:+?ALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
Fiddler of the U S . Department of Agriculture, L. K. Keefer of the National Cancer Institute, and D. H. Fine, T. Y. Fan, and R. Ross of the Thermo Electron Corporation.
LITERATURE CITED (1) J. M. Barnes and P. N. Magee, Er. J . Ind. Med., 11, 167-74 (1954). (2) P. N. Magee and J. M. Barnes, Er. J . Cancer, 10, 114-22 (1956). (3) H. Druckefy,R. Preussman, S. Ivankovic, and D. Schmahi, 2.Krebsfwsch. Klin. Onkol., 69, 103-201 (1967). (4) M. L. Dougiass, B. L. Kobacoff, G. A. Anderson, and M. C. Cheng. J . SOC. Cosmet. Chem., 29, 581-606 (1978). (5) D. H. Fine. D. P. Rounbehler, and P. E. Oettinger, Anal. Chim. Acta, 7 8 , 383-9 (1975). (6) A. E. Wasserman, W. Fiddler, R. C. Doerr, S. F. Osman, and C. J. Dwley, Food Cosmet. Toxicol., 10, 681-4 (1972). (7) T. Fazio, J. N. Damico, J. W. Howard, R. H. White, and J. 0. Watts, J . Auric. Food Chem.. 19. 250-53 119711. (8) RrAl Scanlan, Crit.' Re;. Food Techno/.,5, 357-402 (1975). (9) N. P. Sen, D. C. Smith, L. Schwinghamer, and B. Howsam. Can. Inst. Food Technol., 3(2), 66-9 (1970). (IO) G. Schwuing and D. Ziebarlh, in "Environmental N-Nitroso Compounds, Analysis and Formation", E. A. Walker, P. Bogovski, and Griciute, Ed., Proceedings of Conference at Tallinn, Estonian SSR, October 1-3, 1975, International Agency for Research on Cancer Scientific Publication 14, Lyon. France, 1976, pp 269-77. (11) J. Sander and G. Waly, Ref. IO, pp 291-300. (12) R. M. Hicks, T. A. Gough, and C. L. Waiters, in "Environmental Aspects of N-Nitroso Compounds", E. A. Walker, M. Castegnaro, L. Griciute, and R. E. Lyle, Ed., Proceedings of Conference at Durham, N.H., August 22-24. 1977, InternationalAgency for Research on Cancer Scientific Publication 19, Lyon, France, 1978, pp 465-73. (13) L. Lakritz, A. E. Wasserman, Gates, and A. M. Spinelii, Ref. 12, pp 425-29. (14) E. D. Peliizzari, J. E. Bunch, J. T. Bursey, and R. E. Berkley, Anal. Len., 9(61. 579-94 11976). (15) K: Bretschneider and J. Matz, Ref. IO, pp 395-9. (16) D. H. Fine, D. P. Rounbehier, N. M. Beicher, and S. S. Ipstein, Ref. 10, pp 401-8. (17) D. H. Fine, D. P. Rounbehler, A. Rounbehler, A. Silvergleid, E. Sawicki, K. Krost, and G. A. DeMarrais, Environ. Sci. Technoi., 11, 581-4 (1977). (18) D. H. Fine, D. P. Rounbehier, E. D. Pellizzari J. E. Bunch, R. W. Berkiey, J. McCrae, J. T. Bursey, E. Sawicki, K. Krost, and G. A. DeMarrais, Buil. Environ. Contam. Toxicoi., 15, 739-46 (1976).
