2536
Anal. Chem. 1990, 62,2536-2540
from the 1.5% GdHY sample is due to a shortened proton T1,, causing reduced cross polarization efficiency, rather than undetectably broad 13C resonances (i.e., short T2(C)). In summary, we have performed those experiments necessary to determine if 13C CP/MAS NMR spectra of carbonaceous deposits on lanthanide-exchanged zeolites reliably represent the coke present in the catalyst. We find that the commercial rare-earth-exchanged catalyst studied does not pose any problems, although lanthanide ions with long Tl(e) values, such as Gd3+and D$+,if present in sufficient quantities, have adverse effects. This study suggests that elemental analysis for lanthanide content should be performed on rare-earth-exchanged catalysts before NMR experiments on such materials are planned.
LITERATURE CITED (1) Satterfield, C. N. Hefercveneous Catalvsis in Pracfice: McGraw-Hill: St. Louis, MO, 1980. (2) Gates, 8. C.; Katzer, J. R.; SchuR, G. C. A. Chemlsiry of Catawic Processes;McQraw-H111: St. Louis, MO, 1979. (3) Jacobs, P. A. Carboniogenic Activ/fy of Zeolltes; Elsevier Scientific: New 1977. . Yark. . (4) Venuto, D: 8:; Hablb, E. T. FlW Cata/ytic Cracking with ZeoMe Catalysts; Marcel Dekker: New York, 1979. (5) Wdf. E. E.;Alfani, F. Catal. Rev.-Sci. Eng. 1982, 24, 329-371. (6) Derooane, E. G. I n Proceedings of the Internatbnal Symposium on Catawsis by Acffs and Bases; Imellk, B., et al., Eds.; Elsevier Scientific: New York, 1985; pp 221-240. (7) Derooane. E. G.; Oilson, J. P.; Nagy, J. B. Zdites 1982, 2 , 42-46. (8) Weitkamp, J.; Maixner, S. ZeOmes 1987, 7, 6-6. (9) Richardson, B. R.; Haw, J. F. Anal. Chem. 1989, 67, 1821-1825. (10) Bleaney, B. J . Mgn.Res. 1972, 8, 91-100. (11) La Mar, G. N.; Horrocks, W. Dew.; Holm, R. H. NMR of Paramagnetic
Molecules; Principles and Appkations ; Academic Press: New Yo&, 1973. (12) Gnapathy, S.; Chacko, V. P.; Bryant, R. G.; Etter, M. C. J . Am. Chem. SOC.1988, 708, 3159-3165. (13) Campbell, G. C.; Crosby. R . C.; Haw, J. F. J . Magn. Res. 1988, 69, 191-195. (14) Hagaman, E. W.; Chambers, R. R.; Woody, M. C. Anal. Chem. 1988, 58. 387-394. (15) Vassallo, A. M.; Wilson, M. A.; Collin, P. J.; Oades, J. M.; Waters, A. G.; Malcolm, R. L. Anal. Chem. 1987, 59, 558-562. (16) Torchia, D. A. J. Magn. Res. 1978, 30, 613-616. (17) Eberly, P. E., Jr.; Kimberlin, C. N., Jr. Adv. Chem. Ser. 1971, 702, 374-388. (18) Drago, R. S.; Zink, J. I.; Richmon, R. M.; Perry, W. D. J . Chem. Educ. 1974, 57, 371-376. (19) Drago, R. S.; Zink, J. I.; Richmon, R. M.; Perry, W. D. J. Chem. Educ. 1974, 57, 464-467. (20) Drago, R. S. Physical Methods in Chemistry; W. B. Saunders: New York, 1977. (21) Marrill, T. C. LanfhanMe Shift Reagents in Chemical Analysis; VCH Publlshers: New York, 1986. (22) Breck, D. W. Zeolite Molecukrr Sieves; Wiley-Interscience: New York, 1974. (23) Lee, E. F. T.; Rees, L. V. C. Zeolites 1987. 7, 143-147. (24) Marynen, P.;Maes, A.; Cremers, A. Zeolites 1984, 4, 287-290. (25) Lemos, F.; Ribeiro, F. R.; Kern, M.; Giannetto, G.; Guisnet, M. Appl. Catai. 1988, 39, 227-238. (26) Carvajel, R . C.; Chu, P.; Lunsford, J. H. J . Catal. 1990, 725, 123-131. (27) Wind, R. A. Poster presented at the 31st Experimental Nuclear Magnetic Resonance Spectroscopy Conference, Pacific Grove, CA, April 1-5, 1990.
