its color gradually during the working day, which results in a continuous increase of the chromatogram base line. A second drawback of this reagent used in conjunction with an all-aqueous system is that the reagent-to-effluent ratio has to be exceptionally high (see Table I) to keep an adequate ethanol content in the reaction mixture, hence resulting in a substantial dilution of the sample. In addition, larger volumes of reaction coils exhibiting a higher back pressure are then required for a reaction time similar to other reagents. In spite of these disadvantages caused by an improper eluent/reagent combination, the sensitivity even under those experimental conditions was among the highest (Table 11),but the noise level was higher than that obtained for the ABH reagent (8 X au at 520 nm).
2-Cyanoacetamide Used as a Photometric Reagent (CAA-UV). Experimental conditions and results obtained for this reagent are summarized in Table 11. The absolute level of noise obtained was the lowest (8 x au) and the drift of base line under normal working conditions was negligible (A = 276 nm). Under present instrumental conditions, the minimum detectable quantity of glucose at a 3:l signal to noise ratio was 52 ng. The reagent gave a linear response up to 33 or 40 wg of injected saccharide for sucrose (after hydrolysis in the heterogeneous catalysis reactor) and glucose, respectively. Unfortunately, its response to fructose, in agreement with the literature, was very weak. Fructose gave approximately 2% of the response of glucose. The response of fructose after reaction with CAA reagent showed a Calibration curve which is slightly nonlinear. The use of this reagent is thus limited to those materials not containing ketoses (e.g., glycoprotein hydrolyzates) or those samples, Le., biological fluids whose ketose content is not of primary interest. This is not the case in most agricultural and food samples. Direct comparison of the sensitivities of the CAA and the ABH reagent can be made from Figure 4. However, it should be noted, that in spite of a much poorer absolute response of the CAA reagent for all sugars (the amount of fructose is too low to be detected, its limit of detection being about 2.5 pg), it gives a clearer resolution for raffinose and sucrose. This observation can be explained by the fact that the catalytic reactor packed with the 4% cross-linked resin not only hydrolyses raffinose to melibiose and fructose but also gives a
partial resolution of these reaction products (20). The tail part of the broad peak of “raffinose” detected by the ABH reagent is thus composed of fructose, which is not detected by the 2-cyanoacetamide reagent. 2-Cyanoacetamide Used as a Fluorigenic Reagent (CAA-Fl). Fluorimetric determination of carbohydrates was the original purpose of using the 2-cyanoacetamide reagent (13,14). The CAA reagent optimized for UV photometry (15) was found by us to give only a very weak fluorimetric response with sugars. When a reagent recommended for fluorimetry (13, 14) was prepared (with the concentration 10 times increased and its pH lowered to 8), a poorer signal-to-noise ratio was obtained than for UV photometry. The noise relative to the signal increased 3-fold for raffinose, 4-fold for sucrose, and 5-fold for glucose and fructose with a corresponding increase in obtainable detection limit. The detection limit for glucose was thus about 180 ng a t a 3:l signal-to-noise ratio. The results for a fluorimetric procedure are, however, not directly comparable with photometric reagents and are therefore not reported in Table 11. The operationalconditions used by us for the fluorimetric determination of sugars (reagent flow rates, reactor volume, and reaction time and temperature) were the same as those reported in Table I for the photometric determination of sugars with CAA reagent. The peak heights relative to glucose (=LO) were as follows: raffinose, 0.75; sucrose, 0.75; fructose, 0.07. This relative response of fructose is lower than that reported previously by Honda et al. (13)and, in fact, in contrast to their findings that the fluorigenic reagent appears to be more specific for aldoses than the photometric one. Band Broadening. The contributions of various modules of the complete system operated at optimum conditions have been examined using glucose and fructose as model compounds. The main source of peak dispersion (60-65% of the total peak variance) was the separation column (column I). About half of the detection system contributions (15-20% of the total) was due to the liquid phase reaction system including the capillary coil, whereas the contributions of the solid phase catalytic reactor (column 11) were as low as those caused by the photometric detector (&lo% of the total variance). This observation further emphasizes the advantages of including packed catalytic reactors into postcolumn derivatization systems.
