Anal. Chem. 1983, 55, 1786-1791
1786
shown in this work, analytically useful surface modification strategy. The method appears to be much less specific than other commonly suggested modification approaches and results in an electrode surface whose actual chemical composition has been only incompletely characterized to this point in time. However, the electrochemically pretreated glassy carbon surface possesses suitable characteristics in terms of stability and reproducibility for direct application in LCEC. The hydrazines represent one class of anaytes for which both the sensitivity and the selectivity of the chromatographic determination can be dramatically increased. With the pretreated electrodes, moderate working electrode potentials can be employed to determine numerous hydrazines with a sensitivity comparable to that typically observed for the LCEC of ideally oxidizable analytes. On the other hand, unusually selective detection following electrochemical pretreatment can also be achieved for the hydrazines examined of electrodes by utilization of potentials as low as +0.10 V vs. AgIAgC1. Applications of these electrodes in the LCEC of other activation-controlled oxidations appear promising. Registry No. C, 7440-44-0; 1,2-DMH, 540-73-8; l,l-DMH,
57-14-7;hydrazine, 302-01-2;methylhydrazine, 60-34-4.
LITERATURE CITED (1) Neurath, G.; Luttich, W. J . Chromatogr. 1968, 3 4 , 257-258. (2) Llu, Y.-Y.; Schmeltz, I.; Hoffmann, D. Anal. Chem. 1974, 4 6 , 885-889. (3) Abdou, H. M.; Medwick, T.; Bailey, L. C. Anal. Chim. Acta 1977, 9 3 , 221-226. (4) Butterfield, A. G.; Curran, N. M.; Lovering, E. G.; Matsui, F.: Robertson, D. L; Sears, R. W. Can. J . Pharm. Scl. 1981, 16, 15-19. (5) Matsui, F.; Butterfield, A. G; Curran, N. M; Lovering, E. G.; Sears, R. W.; Robertson, D. L. Can. J . Pharm. Sci. 1981, 16, 20-22. (6) Fiala, E. S.;Kulakis, C. J . Chromatogr. 1981, 214, 229-233. (7) Kester, P. E.; Danielson, N. D. 1983 Pittsburgh Conference on Analyt-
ical Chemistry and Applied Spectroscopy, Atlantic City, NJ, March 1983;Abstract No. 172. (8) Snell, K. D.; Keenan, A. G. Chem. Soc. Rev. 1979, 8 , 259-282. (9) Murray, R. W. Acc. Chern. Res. 1980, 13, 135-141. (IO) Blaedel, W. J.; Jenkins, R. A. Anal. Chem. 1975, 4 7 , 1337-1343. (11) Blaedel, W. J.; Mabbott, G. A. Anal. Chem. 1978, 5 0 , 933-936. (12) Wightman, R. M.; Paik, E. C.; Borman, S.; Dayton, M. A. Anal. Chem. 1978, 50, 1410-1414. (13) Gonon, F. G.; Fombarlet, C. M.; Buda, M. J.; Pujoi, J. F. Anal. Chem. 1981, 53, 1386-1389. (14) Engstrom, R. C.Anal. Chern. 1982, 5 4 , 2310-2314.
RECEIVED for review April 21, 1983. Accepted June 20, 1983.
Liquid Chromatographic Determination of Amino and Imino Acids and Thiols by Postcolumn Derivatization with 4-Fluoro-7-nitrobenzo-2,1,3-oxadiazole Yoshihiko Watanabe and Kazuhiro Imai*
Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan
A new postcolumn derlvatlratlon method wlth use of 4fluoro-7-nltrobenro-2,1,3-oxadlazole (NBD-F) for highly and sensltlve determination of amino and lmlno aclds and thiols has been achieved. These compounds were automatically separated by cation exchange resin, reacted with the reagent to produce highly fluorescent NBD derlvatlves, and detected wlth a spectrofluorometer (A,, = 470 nm, A, = 530 nm for amlno and Imino aclds; A,, = 450 nm, A,, = 520 nm for thlols). As little as 5, 50, and 10 pmol of Pro, Tyr, and Cys can be detected, respectively. This method was applied to the determlnatlon of amlno and lmlno aclds In blood dlsks of normal and pathological newborns (phenylketonurla, maple syrup urine disease, and tyrosinosis). The values obtalned were compared wlth those obtalned by the o-phthalaldehyde (OPA) method.