RECEIVED for review May 17,1990. Accepted August 23,1990. E.J.M. is a National Science Foundation Fellow. The support of the National Science Foundation through Grant CHE 8918741 is gratefully acknowledged.
High-Performance Liquid Chromatographic Postcolumn Reaction Detection Based on a Competitive Binding System Andrzej Przyjazny,' Thea L. Kjellstrom? and Leonidas G . Bachas* Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055
Postcolumn reacUons are typically employed to Improve detectlon In hlghperformance lquM chromatography (HPLC) separation technlques. Thb study prqmses the use of competltlve Mnding prlncrples In deslgnkrg novel portcolumn reaction schemes. The fewiMlity of this approach was tested by udng the HPLC determlnatkm of blotln and blocytln as a model system. The effluent from the HPLC column was merged wHh a reagent stream contalning avldln, whose blndlng dtes were occupied by the dye HABA (244'hydroxyphenylazo#mzokadd). HABA was dleplaced by the analytes from the av#ln-HABA complex and the free dye was detemdned wlth a UV-VIS detector at 345 MI. The promhue was opthnlzed wHh respect to reactor deslgn, reagent concentrations, and the flow rate of reagent solution. Analytical characteristics of the developed procedure were determined and compared with the dlrect detection of blotin and Mocytln at 220 nm. The postcolumn reaction scheme Improved the selecthrlty and 8edMvky oftbe detectbn d bbMn and Mocytin while malntalnlng slmUar detection Ihnlts.
Permanent address: Institute of Inorganic Chemistry and Technical University of Gdahsk, 11/12 Majakowski St., :&%%%~sk, Poland. *Present address: Bioanalytical Development, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285.
INTRODUCTION Over the last two decades high-performance liquid chromatography (HPLC) has become an invaluable tool in separating nonvolatile or thermally labile organic and/or inorganic compounds. The application of HPLC to the determination of trace amounts of compounds, however, has been sometimes limited by the lack of adequate selectivity and sensitivity provided by conventional liquid chromatographic detectors. Commonly employed detection systems include those based on UV-vis absorption, fluorescence, electrochemistry, and changes in refractive index. Some of these detectors are more selective than others, and in fact, selectivity can be improved by employing several detectors in series (1). Both the selectivity and sensitivity of HPLC detectors can be further enhanced by using postcolumn reaction techniques (for examples see review articles 1-3). Many diverse reaction schemes have been used successfully. The simplest postcolumn reactions involve only the addition of energy in the form of irradiation, electrochemical potential, or heat to the column effluent containing analytes to produce detectable species. Other reaction schemes include redox reactions, hydrolytic reactions, ion-pair formation, ligand-exchange and complexation, and true chemical derivatization. This paper describes an evaluation of a novel approach to postcolumn reaction detection based on the competitive
0003-2700/90/0362-2536$02.50/00 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990 MW"g
Pulse
Waste reservoif
Flgure 1. Schematic diagram of an HPLC postcolumn reaction system for the spectrophotometric detection of biotin and biocytin using the
avidin-HABA reagent.
increased sensitivity resulting from the high molar absorptivity of HABA (c = 20.8 X lo3 mol-' L cm-I a t 348 nm (IO)),provided that the displacement reaction proceeds to a significant extent; (3) improved selectivity over potential interferents resulting from the shift in the detection wavelength from 220 to 345 nm (or 500 nm). In principle, only the compounds absorbing at 345 nm (or 500 nm) or those displacing HABA from its complex with avidin should interfere. This feature should considerably facilitate analyses of complex biological samples, where the number of compounds absorbing in the vicinity of 220 nm can be enormous.
