Automated Determination of Carboxylic Acids by Anion-Exchange Chromatography with Specific Color Reaction Yasuhiko Kasai, Takenori Tanimura, and Zenzo Tamura' Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo 7-3- 1, Bunkyo-ku, Tokyo, Japan
Yoshlnori Orawa Central Research Laboratories, Kikkoman Shoyu Co. Ltd., Noda 399, Noda-shi, Chiba-ken, Japan
The speclfic spectrophotometricdeterminationof a carboxyl group Is adopted for the detection in automated anion-exchange chromatography of water-soluble carboxyllc aclds using diluted hydrochloric acid as mobile phase. The method requires simple clean-up procedures for sample preparation. The ease of operation is similar to that of the usual amino add analyzer. The plot of peak area vs. amount of carboxylic aclds was linear between 0.2 and 4 ymol. The method was evaluated on the separatlon of synthetlc mixtures and was applied to the analysis of white wine.
Despite the recent advancement of chromatographic separation of carboxylic acids, the problem of chromatographic determination of water-soluble aliphatic carboxylic acids in biological mixtures remains to be solved. Gas chromatographic analyses are facile and efficient for those carboxylic acids which are extractable into nonpolar organic solvent from aqueous solution, but they require cumbersome sample preparations for hydroxy acids or polyfunctional acids such as those included in the tricarboxylic acid cycle. Since the laborious extraction and derivatization that is essential for gas chromatographic analysis is not required, it has also been extensively studied and automated for the analyses of carboxylic acids (1-16), environmental waters ( I 7, 18),and artificial mixtures (19-28). Optical, chemical, and electrochemical methods of detection were used in these experiments. The uv detector, which has been most widely used in liquid chromatography, was adopted for the analyses of aromatic and unsaturated acids (4,6,8-10, 13,15,20,21).However, it is insensitive to saturated aliphatic acids. A differential refractometer ( 4 , 5 , 2 2 )is responsive to any compound which has a different refractive index from the eluent, but it is seriously inter€ered with by the minor variations of eluting solvent. Kesner (7) introduced the pH indicator method, and it was applied to the analysis of biological fluid on a column of silicic acid. However, this method is interfered with by either acidic or basic compounds, and requires a refined technique for column preparation and elution. The chromic acid oxidation method was devised by Zerfing e t al. (23) and Samuelson et al. (24)for artificial mixtures of hydroxy acids, keto acids, and di- and tricarboxylic acids. In this case, determination is based on the measurement of amount of dichromate consumed or chromium(II1) produced. The cerate oxidation method (14,15) depends upon the fluorescence measurement of cerium(II1) produced from the reaction of cerium(1V). These oxidation methods, however, are not applicable to the persistent carboxylic acids and interfered with other oxidizable compounds. A variety of other monitoring methods have been explored, e.g., conductometric (4), infrared spectrophotometric ( 2 5 ) ,flame ionization (26), high frequency (27),and coulometric (28)methods. However, these methods of detection belong to either acidity measurement or measurement of bulk properties, and are not specific
for carboxyl group in a molecule. Therefore, they are interfered seriously with the presence of other compounds and have limited usefulness for the analysis of biological mixtures. Our previous paper (29) described a spectrophotometric determination of carboxylic acids based upon the reaction sequence of Figure 1. This method is based on the measurement of hydroxamic acid (IV) which is formed through a direct coupling of carboxylic acid (I) and hydroxylamine by aid of dicyclohexylcarbodiimide (DCC)(11).Hydroxamic acid develops a wine-red color with ferric ion in an acidic medium. The absorption spectrum has ,A,, of 500 to 550 nm. In the present paper, the specific color reaction which we have developed is combined with an efficient separation by ion-exchange chromatography. The resulting method was applied for the determination of hydroxyl or polyfunctional carboxylic acids.
