Anal. Chem. 1995,67, 1121-1124
Determination of Amino Acids and Proteins by Dual-Electrode Detection in a Flow System Ying=SingFung* and Song=YingMo Department of Chemistry, The University of Hong Kong, Hong Kong
A new analytical procedure using a serial dual-electrode detector was developed for amino acid and protein analysis. Bromine was generated at the upstream electrode and detected by the downstream electrode. Ammo acids and proteins present in the samples were shown to lower the downstream current but had no apparent effect on the upstream current. The potential of both electrodes, the flow rate of the mobile electrolyte, and the dispersion of the samples were optimized to give satisfactory linear working ranges for seven amino acids ( M) and M). The working range can be total proteins (lo-'extended up to 10-l M amino acid and M protein by using a more anodic upstream potential. Direct determination without preconcentration can thus be performed on all the real food samples analyzed. This method has been applied to the flow injection analysis of total proteins in food, with results comparable to those obtained using the established AOAC procedure. The dual-electrode detector provides a suitable method for detecting analytes after HPLC separation. Compared to the traditional method with W detection, the dualelectrode detector is shown to provide a better selectivity for the detection of ribonuclease and catalase in food. The recent rapid advance in genetic engineering and biotechnology and the need for nutritional assessment necessitate the development of methods for analysis of amino acids and proteins in food and biological material, in particular in the areas of automatic analysis and the determination of amino acids and proteins in a complex Due to its simplicity and high sensitivity, electrochemical detection provides a suitable analytical method for determination of amino acids and proteins in mobile electrolyte after HPLC separation and for direct detection in flow injection analysis for automatic analysis.7-12 (1) Nakahawa, T.; Shibukawa, A; Kaihara, A; Itumochi, T.; Taneka, H. J. Chromatogr. 1986,353, 388. (2) Grego, B.; H e m , M. J. Chromatop. 1983,255, 87. (3) Abecassis, J.; David-Eteve, C.; S u n , A J. Liq. Chromatogr. 1985,8,135. (4) Hayashi, T.; Tsuchiga, H.; Naruse, H. J, Chromatogr. 1983,274,318. (5) Kawashiro, K; Morimoto, S.; Yoshida, H. Bull. Chem. SOC. Jpn. 1983,56, 792. (6) Polta, J.; Johnson, D. C. J. Liq. Chromatogr. 1983,6,1727. (7) Chang, M. Y.; Chen, L. R; Ding, X D.; Selavka, C. M.; Krull, I. S.; Bratin, K J. Chromatogr. Sci. 1987,25,460. (8) Johnson, D. C.; Weber, S. G.; Bond, A M.; Wightman, R M.; Shoup, R E.; Krull, I. S. Anal. Chim. Acta 1986,180,187. (9) Kissinger, P. T. J. Chromatogr. Biomed.Appl. 1989,488,31. (10) LaCourse, W. R; Krull, I. S. Anal. Chem. 1987,59,49. (11) Selavka, C. M.; Krull, I. S. J. Liq.Chromatogr. 1987,10,345. (12) Welch, L.E.; LaCourse, W. R; Mead, D. A, Jr.; Johnson, D. C. Anal. Chem. 1989,61,555.