EXPERIMENTAL SECTION
binding principle. The main goal of the work was to test the feasibility of this approach by using a relatively simple chemical system. Should the approach prove feasible, further research would follow aiming a t additional enhancement of selectivity and sensitivity of detection by incorporation of some chemical amplification step and/or a different detector (e.g., fluorescence detector). A mixture composed of biotin and biocytin was selected as a model system. Since neither of the two compounds possesses a strong chromophore, the spectrophotometric detectors typically used in HPLC systems are unsuitable for the detection of low concentrations of these biomolecules. Indeed, both biotin and biocytin have low absorption coefficients and, therefore, must be monitored at 220 nm ( 4 ) or below. Unfortunately, a variety of other compounds absorb a t this wavelength (5) and may cause interferences. Precolumn derivatization of biotin and its analogues with w,4-dibromoacetophenone (6) (UV detection), 4-((bromomethyl)methoxy)coumarin (6) (fluorometric detection), or 9-anthryldiazomethane (7)(fluorometric detection) has been used to improve their detectability. However, sample derivatization prior to chromatographic separation may sometimes be time-consuming and may not lend itself readily to automation. The competitive binding approach described in this paper took advantage of the change in the optical properties of the dye 2-(4'-hydroxypheny1azo)benzoic acid (HABA) upon binding to avidin (8). The two competing equilibria in this system are shown below: biotin (or biocytin) avidin + avidin-biotin (or avidin-biocytin)
+
,,,A
2537
HABA + avidin + avidin-HABA 345 n m
A,
500 n m
As shown in Figure 1, the effluent stream from an HPLC column was mixed with another stream containing the avidin-HABA complex. Since the dissociation constant of the avidin-biotin (or avidin-biocytin) complex (equal to 1.3 X mol L-' for biotin (9))is substantially lower than that of the avidin-HABA complex (5.8 x lo4 mol L-' (8)),the dye should be displaced from the binding sites of avidin by the analytes. This should result in an increase in the absorbance signal at 345 nm and a decrease in absorbance a t 500 nm. It should be mentioned that this absorbance change has formed the basis for a spectrophotometric assay for biotin (8). In a static system, the reaction is stoichiometric for all practical purposes. On the other hand, under the dynamic conditions of the POstcolumn reaction detection system, the extent of the reaction might be less than 100%. However, to be applicable to POstcolumn detection, a reaction does not need to be complete. The only requirement is reproducibility (3). Application of this competitive binding system to a continuous, on-line reaction detection in HPLC should provide a number of advantages, such as (1) possibility of determination of trace amounts of the analytes in complex matrices which may contain interferents that preclude a direct assay without prior chromatographic separation; (2) potentially
Apparatus. The instrumental setup used in this study (see Figure 1)consisted of a Rainin HPLC system (Rainin Instrument Co., Woburn, MA) interfaced with a Macintosh Plus computer (Apple Computer, Inc., Cupertino, CA). The system included a Rainin Rabbit solvent delivery system capable of gradient elution, a Rheodyne Model 7125 injector with a 20-pL sample loop (Berkeley, CA), and a Knauer Model 87 variable-wavelength UV-vis detector. Reverse-phase separations were achieved with a 5-pm Microsorb C18 column (250 X 4.6 mm i.d.) (Rainin) operated at ambient temperature, which was preceded by a 5-pm Microsorb guard column (15 X 4.6 mm) (Rainin). Elution patterns were monitored at 220 nm to detect the carbonyl functionality of biotin and biocytin. Reagent solution, pumped by a Beckman Model llOB solvent