EXPERIMENTAL Reagents. Hydroxylamineperchlorate and benzohydroxamic acid were prepared in our laboratory (29).Other compounds were obtained from Kanto Chemical Co. Ltd., Tokyo. A 0.02 M hydroxylamine perchlorate solution, a 0.06 M triethylamine solution and a 0.25 M DCC solution were prepared by dissolving each reagent in anhydrous ethanol or isopropanol. A 0.01 M ferric perchlorate solution was prepared by dissolvingFe(C10&.6H20 in 0.5 M HClOh solution which was prepared by diluting commercial 70% perchloric acid with ethanol or isopropanol. Reagent solutions are stable for at least one month at room temperature when kept in a brown bottle. Materials. Strongly basic anion-exchange resin, Diaion CA 08 (16-20 p ) was obtained from Mitsubishi Kasei Kogyo Co. Ltd., Tokyo. A pump for eluent, a sampling device,flow switchingvalve, connection fittings, thick wall chromatographic tubing (made from Pyrex glass, 0.3-cm i.d. and 100 cm in length, which could contain pressure up to 100 kg/cm2)with a jacket for temperature control and a reaction bath with a thermoregulator were obtained from Kyowa Seimitsu Co. Ltd., Tokyo. Pumps for reagents, a photometric detector with a 10-mmflow cell, and a recorder with multipoint pen which prints one point every 6 s, were obtained from JOEL Ltd., Tokyo. Three ranges of absorbance, that is 0-100%, 70-100%, and 90-100% transmittance full scale, are measurable with the recorder, and two of them were chosen depending on the sample concentration. Column Preparation and Sample Application. The column was slurry-packed with Diaion CA 08 in chloride form. Diluted HCI, 0.15 or 0.2 N, was employed as the mobile phase. While conditioning the column, the effluent was diverted to waste, keeping the detection system unaffected.The inlet pressure was monitored during the operation and maintained below 70 kg/cm2. This limitation permitted use of three columns connected in series (total length, 300 cm) with a flowrate of 0.2 ml/min. The pressure fluctuation due to the pulsation of the pump was within 5 kg/cm2. The column temperature was maintained at 50 f 0.1 "C.Sample solution was applied on the column by a sampling device, which had two loops of Teflon tubing. The volumes of these loops were determined as 55.2 and 73 pl by titration, respectively.
RESULTS AND DISCUSSION Silicic acid column chromatography is most widely used for the separation of carboxylic acids, but it requires a freshly prepared chromatographic column in every analysis. Since ANALYTICAL CHEMISTRY, VOL.
49, NO. 4,
APRIL 1977
655
NR'
6
RCOOH
WR' ( 1 )
IV
+
-
NR' RCOO; NHR'
NHR'
HZNOH
RCONHOH +
O=$ NHR'
(IVI
(I!)
(111)
Fe"
e (RCONHOFe)2'
t
( V )
H' R ' = COHI,
Flgure 1. Reaction sequence of ferrlc hydroxamate formation
easy conditioning and reproducibility are preferable for analysis in automated liquid chromatography, strongly basic anion-exchange polystyrene resin was used. Formate or acetate buffer, which has previously been used for the elution of carboxylic acids from anion-exchange columns (30),could not be used, because the presence of such salts leads to the formation of hydroxamic acids, thus interfering with the detection method reported here. As the consequence, hydrochloric acid is used as eluent in this experiment (31). Illustration of Flow Diagram. The flow scheme (Figure 2) of the method was designed to produce reaction conditions similar to the manual procedure (29). The mixture of carboxylic acids was separated by anion-exchange chromatography with diluted hydrochloric acid as eluent. Hydrochloric acid in the effluent was neutralized by triethylamine which did not interfere with the determination. Triethylamine from Pump 2 was mixed with hydroxylamine perchlorate from Pump 3, and the mixture was fed to the effluent stream through Coil I. The pH value of the resultant solution was between 4.0 and 5.0 which was checked with both bromophenol blue and bromocresol green prior to introducing DCC by Pump 4. The reaction bath temperature was maintained at 50 f 0.1 "C and the length of reaction coil (Coil 111) was 50 m (0.5-mm i.d.). The ferric perchlorate was delivered by Pump 5 to form ferric hydroxamate, and this final mixture cooled in the cooling bath. The absorbance was measured a t 520 nm or 536 nm with a filter photometer. The outlet of the detector was attached to Coil V (0.5-mm i.d. X 10 m) which provided enough back pressure to prevent bubble formation in the flow cell and maintain pulseless flow throughout the system.