0003-2700/95/0367-1121$9.00/0 0 1995 American Chemical Society
The problem with the application of electrochemical methods for amino acid and protein analysis is the lack of electrochemically active groups in most of these compounds. Thus, a derivatization procedure must be used prior to determination. Two approaches are adopted. The first approach is to derivatize the analyte with an electrochemically active group prior to determination. A large number of chemical reagents have been used successfully via the reductive?J3 oxidative,14-16phot~chemical>~~ and chemical conversion17 routes prior to electrochemical detection. Although many derivatizing reagents were developed covering a large number of amino acids with suitable sensitivityand selectivity,the procedure suffers inherent dficulties, as it requires additional sample handling procedures, extra instrumentation, and additional connections for on-line or pre- or postcolumn reaction, which led to the problem of peak broadening and the unavoidable dilution and possible interference due to the addition of the derivatizing reagents. The second approach to tackle the above problem is to generate in situ chemical reactions at electrode surfaces to produce electrochemically active products for detection. The use of modified electrodes based on carbon paste,I7nickel oxides,'*J9 copper,"21 platinum,12s"-n and glassy carbon28 is developed along this line. The reactive oxide could be generated in situ in an alkali medium in a triple potential pulse sequence with initial potentiostatic cleaning of the electrode surface, followed by potentiostatic generation of the reactive oxide prior to amperometric or coulometric detection.27Dual electrodes were also used with reactive intermediates generated directly at the electrode surface of the upstream electrode prior to detection by the downstream electrodeSz6Although the electrode approach simplifies instrumenta(13) Jacobs, W. A; Kissinger, P. T. J. Liq.Chromatogr. 1982,5,669. (14) Shmada, K; Kawai, Y.; Oe, T.; Nambara, T. J. Liq. Chromatogr. 1983,12, 359. (15) Lunte, S. M.; Mohabbvat, T.;Wong, 0. S.; Kuwana, T. Anal. Biochem. 1989, 178,202. (16) Oates, M.D.; Jorgenson, J. W. Anal. Chem. 1989,61,432. (17) Mahachi, T.J.; Carlson, R M.; Roe, D. F. J. Chromatogr. 1984,298,279. (18) Hui, B. S.; Huber, C. 0. Anal. Chim. Acta 1982,134,211. (19) Kafil, J. B.; Huber, C. 0. Anal. Chim. Acta 1982,139, 347. (20) Kok, W. Th.; Brinkman, U. k Th.; Frei, R W. J. Chromatogr. 1983,256, 17. (21) Kok, W. Th.;Hanekamp, H. B.; Bos, P.; Frie, R W. Anal. Chim. Acta 1982, 142,31. (22) Krstulovic, A M.; Friedman, M. J.; Colin, H.; Guichon, G.; Gaspar, M.; Pajer, K. A J. Chromatogr. 1984,297, 271. (23) Lahana, k;Liberti, A; Morgia, C.; Tarola, A M.J. Chromatogr. 1986,378, 85. (24) Iriyama, K;Yoshiura, M.; Iwamoto, T. J. Liq. Chmmatogr. 1986,9, 2955. (25) Narasimhachari, N.;Ettigi, P.; Landa, B. J. Liq. Chromatogr. 1985,8, 2081. (26) Allison, .L A; Mayer, G. S.; Shoup, R E. Anal. Chem. 1984,56, 1089. (27) Polta, J. A; Johnson, D. C. J. Liq. Chromafogr. 1983,6,1727. (28) Cox, J. A; Gray, T. J. Anal. Chem. 1989,61,2462.
Analytical Chemistry, Vol. 67, No. 6, March 15, 7995 1121
tion and obviates the requirement of additional reagents, its scope of applicability is limited. In general, direct electrochemicaldetection of amino acids is limited to the following four amino acids and their derivatives: tyrosine, tryptophan, methionine, and cysteine. Though the method may be more sensitive and selective for specific amino acids, it has problems for determination of large amino acids and proteins, as the diffusion of these biomolecules to the electrode surface is slow and the rate of electron transfer is very sluggish or does not occur due to the folding of the large protein molecule blocking the access of its active groups to the electrode surface. Although proteins that include a sulfurcontaining amino acid were shown to be oxidized at certain modified electrodes,28-32 the electrodeswere passivated by absorption when the concentration exceeded 1 x M. To provide a general detector for amino acid and protein analysis, an indirect dualelectrode detector combining the salient features of the derivatization and electrodic approaches was developed in the present work on the basis of detection by the downstream electrode of the lowering of the bromine concentration generated in the upstream electrode. The idea was based on the fact that most amino acids and proteins react quickly with bromine. Because of its small size, bromine is able to penetrate into the large protein structure, and its rapid reaction with the double bond in most amino acids and proteins makes it a general method for the analysis of amino acids and proteins. Thus, in the present work, the use of the dualelectrode detector method with in situ bromine generation was investigated for the determination of a large variety of amino acids and proteins. The analytical procedure developed was optimized, and its applicability for protein and amino acid determination in food samples was investigated. EXPERIMENTAL SECTION Apparatus. (i) FIA-ECD. The flow injection analysis electrochemical detector system CIA-ECD) consists of a syringe pump (Sage Instruments Model 352), a sample injection value (4 way rotary valve, Rheodyne), connection and dispersing tubings (Teflon tubing, 1.5 mm i.d. x 5 cm long), and a thin-layer dualelectrode cell (7 mm (0 x 4 mm (w)) with two platinum planar working electrodes (3 mm (0 x 4 mm (w)) connected in series along the flow path, separated by a distance of 1 mm. A Teflon spacer of 0.045 mm thickness is used to separate the Teflon block with the two platinum working electrodes and the stainless steel counter electrode block which holds the Ag/AgCl reference electrode. The potential of the dual working electrodes is controlled by a bipotentiostat with a common reference electrode. The currents are sampled by a Cromenco system I11 microcomputer and converted into a suitable form for recording using the Houston Hiplot X-Y digital plotter. (ii) HPLC-ECD. The liquid chromatograph electrochemical detection system (HPLC-ECD) consists of a high-pressure pump (Rainin, Model HP), a Rheodyne 7125 injection valve, a Synchropak column (SAX-300, 4.6 x 250 mm2),and a detection system based on a thin-layer dualelectrode cell. The dualelectrode cell, (29) Schlager, J. W.; Baldwin, R P. J. Chromatogr. 1987,390, 379. (30) Ye, J.; Baldwin, R P.; Schlager, J. W. Electroanalysis 1989,1, 333.