delivery system (Fullerton,CA), was added to the column effluent through a tee connector followed by one of three open-tubular reactors: a 50-cm straight PEEK tubing ('/I6 in. o.d., 0.010 in. i.d.), a 2.4-m coiled PEEK tubing ('/le in. o.d., 0.010 in. i.d., 15 cm coil diameter), and a 10.0-m knitted open-tubular (KOT) reactor made from Teflon tubing (0.5 mm i.d., 14 mm helix diameter) prepared as suggested by Krull (KOT2 from ref ll). A free-flow pulse dampener (Alltech Associates, Deerfield, IL) was installed between the reagent pump and the mixing tee to minimize detector noise caused by pressure fluctuations. A backpressure regulator placed at the detector outlet served the same purpose. In one experiment, an ISCO Model LC-2600 syringe pump (Lincoln,NE) replaced the Beckman pump to observe the detector noise reduction resulting from pulseless delivery of the reagent. Postcolumn reaction detection was performed at 345 nm. Spectrophotometric titrations were carried out by a Lambda 6 UV-vis spectrophotometer (Perkin-Elmer, Norwalk, CT) with 1-cm quartz cuvettes. Solvent System. The chromatographic conditions yielding the separation between biotin and biocytin were provided by isocratic elution with a 7822 (solventA-solvent B) mobile phase ratio. Solvent A was a 0.100 mol L-' phosphate buffer, pH 6.0, and solvent B was a mixture of 0.100 mol L-' phosphate buffer, pH 6.0, and acetonitrile (50:50 (v/v)) (12). A flow rate of 0.40 mL/min was used. Solvents were filtered through a 0.4-pm membrane filter (Nuclepore Corp., Pleasanton, CA) prior to use. Reagents. Biotin, biocytin, and monobasic sodium phosphate (reagent grade) were purchased from Sigma Chemical Co. (St. Louis, MO). HABA, NJV-dimethylformamide (DMF) (ACS reagent grade), and acetone (spectrophotometric grade) were obtained from Aldrich Chemical Co. (Milwaukee, WI). Avidin was purchased from Calbiochem (San Diego, CA). Acetonitrile (HPLC reagent grade) and methyl ethyl ketone (certified) were obtained from Fisher Scientific Co. (Fair Lawn, NJ). Deionized distilled water (Milli-Q Water Purification System, Millipore Corp., Bedford, MA) was used to prepare all solutions. mol L-l) were Biotin and biocytin stock solutions (4 X prepared by dissolving the compounds in the mobile phase. Standard solutions were prepared by further dilutions with the mobile phase. The HABA-avidin solutions were made in 0.100 mol L-' phosphate buffer, pH 6.0 or 7.0. Characterization of the Postcolumn Reaction Detection System. The pcstcolumn reaction detection system was optimized with respect to reagent concentrations, reactor design, and the reagent solution flow rate. It was also compared to direct detection of biotin and biocytin at 220 nm in terms of linearity of response, sensitivity and detection limit, precision, and selectivity. The
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
HABA (pM) 12
-
24
36
I
I
48
60
I
I
1.0 -
u)
2
0.8-
0
2 O
x
0.6-
0
a"
0.40.2
I
0.00
Figure 2. Typical chromatograms of 20 pL of 2.0 X lo4 mol L-' biotin (fist peak) and biocytin (second peak) solution with (A) direct detection at 220 nm and (B) postcolumn reaction detection at 345 nm: mobile phase, ratio of solvent A to solvent B = 78:22; solvent A, 0.100 mol L-' phosphate buffer, pH 6.0; solvent B, 0.100 mol L-' phosphate buffer, pH 6.0, in water-acetonitrile (5050(v/v)); mobile phase flow mol L-' HABA and rate, 0.40 mL/min: postcolumn reagent, 4.8 X 0.080 mg mL-' avidin in 0.100 mol L-' phosphate buffer, pH 7.0; reagent flow rate, 0.60 mL/min. A 10.0-m KOT reactor was used for
postcolumn reaction detection. Both Chromatograms are on the same scale. extent of the displacement reaction under dynamic conditions used in the postcolumn reaction detection system was determined. Finally, the reagent stability and cost per analysis of the developed postcolumn reaction detection system were estimated.