1679
D. H. Fine, D. P. Rounbehier, E. Sawicki, K. Krost, and G. A. DeMarrais Anal. Len., 9 , 595-604 (1976). D. H. Fine, D. P. Rounbehler. F. Huffman, A. W. Garrison, N. L. Wolfe, and S. S. Epstein, Bull. Environ. Contam. Toxicol., 14, 404-8 (1975). P. Bogovski, in "N-Nitroso Compounds, Analysis and Formation", P. Bogovski, R. Preussman and E. A. Wakec. Ed., R o c d i n g s of Conference at Heidelberg, Federal Republic of Germany, October 13-15, 1971, International Agency for Research on Cancer Scientific Publication 3, Lyon, France, 1972, pp 1-5. D. H. Fine, D. Lieb. and F. Rufeh, J . Chromatogr.. 107, 351-7 (1975). T. Y . Fan, J. Morrison, D. P. Rounbehler, R. Ross, D. H. Fine, W. Miles, and N. P. Sen, Science, 196, 70-71 (1977). R. D. Ross, D. H. Fine, D. P. Rounbehler,T. Y. Fan, A. Siberglei. L. Song, and J. Morrison, presented at the 173rd ACS National Meeting, New Orleans, La., 1977. Fed. Regist., 42(152), August 8 , 1977, pp 40009-.40015. B. Spiegelhalder, G. Eisenbrand, and R. Preussmann, Angew. Chem., I n t . Ed. Engl., 17. 367-8 (1978). D. C. Havery and T. Fazio. J . Assoc. Off. Anal. Chem., 60, 517-19 (1977). N. T. Crosby, J. K. Foreman, J. F. Palframan and R. Sawyer, Ref. 21, pp 38-42. L. Hedler, H. Kaunitz, P. Marquardt. H. Faies, and R. E. Johnson, Ref. 21, pp 71-3. J. W. Howard, T. Fazio, and J. 0. Watts, J . Assoc. Off. Anal. Chem., 53, 269-74 (1970). W. Fiddler, R. C. Doerr, J. R. Ertel, and A. W. Wasserman, J . Assoc. Off. Anal. Chem., 54, 1160-63 (1971). A. E. Wasserman, Ref. 21, pp 10-15. G. Eisenbrand, in "N-Nboso Compounds in the Environment", P. Bogovski and F. A. Walker, Ed., Proceedings of a Conference at Lyon, France, October 17-20. 1973, International Agency for Research on Cancer Scientific Publication 9, Lyon, France, 1974, pp 6-11. N. P. Sen, J . Chromatogr., 51, 301-4 (1970). W. Lijinsky, W. H. Chris%, and W. T. Rainey. Jr., "Mass Spectra of NN&w Compounds", Oak Ridge National Laboratory, Oak Ridge, Tennessee (1973). J. Polo and Y. L. Chow, Ref. IO, pp 473-86. R. C. Doerr and W. Fiddler, J . Chromatogr., 140, 284-7 (1977). G. M. Telling, J . Chromatogr., 73, 79-87 (1972).
RECEIVED for review July 25,1978. Accepted May 10,1979.
Fluorometric Determination of Pyruvic Acid and a-Ketoglutaric Acid by High Performance Liquid Chromatography Hiroshi Nakamura' and Zenzo Tamura Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3- 1, Hongo, Bunkyo-ku, Tokyo 1 13, Japan
A HPLC method has been developed for the fluorometric determination of pyruvic acid (PA) and a-ketoglutaric acid (a-KG). They were separated by anion-exchange chromatography, reacted with N'-methylnicotinamidechloride (NMN) In the presence of alkali, and heated after the acidification with formic acid to produce fluorophores. By using the optimized conditions for chromatography and the post-column derivatlzatlon, 15 pmol of PA and 75 pmol of a-KG could be determined. The relative standard deviations of the method were 3.82% and 3.32% for the analyses of 500 pmol each of PA and a-KG, respectively.