(31) Salamon, 2.; Gleason, F. IC; Tollin, G. Arch. Biochem. Biophys. 1992,299, 193. (32) Cmanes, M. T.; Rodgers, IC IC;Sligar, S. G.J. Am. Chem. SOC.1992,114, 9660.
1122 Analytical Chemistty, Vol. 67, No. 6, March 75, 7995
Up and Down Stream Current/NA
200 100
-
m-m--m---m--m--tn -100 .
-200 O~ 0
r~
-
0.3
~
L
06
-
0.9
-
d_-_ 1 _
1.2
Upstream PotentiaVV
Figure 1. Effect of upstream potential on the generation current (upstream electrode, upper curves) and collection current (downstream electrode, lower curves).
the bipotentiostat, and the data acquisition system are the same as described for FIA-ECD. Reagents. The 0.25 M KBr mobile electrolyte for FWHPLC studies is prepared by dissolving 15 g of KBr (BDH, AR) in a 0.25 M sodium phosphate @DH, AR)solution buffered at pH 5. All amino acids and protein standard solutions (1.0 x M) are prepared by dissolving suitable amounts of the standard solution in the 0.25 M KBr mobile electrolyte and diluted to specified concentrations with the mobile electrolyte immediately before use. All chemicals used are AR grade from BDH. Procedure. For both the HPLC-ECD and the FIA-ECD method, the flow rate of the mobile electrolyte is maintained at 0.5 mWmin, and a 100 pL injection volume is used. The samples for analysis are filtered prior to injection via the sample loop. The background current is measured prior to the injection of the analytes. The stability of the peak is checked by repetitive injection up to 150 times to within 5%variation. RESULTS AND DISCUSSION
General Characterizationof the Dual-ElectrodeDetector. The efficiency of the dualelectrode detector is highly dependent on the generation efficiency of the upstream electrode and the collection efficiency of the downstream electrode. The results of the investigation of the effect of the upstream electrode potential on the generation and collection efficiency are shown in Figure 1. The anodic current for the generation of bromine at the upstream electrode is found to increase with more anodic potential, up to a flat plateau at 1.5 V. It is flow rate dependent, with larger current at higher flow rate. The cathodic collection current of the downstream electrode controlled at +0.6 V follows the same trend as the upstream electrode, except that it is less affected by flow rate and upstream electrode potential. In general, the effective current collection efficiency is fairly constant at 0.25, with slight dependence on flow rate. When the upstream electrode potential is controlled at 1.0 V, the downstream current-voltage curve shows a flat plateau from -0.6 to +0.6 V. The increase in current when the downstream electrode potential is more negative than -0.6 V is due to the liberation of hydrogen, whereas the decrease in current at potential anodic to +0.6 V is due to the incomplete oxidation of the bromine collected. In order to reduce the liberation of hydrogen and the oxidation of other impurities, the downstream electrode potential is controlled potentiostatically at +0.6 V.