DISCUSSION AND CONCLUSIONS Two main reasons led to the dgvelopment of postcolumn reaction detectors for the determination of saccharides in HPLC: the need for greater senstivity and for greater selectivity. The only tested fluorigenic reagent (CAA-F1) showed in fact a high selectivity but was not applicable to ketoses. The selectivity of other reagents improved with increasing measurement wavelength (see Figures 5 and 6). As regards the sensitivity, its real value in HPLC is determined by the signal-to-noise ratio, which is extremely dependent on instrumental conditions and until now no serious comparison of derivatization reagents under standardized instrumental conditions has been presented in the literature. Whereas ABH reagent was shown to be the optimum one for a separation technique employing water as the mobile phase, TTB reagent may be even more sensitive in conjunction with other mobile phases, composed of ethanol-water (9) or acetonitrile-water (23) mixtures, when the high organic solvent content of the eluent may prevent its precipitation and allow for a lower reagent-to-eluent ratio. We have found the CAA reagent with its exceptionally long optimum reaction times (cf. Table I) and an extreme dependence of the signal intensity upon the flow rate (an increase of the eluent flow rate by about 20% lowered the signal nearly
229
Anal. Chem. 1985, 57. 229-234
two times) to be not useful for our particular purposes. The reaction of sugars with ABH resembles that reported by Lever (IO) for other hydrazides of aromatic and heterocyclic carboxylic acids. However, with p-hydroxybenzoic acid, which was found to give the best signal-to-blankcharacteristics (IO) of all reagents tested till now, we were able to obtain only a detection limit of about 0.5 pg for sucrose as compared to 15 ng for ABH. It could be speculated that a possible hydrogen bonding betweeen the amino group of the ABH reagent and hydroxyl groups of saccharides can positively affect the reaction or the spectral characteristics of the reaction products. The sensitivity of the recommended ABH system can be further improved. Among possible methods is the use of longer reaction times closer to the manually determined optimum, using segmented systems or an increased reaction temperature which would require the use of an adequate back pressure element after the photometric cell. Another alternative is the postcolumn supply of ABH to the detection system as a stable acidic or neutral solution and its mixing on the low-presure or high-pressure part of the reagent delivery system with a highly concentrated NaOH solution, whose concentration in the reaction mixture can then exceed the limits suggested in the present paper. The reagent discussed can be used in conjunctionwith a solid-phasecatalytic reactor for the detection of both reducing saccharides and nonreducing saccharides, hydrolyzable to reducing subunits. The reagent has shown a good specificity for natural samples (plant extracts and wines), for which other detection principles (RI, direct UV photometry, UV photometry after derivatization) are not sufficiently specific to resolve sugars from accompanying interferences. ABH reagent was compared to other postcolumn derivatization reagents and was found to be for a given column packing/mobile phase combination the most specific for UV detection and sensitive with a detection limit of about 4 X lo+ mol of sucrose. It is reasonable to expect that ABH will be compatible with the borate anion-exchange separation method since a similar reagent (24) has been used under comparable reaction conditions in conjunction with the above mentioned separation method. One can also conclude, that the ABH reagent is
applicable to all the sugars investigated by Lever (IO) since a similar reaction mechanism can be expected.