Numerous methods have been reported for the analysis of a-keto acids based on diverse principles. Among them the colorimetric method using 2,4-dinitrophenylhydrazine( I , Z ) , UV absorptiometric (3-5) and the fluorometric (6, 7 ) methods coupled with enzymic reactions are most widely used. However, these spectrophotometric methods do not permit the simultaneous analysis of coexisting a-keto acids. Gasliquid chromatography (8) and gas chromatography-mass spectrometry (8)are suitable for this purpose, but they require 0003-2700/79/0351-1679$01.00/0
in general cumbersome pretreatment of samples to prepare volatile derivatives and inherit instability of derivatives at low concentrations. The application of high performance liquid chromatography (HPLC) to the analysis of 0-keto acids is a recent development. Hayashi et al. (9) reported a HPLC-UV method for the assay of phenylpyruvic acid which involved the pre-column derivatization with naphthalene-2,3-diamine to 3-benzyl2-hydroxybenzoquinoxaline. Terada et al. (IO) determined pyruvic acid (PA) and a-ketoglutaric acid (a-KG) in serum by separating their 2,4-dinitrophenylhydrazonesfollowed by UV detection. Liao et al. (11) reported a HPLC-UV method for the analysis of urinary PA and a-KG by utilizing the reaction of a-dioxo compounds with o-phenylenediamine to form quinoxalones. On the other hand, fluorescence detection coupled with HPLC separation may provide a more specific and sensitive method to analyze a-keto acids. However, in spite of the presence of several fluorometric methods for the analysis of a-keto acids (12-I4), the attempt of such a HPLC-fluorescence detection is rare. Quite recently, Grushka et al. (15) reported the derivatization of a-keto acids with 4-bromomethyl-7-methoxycoumarin and successive reversed-phase HPLC separation of the fluorescent derivatives. 62 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBL
Scheme I
P.JLSE-DAMFINC
+
R-CHz-CO-COOH 7-
CHI NMN
CHs
&-KETO AC13
(1st STEP)
R
S W L I N G DEVICE
4 -ADDUCT GUARD CCLUMN
0
0 A N A L Y T I C A L COLU,\nl
CYCLIC &-ADDUCT
FLUOROPHORE
However, this (15) and other (9, 11) HPLC methods using partition chromatography would be inadequate for the determination of a-keto acids in biological samples because of requirement of some purification procedure in order to obtain reproducible chromatograms. Although a11 the published HPLC methods (9-11, 15) for a-keto acids employed precolumn derivatization, a HPLC method which uses postcolumn derivatization seems t o be more desirable in the analysis of a-keto acids in view of avoiding the appearance of plural peaks (9, IO) from single compounds. In a previous paper (16), we briefly reported that the fluorogenic reaction (17) of a-methylene carbonyl compounds with N'-methylnicotinamide chloride (NMN) was applicable t o the post-column derivatization of some a-keto acids. In the present paper, a HPLC method for the determination of PA and a-KG which are predominant keto acids in both blood and urine is reported. The method involves their separation on a strong anion-exchange column and successive labeling with NMN t o give highly fluorescent products, permitting the assay of authentic PA and a-KG at the picomole level. The post-column derivatization reaction is thought to proceed as shown in Scheme I.
EXPERIMENTAL Materials. The packed column (4.6 mm diameter X 25 cm) of Partisil-10 SAX (10 pm, microparticulate silica-bonded strong anion exchanger) and the resin were purchased from Whatman (Clifton, N.J.). N'-Methylnicotinamide chloride (NMN, EP) (Tokyo Kasei, Tokyo, Japan), formic acid (88%, GR), sodium hydroxide pellets (GR),ammonium phosphate (monobasic, GR), citric acid (GR), and ammonium citrate (dibasic, GR) (Kanto Chemical, Tokyo, Japan) were used as received. Sodium salts of pyruvic acid (99%, GR), a-ketoglutaric acid (GR), a-keton-caproic acid (GR),a-keto-isovaleric acid (GR), a-keto-isocaproic acid (GR), and a-keto-n-butyric acid (GR) (Nakarai Chemicals, Kyoto, Japan), 2-keto-n-valeric acid (GR), levulinic acid (GR), ethyl acetoacetate (EP),phenylpyruvic acid (GR), @-ketoglutaric acid (GR) (Tokyo Kasei), p-hydroxyphenylpyruvic acid (grade 11), &phenylpyruvic acid sodium salt (Type I), DL-a-keto-Pmethyl-n-valeric acid sodium salt, a-ketoadipic acid, @-ketoadipic acid, 3-indolepyruvic acid and acetoacetic acid lithium salt (grade 11) (Sigma, St. Louis, Mo.) were used. Other chemicals and solvents used were of the highest purity commericially available. Glass-distilled water