_
Table I. Llnear Working Ranges for Seven Amlno Acids under Optlmized Condltlons at Two Upstream Electrode Potentlals
amino
acid
statisticsa for 1.0 V linear range a R 10-1 1.83 0.9991 lo-' 1.59 0.9984 lo-' 1.36 0.9987 lo-' 1.25 0.9999 10-1 1.04 0.9973
linear working range at upstream electrode potential (M) 1.0 v 1.1v
2 x 10-6-8 x cysteine 3 x 10-6-9 x tyrosine 5 x 10-6-9 x lysine tryptophan 5 x 10-6-9 x 6 x 1OW6-1 x glycine methionine 6 x 10-6-2 x 7 x 10-"2 x arginine
5x 7x 8x 9x 1x 1x 2x
10-5-3 x 10-5-5 x
10-5-6
x x x x
10-5-6 10-4-8 10-4-9 lo-' 0.94 0.9992 10-4-9 x 10-1 0.80 0.9986
"y = an + b; R is the correlation coefficient for the linearity test with n = 10 and b = 0 for all seven amino acids listed.
Table 3. Linear Worklng Ranges for Seven Proteins under Optlmired Conditlons at Two Upstream Electrode Potentlals
statistics' for 1.0 V linear range a R
linear working range at upstream electrode potential (M) 1.1v
1.0 v
protein cytochrome c hemoglobin HAS a-amylase conalbumin I catalase myoglobin
1x 2x 2x 3x 4x 4x 5x
10-j-5
4.4 0.9991
5 x W6-2 x 6 x 10+-3 x 6 x 10-6-3 x 7 x 10-6-4 x 2 x 1Ow5-8x 2 x 10-5-9 x 2 x 10-5-9 x
10-7-3 x 10-7-4 x x x
10-7-6 10-j-8 x 10-7-8 x 10-j-9 x
3.8 0.9985
3.6 0.9987 3.3 0.9986 3.1 0.9993 3.0 0.9986
2.7 0.9986
+
" y = an b; R is the correlation coefficient for the linearity test = 10 and b = 0 for all seven proteins listed.
with n
~~
Table 2. Appllcablllty Study of the Dual-Electrode Method for the Determination of Amino Acids in Beverages after HPLC Column Separatlon
amino acid
valine
alanine isoleucine leucine glycine proline aspartic acid glutamic acid
phenylalanine lysine cystine
concentration found (ppm)" beer 3 milk tonic drink
beer 1
beer 2
23 26
43 32 13 34 48 140 29 29
3.0 11 51 180
25 12 31 8.0 7.0
Table 4. Applicability Study of the Dual-Electrode FIA Method for the Detenninatlon of Total Proteins in Food Samples
45 4.0 8.0
32 67 6.0 24 39
150 14 33 38
11 5.0
2200
35
1200 3200 310 260 120
560 1300 2400 410
1600
foodsample
N
1100 2600 240 43 21 230 1300
milkpowder 1 milkpowder2 milkpowder3 cereal tonic drink
3 3 3 3 3
34
total proteins found (%) FIA-ECD AOAC mean RSD mean RSD 25 28 23
9.5 20
0.15 0.16 0.17 0.37
0.14
24 28 24
9.1 21
0.40
0.40 0.50 0.42 0.23
1300 530
= 3.