to generate primary amines from the imino acids has been adopted. No facile way of detection of imino acids and other amino acids has yet been found to the best of our knowledge. A possible candidate is the recently developed fluorogenic reagent for amines, NBD-F (4-fluoro-7-nitrobenzo-2,1,3-oxadiazole) (10-12), which has the ability to react with both primary and secondary amines (Figure 1) under attainable reaction conditions using two or three solvent delivering pumps for buffer and reagents. In this paper, we report the postcolumn reaction and fluorometric detection of amino and imino acids with NBD-F after separation by ion exchange chromatography used with the high-performance amino acid analyzer. Some thiols, e.g., cysteine, homocysteine, and glutathione, are also quantitated by a modified detection system. The proposed method is applied to a profile analysis of amino and imino acids in blood samples on paper disks of 3 mm diameter for the diagnosis of inborn errors of metabolism.
In the past, ninhydrin has been used for the colorimetric determination of amino acids in biological fluids with an automatic analyzer (1). With the advent of high-performance liquid chromatography (HPLC) more efficient separations in shorter times have become possible (2,3). Recently, fluorometry has been introduced in the detection system to increase the sensitivity of the method (2-5). The fluorogenic reagents, fluorescamine (6) and o-phthalaldehyde (OPA) (7), are well suited for the sensitive detection of amino acids having primary amino groups; however, the imino acids, such as proline and hydroxyproline do not yield fluorescence with these reagents (2,3). Therefore, the addition of oxidizing reagents, such as N-chlorosuccinimide (8) or sodium hypochlorite (91,
EXPERIMENTAL SECTION was Reagents. NBD-F (4-fluoro-7-nitrobenzo-2,1,3-oxadiazole) synthesized by the method of Nunno et al. (13). Alloisoleucine (ICN-K&K Inc., New York, NY) was kindly donated by K. Suzuki of the University of Tokyo. All the other amino and imino acid standards were purchased from Kyowa Hakko Kogyo Co., Ltd. (Tokyo, Japan). Amino and imino acid standard solution (2.5 fimol/mL) was purchased from Ajinomoto Co., Ltd. (Tokyo, Japan). Filter papers which were individually applied with standard amino acid solutions (0.5-20 mg/dL) of methionine, leucine, tyrosine, phenylalanine, and histidine were purchased from Fuji Zoki Pharmaceutical Co., Ltd. (Tokyo, Japan). A number of filter papers spotted with blood from normal and pathological newborns were kindly donated by H. Naruse
0003-2700/83/0355-178680 1.50/0 0 1983 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983
1787
ELUENT -
Na Cltrate butfer ( 0 3 mlimln)
0 2N Na PH 3.25 hx 23 min 0 2 N Na PH 4 2 5 lor 16 mln INJECTOR
NO2
0 8N Na PH 5.30 for 30 nm
0.2N NaOH for 5 mh
. .
Figure 1. Structure and reaction of NBD-F with amino acids.
Kyowa Gel 62210F ( 4 6 mm I d , 15 cm) T
Table I. Composition of Buffers for Amino Acids and Thiok buffer 1 buffer 2 (0.2 N (0.2N sodium) sodium) sodium citrate, g concn HCl, mL capric acid, mL thioglycol, mL ethanol, mL 25% Brij-35, mL DH wt 25 "C total volume, L
buffer 3 (0.8 N sodium)
19.7
19.7
78.4
13.2
8.4:
5.0
0.1
0.1
5.0
5.0
80 4.0
4.0
4.0
4.2!5 1.0
5.30 1.0
3.25
1.o
'
0.1
O
0
C
-
REAGENT 0.5M Bwate (pH 12.0) (0.2 milmln)
P- 2
I
...
5
-1t.