Chromatographic Conditions. Elution was made with 0.15 N or 0.2 N hydrochloric acid and the column temperature was maintained a t 50 or 60 "C.The separation of lactic and acetic acid which was difficult at other conditions was accomplished with these concentrations of hydrochloric acid, and temperatures. A typical chromatogram of a synthetic mixture of carboxylic acids is shown in Figure 3. The separation of a mixture of usual polyfunctional acids and simple aliphatic acids is facile with this procedure. Reaction Conditions. In the coupling reaction, the presence of water reduced the yield of hydroxamic acid (29),an obvious disadvantage since the effluent from the column was aqueous hydrochloric acid. To reduce the water content in the reaction mixture, therefore, triethylamine, hydroxylamine, and DCC were dissolved in anhydrous ethanol or isopropanol. By adjusting the flow rates of these reagents, the water content was reduced to less than 10%. Ethanol or isopropanol also acted as good solvent for dicyclohexylurea (V in Figure 1) which tends to precipitate from aqueous solution and plug the narrow Teflon tubing in the flow system. No difference was observed in the yield of hydroxamate, whether the coupling reaction was made in ethanol or isopropanol. The concentrations of the hydroxylamine and DCC were adjusted to reproduce the same conditions as the coupling reaction in manual procedure. The adjustment of pH value was not difficult, because 0.2 M hydroxylamine had buffering action in this region. In the actual analysis, the same concentration and flow rate of triethylamine could be used with either 0.2 or 0.15 N hydrochloric acid. Chloride ion is known to suppress the coloration (29),but this effect was negligible a t the concentration of chloride ion in the present experiment. The length of the reaction coil and the temperature of the reaction was adjusted to produce the highest yield of hydroxamate from the same quantity of benzoic acid. The yield of benzohydroxamic acid was determined by injecting a definite amount of benzoic acid at the bottom of the column, and the per cent yield was calculated from peak area (32) vs. the authentic benzohydroxamic acid treated with same conditions (Table I). Since additional peak broadening in the 50-m reaction coil was not significant and the yield was slightly better, the length of reaction coil was chosen as 50 m. The reaction time is much
Pressure gauge
-?
Ch r om a t o gr aph ic
column
1y-y Coil 11
Waste
pH Check
Flgure 2. Flow diagram of Carboxylic acid analysis
856
1
Coil V Reaction bath
ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
Cooling bath
detector
Waste
Table I. Rate of Benzohydroxamic Acid Formation at Different Reaction Temperature and Reaction Time Tube length (0.5-mm i.d.), m Reaction time, min" I
.
.
o
20
110
Flgure 3.
60
80
100
120
iiio
160
~NI
zoo
220
240
260
zm ntn
Elution curve of a synthetic mixture of water-soluble carboxylic
acids
+
+
Column dimensions: 0.3-cm i.d. X (100 100 100) cm; eluent, 0.2 N HCI; wavelength, 536 nm; sample size, 73 pl; other conditions are the same as in Figure 2. Sample amount, @mol:(1) aspartic, 2.02; (2) gluconic, 3.02; (3)glucuronic, 1.0; (4) pyroglvtamic, 1.0; (5) lactic, 1.0; (6) acetic, 1.0 (7) tartaric, 1.57; (8) malic, 1.6; (9) citric, 2.1; (IO)succinic, 1.3; (11) isocitric, 1.8: (12) +butyric, 1.0; (13) a-ketoglutaric, 1.5
Reaction temp., 50 "C 60 "C 70 "C W112 at 50 O C
10
25
50
1.0
2.6
5.2
46%b 54
65% 66 63 20.1
70% 70 65
57
19.0
21.0
Calculated from tube volume and flow rate (1.86 ml/min). b Yield calculated from peak are (H X Wllz)compared with that
of authentic benzohydroxamic acid treated with the same conditions.