was used throughout this work. Aqueous solutions (10 mM) of keto acids were prepared just before use and diluted ad libitum with distilled water. Apparatus. A Toa HM-5 pH meter (Toa Denpa Kogyo, Tokyo, Japan) was used. The HPLC-fluorescence system (16) previously described for the post-column derivatization of aoxomethylene compounds was modified (Figure 1). All tubings, coils, and a loop used were made of stainless steel (0.5 mm i.d. X inch 0.d) and other LC parts used were also made of stainless steel so that the system worked at much higher pressure (250 kg/cm2). A stainless steel tube (2 mm i.d. X 5.5 cm) packed with Partisil-10 SAX was additionally mounted on the analytical column as the guard column. The column temperature was ambient. The eluent, 0.1 M NH4H2P04(pH 4.41, was delivered a t a flow rate of 1.05 mL/min with a Mini-micro pump (P-1,in Figure 1, Type KHD-16; Kyowa Seimitsu, Tokyo, Japan). In-
REACTIOIi C O I L
HERTIKG COIL COOLICjG C O I L FLLORESCENCE EETECTOR BACK-PRESSLRE
COIL
.iEC 3 RDE R
WASTE
Figure 1. Flow
diagram for the
HPLC
post-column derivatization of
a-keto acids stream injection of samples was performed with a 10-pL Hamilton syringe through a line sample injector (Type KLS-3T, Kyowa Seimitsu) which was connected to a 6-way valve (Type KMH-6V; Kyowa Seimitsu) attached with a 20-pL loop. A 10-cm pulsedamping column filled with 40-pm glass beads (1-mm id.; Durrum, Palo Alto, Calif.) was placed between the pump and the 6-way valve. The column outlet was fitted with a one-way valve to prevent sodium hydroxide solution from entering the column. The eluate was mixed in a 3-way tee-piece (Type KYS-16; Kyowa Seimitsu) with 10 M sodium hydroxide solution, delivered at a flow rate of 0.35 mL/min with a Mini-micro pump (P-2, Type KSD-16; Kyowa Seimitsu). The outlet of the tee-piece was connected to a 3-cm length of of tubing, which was connected to the second 3-way tee-piece. The alkaline eluate was mixed in the M hydrochloric acid second tee-piece with 50 mM NMN in solution delivered at a flow rate of 0.92 mL/min with a double plunger-type Mini-micro pump (P-3,Type KHU-W-52; Kyowa Seimitsu). A plunger damper (Type KD-3; Kyowa Seimitsu) was placed between the pump and the second tee-piece. The outlet of the second tee-piece was connected to a 10-m reaction coil which was maintained at 30 "C with a water-bath circulator (Type BT-35; Yamato Scientific, Tokyo, Japan). The end of the coil was connected to the third 3-way tee-piece to which 88% formic acid was delivered at a flow rate of 0.66 mL/min through a 10-m length of tubing with a Mini-micro pump (P-4, Type KSU-45-H; Kyowa Seimitsu). The outlet of the third tee-piece was connected to a 20-m heating coil maintained at 95 " C in a water-bath and was connected to a 30-cm cooling coil. The cooling coil was immersed in an ice-bath (3 "C) and its end was introduced to a 14-pLquartz flow cell in a Shimadzu fluorescence detector (Type FLD-1; Shimadzu Seisakusho, Kyoto, Japan) equipped with a coated low-pressure mercury lamp emitting light a t 300-400 nm (maximum intensity at 360 nm) and an EM-3 secondary filter which cuts off light shorter than 405 nm. The outlet of the flow cell was connected to a 10-m back-pressure coil. The fluorescence intensity was recorded with a Shimadzu recorder (Type R-12; Shimadzu Seisakusho). Three pressure gauges and additional two one-way valves were placed as shown in Figure 1. Quantitation of P A and a-KG. Typically, to 100 pL of aqueous sample solution was added 100 pL of a proper concentration of aqueous solution of a-keto-n-caproic acid (a-KCA) as an internal standard. The mixture was then vortex-mixed and
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
1681
40
30
h
.-+ XI
F
U
20 Y
z .d
IO
6-
5In
0
0.1
0.2
0.3
0.4
0.5
NH4H2P04 ( M )
4-
L1I
Flgure 3. Effect of molarity of NH,H2P0, solution on the apparent retention times and resolution of PA and a-KG. Flow rate: 1.4 mL/min; other conditions are the same as in Figure 2
3-
2t-
inn
PH Figure 2. Effect of pH on the apparent retention times, (tr)ap, and resolution, Rs, of PA and a-KG. Column: Partisil-10 SAX (4.6 m m aliquot diameter X 25 cm); flow rate: 0.38 mL/min; sample size: a ~ - K L containing 2 nmol of PA and 6 nmol of a-KG; eluent: (a) 0 . 1 M ammonium citrate, (b) 0.1 M ammonium phosphate
an aliquot, usually 10 KL,was injected to the HPLC system. The amount of a-keto acid was calculated from the working curve of the peak height ratio to the internal standard vs. pmol of the a-keto acid.