-
P-3
0.2% NBRF in EtOH (0.26 milmin)
REACTOR
3
Coil I : Tellon tube (0.26mm 1.d.. 5 m)
Coil II : Teflon tube (0.25 mm i.d., 1 m)
of the National Center for Nervous, Mental and Muscular Disorders (Tokyo, Japan). These samples had been stored a t -25 O C prior to use. Reagent, grades of trichloroacetic acid, boric acid, potassium hyroxide, sodium hydroxide, ethanol, methanol, hydrochloric acid, glutathione, cysteine, and homocysteine were purchased from Wako Pure Chemical Co., Ltd. (Tokyo, Japan). Water was deionized and double distilled. Solutions of 0.2% and 0.05% NBD-F in ethanol, prepared daily, were used for amino and imino acids and for thiols analysis, respectively, since NBD.F reacts slowly with ethanol to reduce its activity. The pH, sodium concentration, and formulation of three sodium citrate buffers prepared by Daiichi Kagaku Co., Ltd. (Tokyo, Japan), are surnmarized in Table I. The 0.5 M borate buffer (pH 12.0) was prepared from boric acid by titration with pellets of potassium hydroxide. The amino and imino acid standard solution was diluted before injection with an appropriate volume of water contaiining the internal standard (norleucine). The 5% trichloroacetic acid solution containing the internal standard was prepared as follows: 10 mI, of 25 pM norleucine was mixed with 50 mL of 10% trichloroacetic acid and adjusted with water to 100 mL. A aeries of standard solutions was prepared for each amino acid by appropriate dilution of 10 pmol/pL stock solution. Apparatus and Conditions. Schematic diagrams for the separation of amino and imino acids and thiols by ion exchange chromatography and for their quantitation by the postcolumn derivatization with NBD-F are depicted i~nFigures 2 and 3, respectively. A series of samples (20 pL) was applied tQ the column using a sample injector (KHP-Ul-130A;K,yowa Seimitsu, Tokyo, Japan). The separations of amino and imino acids and thiols were performed on a Kyowa Gel 62210F (Kyowa Seimitsu), a strong cation exchanger, the matrix of which was a 10% cross-linked sulfonated polystyrene resin (7 pm, 4.6 X 150 mm), which gave the theoretical plate numbers of 1600 and 4OOOO for aspartic acid and phenylalanine, respectively. The column temperature was maintained at 50 "C by a thermo-regulator KWA-7E (Kyowa Seimitsu). Four pumps (type KHD-16, Kyowa Seimitsu) were used for delivering the elution buffers, sodium hydroxide solution, borate buffer, reagent solution, and 50% methanol-3 M hydrochloric acid solution at the respective flow rates of 0.3,0.2,0.26, and 0.8 mL/min. At 23,39, and 69 min after the sample injection, the elution buffers 2 ancl 3 and the sodium hyroxide solution were introduced successively. The buffer changes were timed so that the second elution buffer would appear just ahead of the valine peak (Figure 5, peak i) and the third just after the phenylalanine peak (Figure 5, peak 0). After the arginine peak (Figure 5 , peak
Temp : 5-10'C
~
50% MaOH in 3M HCI (0.8 Wmh)
DETECTOR Hitachi 650-10s spectrofluorometer Em 530 n m l Ex 470 nm
Figure 2. Flow diagram for the detection of amino and imino acids. ELUENT 0.2N Na Citrate buffer (pH 4.25) (0.3 Wmh)
Temp : 50'C
COIL I
,
--1
coli I : Teflon tube (0.25 mn id- 1
I---++
m)
60% MeOH in 1.5M HCI (0.8 mllmin)
[I-> WASTE
DETECTOR Hltachl 650-10s spectrofluorometel
Em 520 nml Ex 450 nm
Figure 3. Flow diagram for the detection of thiols.
r) appeared, the column was washed with 0.2 N sodium hydroxide solution for 5 min and then equilibrated with the first elution buffer for 20 min before the onset of the next analysis. All the flow lines after the column were made with 0.25 mm i.d. Teflon tubing, the connections of which were made with three-way tees (type K3T-l6S, Kyowa Seimitsu). The temperature of the reaction coil was controlled by a thermoregulated water bath. The cooling coil following the reaction coil was set to decrease the temperature of the reaction mixture. The detector was a HitachE 650-10s spectrofluorometer (Tokyo, Japan) equipped with an 18-pL quartz flow cell. A Hitachi 056 recorder was used with sensitivity set at 10 mV. The excitation and
1788
.'
ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983
R F.I. l0Oi
,o---
0-0
l,e I
0
501
01
x'
9.0
10.0 11.0
11
,
P k
12.0
1
13.0 PH
Figure 4. Effect of pH of the borate buffer on the reaction of amino and imino acids with NBD-F determined by flow injection analysis: 0, proline (1.0 nmol); X aspartic acid (1.0 nmol).
emission wavelengths were set at 470 nm (450 nm) and 530 nm (520 nm), respectively, with 10 nm slit width. The coil (0.25 X 1000 mm) behind the fluorometer was needed to provide back pressure. The reaction conditions (pH, reagent concentration, ethanol concentration, temperature, time) of the postcolumn reaction were examined to study their effect on the fluorescence produced by the reaction of amino acids and thiols with NBD-F. To compare the amounts of amino acids in the blood disks of inborn errors of metabolism, conventional amino acid analysis using OPA reaction according to the method of Benson and Hare (2) was adopted. Sample Preparation and Extraction. The filter paper applied with blood of the newborn was punched out to a disk (3 mm diameter) which contains 2.6-2.8 pL of blood. The disk was set into a microtube (5 X 50 mm) with a Teflon seal and extracted by addition of 20 pL of 5% trichloroacetic acid solution containing 500 pmol of internal standard (norleucine). After sonication for 15 min at room temperature, a 15-pL aliquot of the solution was applied onto a column.
RESULTS AND DISCUSSION The apparatus construction and flow diagram used in the present work for the determination of amino and imino acids and thiols are similar to that described in the postcolumn derivatization of proline and hydroxyproline with NBD-Cl(5). However, the introduction of NBD-F instead of NBD-C1 necessitated certain changes of the system as described in Figures 2 and 3. In the preliminary experiment, the flow injection analysis was adopted to obtain optimal reaction conditions in the postcolumn reaction, where the sample was injected after the column. The elution buffer 1 (0.3 mL/min) was selected as the representative carrier solution (Figure 2). Since the pH of buffer 1 is the lowest among the elution buffers, the proposed pH and concentration of the borate buffer to keep the appropriate reaction are sufficient also for the other two buffers in the amino and imino acid analysis. The reaction rates of acidic amino acids with NBD-F are the smallest among the amino and imino acids (14),and then aspartic acid was chosen as a model amino acid in this reaction experiment. With proline and aspartic acid, the optimal initial pH of the borate buffer (0.2 mL/min) examined is about 12.0 as shown in Figure 4. Among the various solvents evaluated to maximize NBD-F fluorescence intensity, ethanol was found to be the best (IO). To prevent precipitation of citrate in the flow line, a flow rate of 0.26 mL/min was employed to keep the ethanol concentration below 40%. Although the higher concentration of NBD-F gave stronger fluorescence intensity, an 0.2% concentration was selected considering its solubility in ethanol. At room temperature the reaction of acidic amino acids with NBD-F was very slow, whereas reaction temperature higher than 70 "C resulted in a large bubble formation in the flow
Omin
2o
40
60
Flgure 5. Elution profile of amino and imino acid standards. Each peak represents 625 pmol except for (m) Nle (500 pmol). Conditions were the same as those given In Figure 2: a, Asp; b, Thr; c, Ser; d, Glu; e, Pro; f, Giy; g, Ala; h, (Cys),; i, Val; j, Met; k, Iie; I, Leu; m, Nle; n, Tyr; 0,Phe; p, Lys; q, His; r, Arg.