RESULTS AND DISCUSSION Feasibility of using the competitive binding principle for postcolumn detection of biotin and biocytin was established by spectrophotometric titration of the HABA-avidin complex with biotin at 345 nm in a solvent system corresponding to the mobile phase composition. The titration resulted in the displacement of the dye by biotin with an accompanying increase in the absorbance at 345 nm as revealed by the sharp end point, which was to be expected from the low dissociation constant (Kd)of the avidin-biotin complex. The choice of HPLC conditions that provide resolved peaks for biotin and biocytin was based on a previous work (12). No further attempt was undertaken to optimize the separation in terms of time of analysis and resolution. A chromatogram of a mixture of biotin and biocytin with a direct detection at 220 nm obtained under these conditions is shown in Figure 2A. An example of a postcolumn reaction detection of biotin and biocytin using the avidin-dye complex is depicted in Figure 2B. From a comparison of the two chromatograms obtained at the same sensitivity, it may be concluded that the competitive binding principle can indeed be employed for the detection of the analytes in question. The effect of the length and design of the reactor on the detector response at 345 nm was investigated by installing each of the three reactors (straight, coiled, and knitted) between the tee connector and the detector and performing the separation of biotin and biocytin under identical chromatographic conditions. The 50-cm straight tubing was found to be unsuitable for postcolumn reaction detection due to excessive noise generated by the pulsatile reagent pump. The remaining two reactors (2.4-m coiled tubing and 10.0-m knitted opentubular reactor) yielded almost identical detector response (within the limits of experimental error); however, the noise level was significantly higher in the former case. Apparently, the capillary tubing used for constructing the reactor acts as an additional pulse dampener connected in series with the free-flow pulse dampener. Comparison of chromatograms
0.02
0.04 0.06
0.08 0.10
0.12
Avidin (mg/mL) Figure 3. Effect of the concentration of HABA and avidin in the postcolumn reagent on the detector response for 2.67 nmol of biotin (0) and biocytin (0)at 345 nm. Reagent flow rate: 1.0 mL/min. For the remaining conditions, see Figure 2.
obtained when using the two reactors revealed that band broadening, measured as the peak width, was very similar in both cases. Consequently, the 10.0-m KOT reactor was used in further experiments. The dependence of detector response on the concentration of the reagents was studied by varying the HABA and avidin concentrations in the 1.2 x to 6.0 X mol L-' and 0.020-0.10 mg mL-' range, respectively; the solutions were prepared in 0.100 mol L-' phosphate buffer (pH 7.0). HABA was used in a 10-fold molar excess with respect to avidin (assuming four binding sites per avidin molecule) to saturate all of the binding sites of avidin. During these experiments the ratio of concentrations of the two reagents, the amount of biotin and biocytin injected, as well as all other chromatographic conditions were maintained constant and the peak areas that corresponded to the two analytes were measured. The results are shown in Figure 3. The shape of the curve for biotin would apparently indicate the uptake of all available biotin by avidin at the biotin concentration used in the study. This hypothesis was checked experimentally by injecting varying amounts of HABA (0.20-20 nmol) into the mobile phase using a sampling valve installed between the HPLC column and the mixing tee. The mobile phase was then merged with the reagent stream and the detector response monitored at 345 nm. The resulting HABA peak areas were compared with those corresponding to biotin and biocytin, the last two being a measure of the amount of HABA displaced by the two analytes. The data for biotin and biocytin were obtained when determining calibration curves (see further text). All chromatograms were recorded under identical, optimized chromatographic conditions. The peak areas corresponding to biotin and biocytin were found to be consistently smaller than those corresponding to an identical amount of HABA. The ratio of biotin and biocytin peak areas to HABA peak area indicated that the extent of HABA displacement is about 27% for biotin and about 20% for biocytin. Thus, the postcolumn reaction system does not attain thermodynamic equilibrium, although there exists some unidentified factor which limits the extent of displacement of HABA from its complex with avidin as revealed by the shape of the curves in Figure 3. It should be noted that an increase in the reagent concentrations over 0.080 mg mL-l avidin and 4.8 X mol L-' HABA results in a nonpractical system, since the sensitivity remains almost constant (Figure 3) while the cost of reagents increases steadily. Accordingly, the reagent concentrations
ANALYTICAL CHEMISTRY, VOL. 62, NO. 23,DECEMBER 1, 1990 2538
-
1.0 -
C .E
h
0 s 0.6 0 2 0.4 0.2 -
P
Y
I
Y
0.8
E
.-
c
C
0 .c C
a
c
6? I.o
0.0
2.0
0.0
Figure 4. Dependence of detector response for 2.67 nmol of biotin
(0) and biocytin (0)on the reagent flow rate: reagent concentrations, 4.8X lod mol L-' HABA and 0.080 mg mL-l avidin. For the remaining
conditions, see Figure 2.