RESULTS Factors Influencing the Separation of PA from a-KG. The conditions for the separation of these a-keto acids on a Partisil-10 SAX column (4.6 mm diameter X 25 cm) were investigated with 2 nmol of PA and 6 nmol of a-KG. Figure 2 shows the effect of pH of the eluent, 0.1 M ammonium citrate (a) or 0.1 M ammonium phosphate (b), on the retention and resolution (Rs) of PA and a-KG. In both cases, apparent retention times, (tr)ap, expressed by the period between injection and detection of solutes, and the resolution became larger with decreasing pH of buffers. As can be seen clearly by the use of the same molarity and the counterion (NH4+),citrate is a stronger eluent than phosphate, giving much lower resolution. In addition to the anion effect, cation also affected the separation of these a-keto acids: the replacement of sodium phosphate for ammonium phosphate resulted in unsatisfactory results in any combinations of pH and molarity. The practical pH range of an eluent for the resin is limited between 1.5 to 7.5 owing to the stability of silica matrix; therefore, ammonium phosphate seemed to b e proper as an eluent for high resolution of PA and a-KG as shown in Figure 2b. Because of easiness of preparation and highest resolution, NH4H2P04solution was chosen as the eluent. When aqueous solutions of NH4H2P04ranging from 0.01 M to 0.5 M were employed, they showed almost identical pH values and the apparent retention times and the resolution increased with decreasing molarity (Figure 3). In view of analytical time, the molarity of NH4H2P04was settled to be 0.1 M. The largest flow rate of 0.1 M NH4H2P04(pH 4.4) was used thereafter to shorten the analytical time, though the
w
V
cn w
U
9
1
LL
1'
?' 10
20
30
40 50
TEMPERATURE( " c )
Figure 4. Effect of temperature on the first step. Sample size: a 2-hL aliquot containing 2 nmol of PA and 8 nmol of a-KG; eluent: 0.1 M NH,H2P0, (pH 4.4), 1.05 mL/min; derivatization conditions except reaction temperature are the same as described in the Experimental
resolution was the lowest level but satisfactory enough to separate PA from a-KG. Factors Influencing the First Step of the Reaction. Temperature. As shown in Figure 4, the nucleophilic reaction of a-keto acids with NMN was remarkably dependent on the temperature, giving optima a t 40 OC with PA and at around 7 "C with a-KG. The reaction temperature of the first step was tentatively set at 30 "C to make a compromise with the different optimal temperatures. NaOH Concentration. The final fluorescence produced was almost proportional to the NaOH concentration to be added, and even 10 M NaOH was not a sufficiently high concentration to obtain maximal fluorescence with PA and a-KG (Figure 5). NMN Concentration. The formation of fluorescent products from the a-keto acids also increased with increasing concentrations of NMN up to 50 mM (Figure 6). The higher concentrations of the reagent caused fluorescence quenching by absorbing the exciting light. Reaction Time. Increasing the reaction time of the first step did not bring out the expected increased fluorescence because of alkaline hydrolysis of the -CONH2 group of NMN and a-adduct to the -COO- group, the former is essential for the formation of the fluorophores. For example, doubling the
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
100W
0
- 6
w
"E
m
v
Z
7t
u 5-
0 W L1: 50-
Q 4W
LT
0
3 LL
Q
3-
a
2-
W
a 0
0
2
4
6
8
NaOH ( M )
1
0
Effect of NaOH concentration on the first step. Conditions are the same as in Figure 4 Figure 5.
'tl
HEATING COIL ( m ) Effect of the length of heating coil on the formation of fluorophores. Sample size: a 6-pL aliquot containing 0.6 nmol of PA and 1.8 nmol of a-KG; other conditions are the same as described in the Experimental Figure 7.