Table 11. Relative Peak Height Intensities of NBD-Amino and -Imino Acids RPHU compound compound ASP Thr Ser Glu Pro GlY Ala (CYS), Val
2.20
2.05 1.82 1.55 6.96 2.65 1.00 1.49 2.38
Met Ile Leu T Y ~ Phe
RPH a
LYS
0.62 3.05 1.89 0.58 1.71 3.55
His Arg
0.75
2.45
RPH, relative peak height, Ala is set as 1.00. Amino and imino acids, 625 pmol each, were injected into the column and detected; see Figure 2. line which caused noises on the chromatogram. The temperature was then set at 50 "C. The use of a reaction coil longer than 10 m (0.25 mm i.d.) led to peak broadening and consequent loss of resolution of each amino and imino acid on the chromatogram. Accordingly, the reaction conditions (at 50 "C, for 0.5 min) cited in Figure 2 were selected. The hydrolyzed product (NBD-OH; 4-hydroxy-7-nitrobenzo-2,1,3-oxadiazole)of the reagent has a weak fluorescence but loses its intensity at an acidic pH of about 1 (IO). Therefore, in order to reduce the background fluorescence, hydrochloric acid in methanol was added to the flow line after the reaction. The chromatogram for a standard mixture of amino and imino acids (625 pmol each) is shown in Figure 5. The pH variation of 9 to 9.5 of the reaction mixture caused by mixing the elution buffers and the borate buffer resulted in no base line shift on the chromatogram. The coefficient of variation (CV) of the retention times for the 18 amino and imino acids was 0.73% (n = 5). The relative peak heights of the equimolar amounts of the two types of acids are shown in Table 11. The peak height of proline is the largest while that of tyrosine is the smallest. A calibration curve for each amino and imino acid tested shows good linearity over the range from 50 to 2500 pmol with a peak height ratio of the internal standard, norleucine. The CV of the relative peak heights of 625 pmol of all the acids was 3.06% (n = 5). Detection limits were about 5 pmol for proline (the smallest) and 50 pmol for tyrosine (the largest) at the signal to noise ratio of 2. A chromatogram obtained from one blood disk (3 mm id., containing 2.6-2.8 WLof blood) of a normal newborn is shown
ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983
Ollli"
20;
1789
-
40
BO
Flgure 6. Amino and imino acid elution profile for a blood disk (3 mm i.d.) from a normal newborn. Extraction procedure is noted in the text. Conditions were the same as those give in Fl!gure 2. Abbreviations are given in Figure 5 except for (s)Om.
L
0
rnin
Table 111. Amino and Imino Acid Contents in Blood Disks from Normal Newborns (pmol/L) amino acid Pro Gly Ala Ile Leu Tyr Phe LYS His
no. I
no. 2
no. 3
no. 4
no. Ea
265 370 388
!231 450 330 92 l40 55 59 1220 76
270 360 370 83 155 70
205 400 395 89 123 66 51 190 73
190 393 320 79 157 72 55 20 5
111 14 7 80 71
229 87
68
198 94
Phe
maple syrup urine disease
Val Leu
tyrosinosis
Tkr
1 2 3 4 5 6 4 5 6 7 8 9
2750 2300 1650 400 400 380 1320 1760 590 2130 900 1150
40
60
20
40
60
20
40
88
Table IV. Amino Acid Contents Obtained by the NBD-F and OPA Methods content, pM amino sample disease acid no. NBD-F OP A: phenylketonuria
I
20
0
in i n
2720 2280 1510 420 400 360 1330 1690 560 2140 960 1250
in Figure 6. On this chromatogram, the peaks of some amino acids were not completely separated; the peaks of the aspartic acid and asparagine pab and glutamic acid and glutamine pair overlapped each other. The CV of amino and imino acids in the blood disks of a normal newborn was 6.7% (n = 5) and was caused mainly by the sample preparation procedure includiing the punching out of the filter paper; the CV of the weight of the blood disks after cutting from the paper was 4.1%. Decreasing the extraction time decreased the recovery of amino and imino acids. Table I11 shows the contents of the two types of acid in blood disks obtained by the proposed method. These values coincide with those by the other investigators (15). Figure 7 shows the chromatograms obtained from blood disks of inborn errors of' metabolism, such1 as phenylketonuria, maple syrup urine disease, and tyrosinosis. The size of the phenylalanine peak in Figure 7a, the isoleucine and the leucine peaks in Figure 7b, and the tyrosine peak in Figure 7c were increased as compared to a normal profile (Figure 6). Table
L
I
O min
60
Flgure 7. Amino and imino acid elution proflies for blood disks from phenylketonuria (a), mapie syrup urine disease (b), and tyrosinosis (c). The extraction procedure is noted in the text. Conditions are given in Figure 2. Abbrevlations are given in Flgures 5 and 6 except for (t) aiioisoleucine.