were kept at 0.080 mg mL-' avidin and 4.8 X mol L-l HABA throughout the rest of the work. The effect of reagent solution flow rate on the detector response (reflected by the peak area) for biotin and biocytin at 345 nm was examined by varying the reagent flow rate in the 0.20-2.5 mL/min range at different reagent concentration levels, while keeping constant the amount of biotin and biocytin injected (2.67 nmol), the mobile phase flow rate (0.40 mL/min), as well as the other chromatographic conditions. An example of such a dependence for the optimum reagent concentrations is shown in Figure 4. Similarly shaped plots were obtained for other concentrations of the reagents; however, a decrease in the reagent concentrations was accompanied by a shift of the curve maxima toward higher flow rate values. The presence of a maximum may be explained by considering two opposing effecb which influence the detector response: (1)the supply of reagents which increases with the flow rate and, according to the law of mass action, causes a larger release of HABA from the avidin-dye complex, thus enhancing the sensitivity of the detection; (2) the dilution of a combined stream of the column effluent and the reagent solution which tends to lower the concentration of the released HABA at higher flow rates of the reagent solution. This reasoning also indicates that, for a given constant reagent consumption per unit time, one should rather use high reagent concentrations at lower flow rates than low reagent concentrations a t higher flow rates. This hypothesis was verified experimentally and found to be valid. The choice of a proper reagent flow rate is essential, as it is directly related to the reagent consumption and hence to the cost of analysis. On the basis of Figure 4, the reagent flow rate selected for further experiments was 0.60 mL/min. In most experiments, the pH of the mobile phase was 6.0 whereas that of the reagent solution was maintained at a value of 7.0. However, a separate study was performed to establish whether the change in pH of the reagent solution to 6.0 would have an effect on the detection sensitivity. By use of the same amounts of the analytes and identical chromatographic conditions, it was found that within the limits of experimental error the detector response at 345 nm was the same at both pH values (7.0 and 6.0) of the reagent solution. Thus, the pH of the reagent solution does not need fine adjustment but should remain within the 6.0-7.0 region. The introduction of a postcolumn reactor with its additional dead volume into a chromatographic system affects both retention times and peak widths. Retention time should be inversely related to the reagent solution flow rate and hence, a t a constant mobile phase flow rate, to the total flow rate.
I .o
2.0
Reagent flow rate (mL/min)
Reagent flow rate (mL/min)
Flgure 5. Dependence of retention time of biotin (0) and biocytin (0) on the reagent flow rate: mobile phase flow rate, 0.40 mL/min. This is substantiated by the experimental results shown in Figure 5. The volume of the KOT reactor was calculated to be about 2 mL. Consequently, at a total flow rate of 1.0 mL/min (0.40 mL/min for the column effluent and 0.60 mL/min for the reagent solution) the retention times of biotin and biocytin in the postcolumn reaction detection scheme should be longer by about 2 min compared to those obtained with the direct detection at 220 nm. This estimate is in full agreement with the experimental results (compare parts A and B of Figure 2). Furthermore, it was also noted that the introduction of a knitted open-tubular reactor into the HPLC system employed did not result in a considerable band broadening unless the analyte concentrations exceeded 2.0 X lo4 mol L-l; typically, the peak width increase due to reaction detection was approximately 15%. This observation is consistent with the literature reports indicating that the band broadening caused by an open-tubular reactor is minimized by reversing the flow direction frequently (e.g., knitting) (1-3, 11). To determine the analytical characteristics of the developed method, calibration curves for biotin and biocytin were constructed by using both direct detection at 220 nm and the postcolumn reaction detection at 345 nm. The curves were prepared by using chromatographic conditions that had been previously found to be optimal (reagent flow rate, 0.60 mL/min; reagent concentrations, HABA 4.8 X mol L-l, avidin 0.080 mg mL-l) and covered a range of 2 orders of magnitude, from 1.0 x mol L-' to 1.0 x mol L-' (i-e., 0.20 to 20 nmol for a 20-pL sampling loop). Linear regression by a least-squares method yielded the following equations: (1)direct detection a t 220 nm