1-
^ L
/"\PA
W
i
1
*-KG
5
10
25
50
-
01
100 200
NMN(mM) Flgure 6. Effect of NMN concentration on the first step. Conditions are the same as in Figure 4
length of the reaction coil (10 m) resulted in 10% increase in the peak height of PA but 30% decrease in that of a-KG, furthermore, it caused 15% decrease in the resolution. The unstability of the a-adduct of a-KG was already seen in Figure 4. F a c t o r s Influencing the Second Step. The acidification of the alkaline reaction mixture was essential for the transformation of a-adducts into fluorophores. When 88 % formic acid was introduced and heated at 95 OC, the fluorescence yield increased with the length of the heating coil to reach almost a maximum with a 20-m coil (Figure 7). The second step of the reaction also proceeded proportionally with increasing temperature and acidity as well as the reaction time (data not shown). Analysis of P A a n d a-KG. Based on the above findings, a HPLC system for post-column derivatization of PA and a-KG was established as described in the Experimental. Table I summarizes the relative retention times of PA, a-KG, and other keto acids obtained with the Partisil-10 SAX column and 0.1 M NH4H2P04(pH 4.4) as the eluent. The separation of PA from other keto monocarboxylic acids was incomplete under the chromatographic condition designed only to resolve PA from a-KG. The typical HPLC chromatogram of PA and a-KG using a-keto-n-caproic acid (a-KCA) as an internal standard is shown in Figure 8. The lower limits of determination were 15 pmol for P A and 75 pmol for a-KG as an injected amount. By increasing the sensitivity of the detector, 3 pmol of PA and 20 pmol of a-KG were detectable. In contrast, at the lowest sensitivity of the detector, the system gave a linear relationship between fluorescence intensity (peak height) and the amounts up to 30 nmol for PA and 120 nmol for a-KG. The precision of the method at two levels was
I
-I
Figure 8. Elution curve of a synthetic mixture of aketo acids. Sample size: a 2-pL aliquot containing 2 nmol of PA, 3 nmol of a-KCA, and 6 nmol of a-KG; other conditions are the same as described in the Experimental
Table I. Relative Retention Times of Various Keto Acids
keto acid pyruvic or-keto-n-butyric levulinic phenylpyruvic acetoacetic a-keto-isocaproic p-ketoglutaric 2-keto-n-valeric a-keto-n-caproic a-ketoadipic a -ketoglutaric 3-in dole pyruvic p-ketoadipic oxalacetic
re1 retention time 1.00
1.06 1.07
1.09 1.10 1.12
1.13 1.16 1.36 1.41
1.58 1.80
1.85 1.86
evaluated by analyzing mixtures of PA and a-KG five times. The standard deviations were 0.034 X mol and 0.030 X mol for 1 X mol each of PA and a-KG, respectively,
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
mol and 0.166 X and 0.191 X each of PA and a-KG, respectively.
LITERATURE CITED
mol for 5 X lo-'' mol
DISCUSSION The present method is the first HPLC method involving the separation of native a-keto acids and the post-column derivatization. As to the HPLC-fluorescence detection of keto acids, only one report has been published by Grushka et al. (15) who used 4-bromomethyl-7-methoxycoumarin(Br-Mmc), a fluorogenic reagent for carboxylic acid. The HPLC system described here enabled fluorometric determination of keto acids at the picomole level, which is at least 1000-fold, 10-fold, and 5-fold more sensitive than the 2,4-dinitrophenylhydrazone (IO), the Br-Mmc (1.9, and the quinoxalone (11) methods, respectively. The fluorogenic NMN reaction used is not always specific for a-keto acids; however, the present HPLC method which combined the anion-exchange chromatography and the NMN reaction is selective for keto acids. The high sensitivity of the method may permit the direct injection of samples such as urine and deproteinized blood for the determination of a-keto acids. Determination of activities of transaminases by the present method is being studied in our laboratory.