When various amino acids were introduced to the mobile electrolyte, they did not affect the generation efficiency of the upper electrode but led to a flow rate-dependent depression of the current of the downstream electrode. In general, the lower the flow rate, the bigger the difference between the curves at a given potential at the plateau region. The variation of the upstream electrode potential would lead to the generation of different amounts of bromine and hence would affect the working range of the method and give the expected results: the higher the potential, the larger the linear range. There are practical considerations in choosing suitable flow rate and upstream electrode potential. Lowering the flow rate, though it increases the sensitivity, could affect the dispersion of the flow injection system (see next section) and reduce the number of samples analyzed per hour. Use of a higher potential, though it extends the working range, could lead to the reduction of unwanted impurities, and a high flow rate could also affect the dispersion of the flow injection system, as indicated in the next session. Thus, the upstream electrode potential is selected at 1.0 V, the flow rate at 0.5 mWmin, and the downstream electrode potential at 0.6 V. Study of the Flow Injection System. The injection volume, length of the connection tubing,dimensions of the electrochemical cell, and flow rate of the mobile electrolyte can affect the dispersion of the injected sample solution, with their effects apparent in the peak width and peak height of the FIA signal. In
order to reduce the dispersion and increase the peak height, the connection tubing is kept to a practical minimum and the internal volume of the electrochemical cell to about 1.3pL. Results for studying the effect of the injection volume on peak height and peak width (at half-peak height) indicate that both the peak height and the peak width increase with the injection volume. Thus, a compromise between sensitivity and selectivity needs to be established. Higher flow rate leads to lowering of both peak height and peak width, though the effect is somewhat leveled off at flow rate greater than 0.6 mWmin. The above effect may be due to the reduction of the time of the reaction between the analyte and the bromine generated by the upstream electrode, as faster flow rate leads to less contact time between the analyte and the bromine generated. In consideration of the above factors, the flow rate is chosen at 0.5 mL/min and the injection volume at 100 pL. The repeatability of the FIA signal traces for the blank and analyte under repetitive injection is satisfactory. The background current is shown to be about 1%of the sample signal, and the maximum sample throughput using the FIA procedure prior to overlapping of the sampling peaks is 60 samples/h. The stability of the FIA peak upon repetitive injection under different concentrations of L-cystine is satisfactory, with variation of the peak current less than 2%of the analytical signal upon repetitive injection up to 150 times. Applicability Studies. For studying the applicability of the analytical procedure developed for the analysis of amino acids, the linear working ranges of seven amino acids studied are shown in Table 1. The working range can be extended up to 2 orders more by increasing the upstream potential to 1.1V, though the lower limits also increase at the same time. Thus, the scope of Analytical Chemistry, Vol. 67, No. 6, March 15, 1995
1123
the method can be extended to cover samples with Merent amino acid contents. For real sample analysis, separation by HPLC needs to be done prior to detection using the dual-electrode cell. The results for five beverage samples are shown in Table 2. The FIA-ECD method has been applied for the analysis of total protein. The linear working ranges for seven proteins are shown in Table 3. Again, the use of a higher upstream potential extends the upper working range of the method, but it also increases the detection limits. Seven real samples obtained from supermarkets are analyzed using the FIA-ECD method in parallel with the AOAC method,33with results given in Table 4. The difference of results observed in the two methods is shown to be insigniticant statistically within the 95% contident level. Thus, the method developed is shown to give the same results as the AOAC method. Protein mixtures in complicated matrixes can also be analyzed after HPLC separation prior to dual-electrode detection. The chromatograms obtained using the dual-electrode detector and the conventional W detectors are shown in Figure 2, which indicate a better selectivity using the dual-electrode detector as compared to the conventional W detector for the detection of ribonuclease and catalase. In summary, the serial dual-electrode detection with in situ generation and detection of bromine is shown to provide a simple, sensitive, and selective method for the determination of amino acids and proteins. It could be used directly as the detector in flow injection analysis for the analysis of total protein or after HPLC separations for the analysis of amino acids. Up to 60 samples can be analyzed using the FIA-ECD procedure prior to overlapping of the FIA peaks. The sensitivity of both the HPLCECD and the FIA-ECD method is shown to be sufficient for (33) Association of Official Analytical Chemists, Inc. In AOAC OficialMethod of Analysis, 15th ed.; Helrich, IC, Ed.; AOAC: Arlington, VA, 1990 Vol. 2, 930.29, p 834.
1124 Analytical Chemistry, Vol. 67, No. 6, March 15, 1995
/I
-
0.26ml/min
O.BOml/min
A
II
l.Oml/min
Figure 2. Chromatograms obtained using dual-electrode detection (A) and UV detector (6)after HPLC separation of ribonuclease and catalase at different flow rates (injection Volume, 100 pL; column, SAX-300, 4.6 x 25 mm2; upstream potential, 1.O V; eluent, 0.25 M KBr 0.25 M Na3(P04)2 at pH 5).
+
analyzing real samples without preconcentration, and the sensitivity can be easily enhanced to 2 orders of magnitude or more by imposing higher anodic potential at the upstream electrode. Thus, the method provides a versatile method for the analysis of a wide range of amino acids and proteins at differing concentrations. ACKNOWLEDGMENT We would like to acknowledgefinancial support for this project from the Committee on Research and Conference Grants of Hong Kong University. Received for review July 18, 1994. Accepted December 26, 1994.@
AC940713Y @
Abstract published in Advance ACS Abstracts, February 1, 1995.