A = 632
+ (9.63 X 108)cM(biotin), r2 = 0.998, RSD = 2.2%
A = -2420
+ (1.26 X 109)cM(biocytin), r2 = 0.999, RSD = 1.2%
(2) postcolumn reaction detection a t 345 nm A = 3610
+ (7.73 X 109)cM(biotin), r2 = 1.000, RSD = 0.66%
A = 4890
+ (5.70 X 109)cM(biocytin), r2 = 0.996, RSD = 2.2%
where A is the peak area in [ p V SI, CM is the molar concentration, and RSD is the relative standard deviation. The results indicate that the linear dynamic range for both the direct and postcolumn reaction detection systems is about 2 decades. However, an increase in concentration of the analytes over 2.0 x mol L-' was accompanied by a marked peak
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
broadening (the peak width increased by approximately a factor of 2 when changing the analyte concentration from 2.0 X lo4 to 1.0 X mol L-'), and above 1.0 X mol L-' the calibration curves leveled off. This is indicative of a local depletion of the available avidin binding sites. Consequently, the direct injection of samples with the analyte concentrations higher than about 2.0 X lo4 mol L-' is not recommended when the chromatographic resolution might constitute a problem. Comparison of slopes of the calibration curves, which are a measure of the sensitivity of detection, reveals that the postcolumn reaction detection is more sensitive than the direct detection of biotin and biocytin by factors of 8.1 and 4.5, respectively. On average, the ratio of peak areas of biotin to biocytin was about 0.76 for the direct detection and 1.36 for the postcolumn reaction detection. In the former case, higher sensitivity for biocytin can be attributed to one more carbonyl bond in the biocytin molecule compared to biotin, whereas in the latter case lower sensitivity for biocytin is presumably due to a lower value of the binding constant for the avidinbiocytin complex compared to the avidin-biotin complex. Similar differences in the binding constants for the imino analogues of biotin and biocytin, iminobiotin, and "-iminobiotinyllysine have been reported and attributed to repulsion of the positively charged a-amino group of lysine by the positively charged avidin (13). The same authors have also demonstrated that the dissociation constants (Kd values) of avidin complexes with a number of iminobiotin analogues differed little from that of iminobiotin; i.e., modifications a t the carboxyl group had little effect on the binding of biotin derivatives to avidin (14).If the same were also true for the biotin derivatives other than biocytin, molar responses of the reaction detector should be similar for these analogues. Estimates of the detection limits were based on slopes of the calibration curves and the base line noise, assuming S I N = 3. The following values were obtained: (a) for direct detection at 220 nm, biotin 1.2 X lo4 mol L-' (6 ng, assuming a 20-pL sample size), biocyin 3.4 X lo4 mol L-' (25 ng); (b) for postcolumn reaction detection, biotin 7.3 X lo4 mol L-' (36 ng), biocytin 9.7 X lo4 mol L-' (72 ng). The worse detection limits in the case of postcolumn reaction detection, despite the better sensitivity of this approach, result from a significantly higher noise level (by over an order of magnitude) caused by a pulsatile pump used to supply the reagent stream and, possibly, by an imperfect mixing of the effluent with the reagent stream. Replacement of a reciprocating pump by an ISCO syringe pump resulted in a reduction of the noise level and, hence, in an improvement of the detection limits, by a factor of 6. As a result, the detection limits of the two procedures became similar. Repeatability of the results, as measured by the relative standard deviation, was similar for the two detection approaches. In the case of retention times, RSD varied from about 0.35% to 0.7%. For the peak areas, RSD depended on the