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(1) Friedemann, T. E.; Haugen, G. E. J . Biol. Chem. 1943, 147, 415-42. (2) Friedemann, T. E. "Methods in Enzymology", Colowick, S. P., Kaplan, N. 0.Eds.; Academic Press: New York, 1957; Vol. 111, pp 414-18. (3) Kubowitz, F.; OR,P. Biochem. Z. 1943, 314, 94-117. (4) Ochoa, S,; Mehler, A. H.; Kornberg, A . J . Biol. Chem. 1946, 174, 979-1000. ( 5 ) Marbach, E. P.; Weil, M. H. Clin. Chem. ( Winston-Salem, N.C.) 1987, 13, 314-25. (6) Greengard, P. Nature (London) 1956, 178, 632-34. (7) Lowry, 0. H.: Roberts, N. R.; Lewis, C. J. Biol. Chem. 1958, 220,879-92. (8) Langenbeck, U.; Hoinowski, A.; Mantel, K.; MWing. H.--U. J. C k m t o g r . 1977, 143, 39-50. (9) Hayashi, T.; Sugiura, T.; Terada, H.; Kawai, S.; Ohno, T. J . Chromatogr. 1976, 118, 403-408. (IO) Terada, H.; Hayashi, T.; Kawai, S.;Ohno, T. J . Chromatogr. 1977, 130, 281-86. ( 1 1 ) Liao, J. C.: Hoffman, N. E.; Barboriak, J. J.; Roth, D. A. Clin. Chem. ( Winston-Salem, N.C.) 1977, 23, 802-805. (12) Spikner. J. E.; Towne, J . C. Anal. Chem. 1962, 34, 1468-71. (13) Mizutani, S.;Wakuri, Y.; Yoshida, N.; Nakajima, T.; Tamura, 2 . Chem. Pharm. Bull. 1989, 17, 2340-48. (14) Takeda, M.; Kinoshita, T.; Tsuji, A. Anal. Biochem. 1976, 72, 184-90. (15) Grushka, E.; Lam, S . ; Chassin, J. Anal. Chem. 1978, 50, 1398-99. (16) Nakamura, H.; Tamura, 2. J . Chromatogr. 1979, 168, 481-87. (17) Nakamura, H.; Tamura, 2 . Anal. Chem. 1976, 50, 2047-51.
RECEIVED for review February 28, 1979. Accepted June 1, 1979.
Multiwavelength Detection for Liquid Chromatography with a Repeat-Scanning Ultraviolet-Visible Spectrophotometer Koichi Saitoh and Nobuo Suzuki" Department of Chemistry, Faculty of Science, Tohoku University, Sendai, 980,Japan
A repeat-scanning spectrophotometer was designed to scan the 200-800 nm spectral range In 375 ms with a repetition rate of 2 Hz, or in 750 ms with a repetition rate of 1 Hz. The flow cell used for chromatographic experiment had a sample path of 10 mm and a volume of 8 kL. The spectrophotometer was interfaced to a small computer to perform multiwavelength detection. The simultaneous recording of chromatograms at different monitoring wavelengths, and instantaneous recording of absorption spectra were performed. The capability of multiwavelength detection has been demonstrated with an experiment on the gel chromatography of benroylacetone and its Be(I1) and Cr(II1) chelates.
High performance liquid chromatography (HPLC) currently offers efficient separation and reduced analysis time for a variety of compounds. In order to take advantage of these capabilities of liquid chromatography (LC), there is an urgent need for a detector which make possible instantaneously the confirmation of the chemical species eluted from a separation column. Although different types of detectors based on a variety of principles have been introduced into LC, the ultraviolet (UV) or visible light absorption detector is still the most widely used. Most of the commonly used detectors are based on light absorptivity measurement and operate at single and fixed wavelength. Such a fixed wavelength instrument is limited to detecting components which have noticeable absorption at the monitoring wavelength. In addition, it does not furnish necessary information on the identity of the 0003-2700/79/0351-1683$01 .OO/O
compound. The introduction of an adjustable wavelength device would solve the former limitation. However, it is, in practice, troublesome to adjust the monitoring wavelength a t every turn to ensure detection with optimum sensitivity for individual components eluted. A commercial type of recording spectrophotometer has been used for recording the absorption spectra of components in eluate. However, so long as a conventional recording spectrophotometer is used, the continuous elution process has to be temporarily interrupted while the spectrum is being recorded because of the restricted recording speed of the spectrophotometer, and this is the so-called stop-scan technique. In recent years, some types of multiwavelength absorption detectors (MWD) for instantaneous recording of the spectra of components have been introduced into LC systems. Bylina et al. ( I ) employed a cathode-ray tube (CRT) whose phosphor screen was located in the focal plane of a monochromator. The luminous spot, whose deflection was electronically controlled on the screen of the CRT, was a movable light source. Denton et al. ( 2 ) used an oscillating mirror rapid scanning spectrometer. Their MWD exhibited rapid scanning capability up to 4.25-kHz scanning rate and 218-Hz repetition rate. However, the light source (xenon arc lamp) would not be practical, and the flow cell was relatively large (87 pL). The use of multichannel integrated photosensors, such as the photodiode array (3)and silicon target vidicon ( 4 , 5 )were also reported. These devices are readily adapted to computer processing techniques. They are, however, less sensitive than photomultiplier tube, and a compromise has to be made between the spectral resolution and the desired wavelength 0 1979 American Chemical Society