IV summarizes the contents of amino acids related to the disease in nine blood samples. The results are in an excellent agreement with those obtained by detection with OPA after separation by conventional ion exchange chromatography (5). Although NBD-F reacts with both amino and imino acids and thiols in an alkaline medium, the thiol adduct (S-NBD) is unstable and S N migration of NBD occurs (16). In the acidic medium, NBD-F reacts only with thiols, giving stronger fluorescence. Therefore, the reaction of NBD-F with thiols,
-
1790
ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983
b
1 I I
I I =
I
I
1
0
10
20
min Elution profile for thiol standards: (a) gluctathione, (b) cysteine, and (c) homocysteine represent 1.0, 0.2, and 1.0 nmol, respectively. Conditions are given as in Figure 3. Figure 8.
which are related to the amino acids tested here, was studied with elution buffer 2 as the eluent (Figure 3). The reaction condition for thiols is selected as shown in Figure 3. Although individual NBD-thiols had different excitation and emission maxima between 430 and 460 and 520 and 540 nm, respectively, we chose 450 and 520 nm, respectively, as a good compromise for the assay of thiols. Figure 8 illustrates the profile of standard glutathione, cysteine, and homocysteine, the retention times of which were 6.1,11.3, and 13.9 min, respectively. The linear relationship between the peak heights and the concentration of cysteine was observed within the range from 0.05 to 10 nmol. The respective detection limits for glutathione, cysteine, and homocysteine were 150,10.0, and 100 pmol. For five runs, the CV of peak heights of glutathione, cysteine, and homocysteine (1.0 nmol each) were 3.1, 2.7, and 3.0%, respectively. This is the first report on the use of NBD-F for the detection of amino and imino acids and thiols in the column effluent. The first advantage of this system is that it detects directly both amino and imino acids owing to their reaction with NBD-F, while fluorescamine (6) and OPA (7) react to give fluorescence with amino acids but not imino acids. In the present work, the detection level of proline is 5 pmol while the other amino acids can be detected a t ca. 20 pmol. According to the flow injection analysis using authentic NBDproline (14,the percentage of derivatization of proline under the selected condition is about 90% while that of NBD-aspartic acid was presumed to less than 30%. The reason that the yield is so low is not clear; reaction time may not be sufficient or the presence of citrate in the reaction medium could affect the reaction yield. The use of recently reported column separation for HPLC with an elution solvent of acetonitrile/phosphate might give a better reaction yield (17). Further examination of the system may result in a more sensitive detection ofamino acids, up to the order of proline. The second advantage of the system is that there is no ammonia peak on the chromatogram (Figure 5) caused by the slow reaction of ammonia with NBD-F. With OPA or ninhydrin, contamination of ammonia in the solvent gives a troublesome peak between lysine and histidine, requiring the purification of buffers and a degassing procedure, which are tedious and time-consuming (18). The third advantage is that the base line increase caused by the hydrolyzed product, in the present case NBD-OH, can be suppressed by the addition of hydrochloric acid in the flow line to lower the pH to reduce its fluorescence (IO). In the case of OPA, background fluorescence (450 nm) cannot be quenched owing to the similarity of the blank fluorophore(s)
with the sample fluorophores. The fourth advantage of this system is that it can be used to detect thiols with a minor modification of the reaction used for amino and imino acid analysis. As shown in Figure 3, the second elution buffer (pH 4.25) has been tentatively adopted for the separation and reaction of low molecular weight thiols such as glutathone, cysteine, and homocysteine since the optimal reaction occurs at an acidic pH of around 4.5. Their reaction rates are so fast that only 14 s was needed. The fluorescence intensities of glutathione and homocysteine were less that that of cysteine, which was almost the same as those of amino acids. Since the intensity of S-NBD is known to be less than that of N-NBD ( I 6 ) ,the reduced fluorescence for glutathione and homocysteine seems to be reasonable. However, in the case of cysteine, because of the S N migration of the NBD moiety (16),the generated N-NBD-cysteine partially affect the larger fluorescence. We are currently investigating the best performance for their reaction to increase sensitivity and to better separate thiols in biological fluids. One of the disadvantages of the system is that it is unable to detect tryptophan on account of its low fluorescence reaction with NBD-F; the reason for this has yet to be clarified. Another disadvantage of the system is that it easily generates bubbles in the flow line, probably due to the usage of ethanol and methanol, although the reaction temperature is at 50 OC. In the present study a Hitachi spectrofluorometer was used, the flow cell of which is a cuvette type unable to tolerate the rather high pressure used in the present experiment. If a different cell were used, suppression of bubble formation with addition of larger back pressure at the end of the flow may be possible. This might lead to a reduction of the base line noise. Guthrie's method which uses some amino acid deficient microorganisms has been adopted for the mass screening of inborn errors of metabolism (19). After the finding of some amino acid abnormalities by Guthrie's method, complete determination of the components and levels of amino and imino acids in the blood is required. Recently, the OPA reaction has been used for this because of its higher sensitivity than ninhydrin, but OPA does not react with imino acids. As indicated in this work, the proposed system may be effective in enabling detection of all the amino and imino acids except tryptophan by using one blood disk of 3 mm in diameter and may also allow the fiiding of other inborn errors of metabolism such as hyperprolinemia (20). In conclusion, the postcolumn detection system may be applicable to other HPLC methods.