concentration level and varied from approximately 2.54% at the 1 X lo-* mol L-' level to about 8% near the detection limit. The selectivity of the proposed postcolumn reaction method was studied by including three would-be interferents, DMF, acetone, and methyl ethyl ketone, in the sample mixture. The three compounds were selected so as to have retention times similar to those of the analytes (9.4, 10.6, and 18.1 min for DMF, acetone, and methyl ethyl ketone, respectively) and, at the same time, to represent typical functionalities absorbing a t 220 nm (the carbonyl or amide bonds). It was established that an addition of up to 0.07 mol L-' of DMF, acetone, and
methyl ethyl ketone did not affect the detector response for biotin and biocytin. In contrast, direct detection a t 220 nm suffered from severe peak overlapping which precluded correct quantification. This indicates that the developed procedure based on the competitive binding principle is indeed selective. One of the basic requirements for postcolumn derivatization is that the reagent be stable for a t least 1 day. It was found that the stability of the avidin-HABA complex, as measured by the detector response for a given sample, depended on the concentration of the complex and varied from 24 h for 1.2 X mol L-' HABA and 0.020 mg mL-l avidin to 48 h for 4.8 x mol L-' and 0.080 mg mL-l avidin, thus meeting the requirement of minimum reagent stability. However, in all the other experiments fresh reagents were used daily. The cost of reagents per analysis was estimated for the optimum conditions, that is the reagent flow rate of 0.60 mL/min, avidin concentration of 0.080 mg mL-', and assuming an analysis time of 20 min. On the basis of a $1.4/mg price for avidin (Calbiochem Catalog, 1990) (the cost of the remaining reagents was insignificant compared to that of avidin and could thus be neglected), the calculated cost per analysis was about $1.3. This estimate does not imply that the developed procedure is less expensive compared to other methods of determination of biotin or biocytin. Rather, this calculation was done to demonstrate that the cost of one analysis is not prohibitive.
CONCLUSION The present, study has demonstrated the feasibility of postcolumn reaction detection of biotin and biocytin based on the competitive binding principle. Shifting the spectrophotometric detection from 220 to 345 nm has resulted in improved selectivity and sensitivity. The avidin-HABA reagent has been found to be sufficiently stable for routine analyses. The calibration curves are linear over 2 orders of magnitude and the detection limit is below 100 ng. By use of a pulseless reagent delivery system, the detection limit can be further improved to about 10 ng. Finally, by use of a chemical amplification step and/or a more sensitive detector, it is anticipated that even better detection limits may be achieved. Research along these lines is currently underway in our laboratory.
LITERATURE CITED (1) DiCesare, J. L.;Ettre, L. S. J. Chromafogr. 1982, 257, 1-16. (2) Brinkman, U. A. T.; Frei, R. W.; Lingeman, H. J. Chromafogr. 1989, 492, 251-298. (3) Frei, R. W.; Jansen, H.; Brinkman, U. A. T. Anal. Chem. 1985, 57, 1529A-1539A. (4) Chastain, J. L.; Bowers-Komro, D. M.; McCormick, D. B. J. Chromatogr. 1985, 330, 153-158. (5) Poole, C. F.; Schuette, S. A. Contemporary Practice of Chromatography; Elsevier: New York, 1984; p 375. (6) Desbene, P. L.; Coustal, S.; Frappier, F. Anal. Biochem. 1983, 128, 359-362. ( 7 ) Rijder, E.; Engelbert, U.; Troschiitz, J. Fresenlus' 2. Anal. Chem. 1984, 379, 426-427. ( 8 ) Green, N. M. Biochem. J . 1985, 94, 23c-24c. (9) Green, N. M. Adv. Protein Chem. 1075, 2 9 , 85-133. (10) Thomas E. W.; Merlin, J. C. SDecfrochim. Acta, Part A 1985, 354, 1251- 1255. (11) Selavka, C. M.; Jiao, K-S.; Krull, I. S. Anal. Chem. 1987, 59, 2221-2224. (12) Kjellstrom, T. L. M.S. Thesis, University of Kentucky, Lexington, 1989. (13) Hofmann, K.; Titus. G.; Montibeller. J. A,: Finn. F. M. Blochemlsfrv 1982. 27, 978-984. (14) Finn, F. M.; Hofmann, K. Pept.: Synth., Sfruct. Funct., Proc. Am. Pept. Symp., 7th 1981, 1-18.
RECEIVED for review May 15,1990. Accepted August 28,1990. This research was supported by a grant from the National Institutes of Health (Grant No. GM40510).