ACKNOWLEDGMENT We are grateful to Kratos, Ltd., for the loan of a postcolumn reaction unit. M. W. F. Nielen is thanked for assistance in setting up the instruments. Registry No. BCA, 979-88-4; CAA, 107-91-5; TTB, 1871-22-3; HCF, 13746-66-2; AHB, 5351-17-7; raffinose, 512-69-6; sucrose, 57-50-1; glucose, 50-99-7; fructose, 57-48-7. LITERATURE CITED (1) Green, J. 0. Natl. Cancer Inst. Monogr. 1988, 2 7 , 447. (2) Kesler, P. B. Anal. Chem. 1987, 3 9 , 1416. (3) Ohms, J. I.; Zeg, J.; Benson, J. V. Anal. Biochern. 1987, 2 0 , 51. (4) Sinner, M.; Simatupang, M. H.; Dletrlchs, H. H. Wood Sci. Techno/. 1975, 9 , 307. (5) Frel, R. W. I n “Chemlcal Derlvatization in Analytical Chemlstry”; Frel, R. W., Lawrence, J. F., Eds.; Plenum: New York, 1981; Vol. I, pp 2 11-340. (6) Lawrence, J. F.; Frel, R. W. “Chemical Derlvatizatlon In Llquld Chromatography”; Elsevier: Amsterdam, 1976. (7) Deekler, R. S.; Kroll, M. 0. F.; Beeren. A. J. B.; Van den Berg, J. H. M. J . Chromatogr. 1978, 749, 669. (6) Scholten, A. H. M. T. Thesis, Free University, Amsterdam, 1981. (9) Mopper, K.; Degens. E. T. Anal. Blochem. 1972, 45, 147. (10) Lever, M. Anal. Biochern. 1982, 4 7 , 273. (11) Kldby, D. K.; Davldson, D. J. Anal. Blochem. 1973, 55, 321. (12) Sinner, M.; PUIS,J. J . Chromatogr. 1978, 756, 197. (13) Honda, S.; Matsuda, Y.; Takahashl, M.; Kakehi, K.; Ganno, S. Anal. Chem. 1980, 52, 1079. (14) Honda, S.; Takahashl, M.; Kakehl, K.; Ganno, S. Anal. Biochem. 1981, 773, 130. (15) Honda, S.; Takahashi, M.; Nishimura. Y.; Kakehl, K.; Ganno, S. Anal. Blochem. 1981, 778, 162. (16) Kato, T.; Klnoshlta, T. Anal. Blochem. 1980, 706,238. (17) Grlmble, 0. K.; Barker, H. M.; Taylor, R. H. Anal. Biochem. 1983, 728, 422. (18) Nardln, P. Anal. Biochern. 1983, 737,492. P.; Ouhrabkovl, J.; Coplkovl, J. J. Chromatogr. 1980, 797 (19) Y!tng, 3 IO.
(20) Vrltnq, P.; Frei, R. W.; Brinkman, U. A. Th.; Nielen, M. W. F. J . Chro matogr. 1984, 295, 355. (21) Scobell, H. D.; Brobst, K. M.; Steele, E. M. Cereal Chem. 1977, 54 905.
(22) Kiatos HPLC Report, Carbohydrates, undated. (23) Davies, A. M. C.; Robinson, D. S.; Couchman, R. J . Chromatogr. 1974, 707,307.
RECEIVED for review March 26, 1984. Resubmitted and accepted September 4,1984.
7- [ (Chlorocarbonyl)methoxy1-4-methylcoumarin: A Novel Fluorescent Reagent for the Precolumn Derivatization of Hydroxy Compounds in Liquid Chromatography Karl-Erik Karlsson,l Donald Wiesler, Mark Alasandro, and Milos Novotny* Department of Chemistry, Indiana University, Bloomington, Indiana 47405
A new reagent, 7-[(chlorocarbonyl)methoxy]-4-methyicoumarin, has been syntheslzed to form fluorescent derlvatives for the llquld chromatography of various hydroxy compounds. Reactlon condltlons were optlmlzed wlth model hydroxy sterolds and prostaglandins. Microcolumns of high chromatographlc efflclency were employed to resolve Isomeric compounds In both standard and blologlcal mixtures. On leave from the Department of Analytical Pharmaceutical Chemistry,Biomedical Center, University of Uppsala, Box 574 5-751 23 Uppsala, Sweden. 0003-2700/85/0357-0229$01.50/0
A sample derivatization step prior to chromatography has become a common part of numerous analytical determinations. In the area of gas chromatography (GC),such a step is primarily sought to improve volatility, thermal stability, and quantitation of polar compounds. In liquid chromatography (LC), the most frequent reason for sample derivatization is improving detection. As widely documented in the review literature (I-3), numerous precolumn reactions have now been successfully explored to enhance sensitivity and/or selectivity of chromatographic measurements. Formation of various fluorescent derivatives for LC de@ 1984 American Chemical Society