-
ACKNOWLEDGMENT The authors express their thanks to Z. Tamura, University of Tokyo, for his interest and support. Thanks are also due to K. Suzuki of the University of Tokyo, H. Naruse of the National Center for Nervous, Mental and Muscular Disorders, and Kyowa Seimitsu Co., Ltd., in Tokyo for their donation of samples and equipment. Registry No. NBD-F, 29270-56-2;Pro, 147-85-3;Gly, 56-40-6; Ala, 56-41-7;Ile, 73-32-5;Leu, 61-90-5;Tyr, 60-18-4;Phe, 63-91-2; Lys, 56-87-1;His, 71-00-1;Asp, 56-84-8;Thr, 72-19-5;Ser, 56-45-1; Glu, 56-86-0;Val, 72-18-4; Met, 63-68-3; Nle, 327-57-1;Arg, 74-79-3; Om, 70-26-8; alloisoleucine, 1509-34-8; glutathione, 70-18-8; cysteine, 52-90-4; homocysteine, 6027-13-0;cystine, 56-89-3. LITERATURE CITED (1) Moore,
S.;Spackman, D. H.; Stein, W.
H. Anal. Chem. 1958, 3 0 ,
1 185-1 190. (2) Benson, J. R.; Hare, P. E. Proc. Narl. Acad. Sci. U . S . A . 1975, 72, 6 19-622. (3) Drescher, M. J.; Media, J. E.; Drescher, D. Anal. Biochem. 1981, 116, 280-286. (4) Udenfriend, S.;Stein, S.; Boehien, P.; Dairman, W.; Leimgruber, W.; Weigele, M. Science 1972, 178, 871-872. (5) Roth, M.; Hampi, A. J. Chromatogr. 1973, 8 3 , 353-356.
Anal. Chem. 1983, 55, 1791-1796 (6) Stein, S.;Boehlen, P.; Dairrnan, W.; Udenfriond, S. Arch. Blochem. Blophys. 1973, 755, 213-220. (7) Roth, M. Anal. Chem. 1971, 4 3 , 880-882. (8) Felix, A. M.; Terkelson, G. Arch. Biochem. Biophys. 1973, 757, 177- 182. (9) Ishida, Y.; Fujita, T.; Asai, K. J . Chromatogr. 1981, 2 0 4 , 143-148. (IO) Irnai, K.; Watanabe, Y. Anal. Chim. Acta 18181, 130, 377-383. (11) Watanabe, Y.; Imai, K. Anal. Blochem. 1981, 166, 471-1172, (12) Watanabe, Y.; Irnai, K. J . Chromatogr. 1982, 2 3 9 , 723-732. (13) Nunno. L. D.; Florio, $5.; Todesco, P. E. J Chem. Soc. C 1970, 1433-1 434. (14) Toyo'oka, T.; Watanabia, Y.; Imai, K. Anal. Chim. Acta 1983, 749, 305-312.
1791
(15) Rossini, A. A,; Aoki, T. T.; Ganda, 0. P.; Soeldner, J. S.; Gahill, G. F. Metabolism 1975. 2 4 . 1185-1192. (18) Biket, D. J.; Price, N. C.; Radda, G. K.; Salmon, A. G. FEBS Lett. 1970, 6,346-348. (17) Schuster, R . Anal. Chem. 1980, 5 2 , 617-620. (18) Hamilton, P. H. Anal. Chem. 1963, 35, 2055-2064. (19) Guthrie, R.; Susi, A. Pediatrics 1983, 32, 338-343. (20) Scriver, C. R J . Clin. Invest. 1964, 43,374-385.
RECEIVED for review February 16, 1983. Accepted June 1, 1983.
Separation of Hydroaromatic Compounds in Synfuels by Picric Acid Columns Timothy J. Wozniak rand Ronald A. :Bites* School of Public and Environmental Aiffairs and Department of Chemistry, Indiana University, Bloomington, Indiana 47405
A separation method foir the Isolation of hydroaromatlc compounds from synthetic fuiels was developed The method uses a dual column of plcrlc acld coated alumina over alumina to achieve class separatlon Into allphatlc hydrocarbons, hydroaromatic compounds, polycycllc aromatlc: hydrocarbons, and nitrogen-contalnlng polycyclic hydrocarbons. Column parameters, lncludlng percentage plcrlc acld and water, were optlmlred. The procedure was applled to LI moderately hydrotreated coal liquid prodluct.
Recent energy shortages have prompted the development of synthetic liquid fuek from coal. The increased aromatic and heteroatomic content of these synthetiic fuels, as compared to petroleum, has raised some concern about their toxicity and mutagenicity (1-3). Identification and elimination of specific mutagenic compounds have led to process changes which reduce the levels of primary aromatic amines (PAA) and polycyclic aromatic hydrocarbons (PAH[) ( 4 , 5). Wilson et al. found that fractional distiillation removed the PAA and the larger PAH, but a t the sacrifice of end product yield ( 5 ) . Additional hydrotreatment, however, reduced the PAA and PAH content while upgrading the quality of the fuel. This extra hydrotreatment dramatically increased the levels of partially hydrogenated polycyclic aromatic hydrocarbons (hydroaromatics); deplending on the amount of hydrotreatment, the hydroaromatic fraction increased from 40% (w/w) up to 80% (w/ur) of the end product (4). Thus, hydroaromatics are a major class of compounds in synfuels; unfortunately, they are relatively unstudied in terms of their analytical data (retention indexes, mass spectra, and NMR spectra) and biological (activity (acute toxicity and mutagenicity). The latter is significant; initial studies of the biological activity of selected hydroaromatics have shown many of them to be acutely toxic at parts-per-million levels (6). Knowledge of the isomeric composition of hydroaromatics is also important from the viewpoint of process development and production of synfuels. Hydroaromatic structures are well-known hydrogen donors (7,8). Certain isomers donate up to half their hydrogen atoms, leaving fully aromatic com-
pounds which are hydrotreated to regenerate the original species (9). A better understanding of the isomeric composition of hydroaromatic compounds could be used to improve the efficiency of liquefaction processes. The complete chemical and biological characterization of hydroaromatics requires suitable class fractionation to remove interfering aliphatics, PAH, and PAA. Previously reported separation methods for synfuels are based on resolution of the fully aromatic, nonvolatile components into discrete fractions. Guerin has used Sephadex LH-20 to separate aliphatics, aromatic hydrocarbons, and nitrogen heterocyclic compounds in synfuels (IO). High-pressure liquid chromatography on C18and amino-bonded phases has been used to separate fully aromatic PAH and PAA by carbon number and aromatic ring number, respectively (11-13). Later et al. reported the modification of a chromatographic procedure using neutral alumina to fractionate several synfuels (14). The major classes were saturated hydrocarbons, aromatic hydrocarbons, nitrogen compounds, and hydroxyl compounds. Chargetransfer agents absorbed on silica gel had been used for separation of saturates and aromatics as early as 1949 by Goldewicz (15). Several researchers have used trinitrofluorenone, trinitrobenzene, or silver nitrate coated on silica gel to achieve similar results (16-19). Several problems exist with the previously described, separation schemes. Most of the methods are based on separation of planar aromatic molecules and involve analysis of the nonvolatile portion of the fractions. Because hydrogenation tends to increase the volatility and distort the planar nature of the resulting compounds, there has been considerable crossover of saturates, hydroaromatics, and aromatics using previous techniques. The use of complexing agents on silica gel offers better resolution but is not amenable to gradient elution because polar solvents remove the complexing agent before adequate fractionation can be achieved. Isocratic elution with nonpolar solvents tends to increase overlap of the hydroaromatic and aromatic components and requires substantial time for elution of all components. This paper will discuss the development of a procedure for the separation and characterization of hydroaromatic compounds in synfuels. We will show that picric acid coated on
0003-2700/83/0355-1791$01.50/0 @ 1983 American Chemical Society