Table 11. Relative Peak Areas of Water-Soluble Carboxylic Acids
2
Acid Acetic Aspartic n -Butyric Citric Formic G1uconic Glucuronic Glutamic G1y co1ic Isocitric
Relative peak area" 1.00
0.43 0.90 0.56
1.19 0.23 0.57 1.01 1.43 0.73
Acid a-Ketoglutaric Lactic Malic Mesotartaric Oxalacetic n-Propionic Pyroglutamic Pyruvic Succinic Tartaric
Relative peak area 0.92 1.24 1.25 1.43
* . .b
0.94 1.08 0.72 1.29 0.97
a Relative peak area = Peak area of one Kmol of acidpeak area of one pmol of acetic acid. Peak area was calculated the same as
0
10
20
Figure 4.
30
40
50
60
70
80
90
100
110
120
1%
140 MIN
Elution curve of white wine
+
Column dimensions, 0.3-cm i.d. X (100 100) cm: eluent, 0.2 N HCI; column temp., 60°C; reaction bath temp., 6OoC; sample size, 55.2 @I; other conditions are the same as in Figure 2. (1) amino acids; (2) lactic acid; (3) acetic acid: (4) tartaric acid: (5)malic acid; (6) citric acid; (7) succinic acid
shorter here than in the manual procedure, but the yield obtained is sufficient for analysis. A difference of yield was not observed a t 50 and 60 OC, thus the reaction temperature was set a t 50 O C , the same as the column temperature. Since the coloration of ferric hydroxamate is reduced at elevated temperature, the reaction mixture was cooled to ambient temperature after the addition of ferric perchlorate. Final concentration of perchloric acid and ethanol or isopropanol became 0.05 N and 90%, respectively, and the conditions were suitable for the determination of ferric hydroxamate. Sensitivity. The limit of determination for various carboxylic acids is approximately 0.2 pmol which is comparable with the sensitive detectors used by Kesner (7) or Tanaka (18). The peak area obtained by multiplying peak height times its width at half height was proportional to the amount of carboxylic acids. One of the factors which determines limits of detection is the molecular extinction coefficient of ferric hydroxamate which is about one thousand. This value was one twentieth of that of Ruhemann purple in ninhydrin reaction. The intensity of coloration for various acids as expressed in relative peak area (Table 11) demonstrated that the absorbance was not proportional to the number of carboxyl groups in a molecule. The difference in the absorbances for various acids is due to the unequal extinction coefficients of individual carboxylic acids and the difference in the yield of the coupling
in Table I. Oxalacetic acid was unstable in the present conditions. Conditions: Eluent, 0.2 N HCl; column temp., 50 "C;reaction temp., 50 "C; column dimensions, 0.3-cm i.d. X (100 + 100) cm; wavelength, 520 nm; other conditions were the same a8 in Figure 2.
reaction. However, the reason for low absorbances of glucuronic acid and gluconic acid seems to be different. The relative absorbances of glucuronic and gluconic acid to acetic acid in manual procedure were 0.825 and 0.355, respectively. However, these ratios further decreased to approximately 65% in the automated analysis. This reduction of relative absorbance in the present system seems to depend on the partial conversion of the acids into lactones in acidic media during chromatographic separation, for esters were inert to the reaction (29).For monobasic acids, the spectra obtained on the reaction products were qualitatively identical with those of authentic hydroxamic acids. But comparison of the spectra could not be made for di- or tribasic acids, since their authentic hydroxamates were not available. The reason for the differences in relative absorbances of various carboxylic acids was not investigated, but the differences were not so serious as to give difficulties for the analytical purpose. Selectivity. As described in the previous paper, all the carboxylic acids, except oxalic acid, studied developed color under the reaction conditions used. The wavelength of maximum absorption of ferric hydroxamate is in the visible region and few compounds which chromatograph similarly on ionexchange columns have an absorption in this region. Consequently, little interference is expected by the presence of colored compounds in the reaction mixture except ferric hydroxamate. Conversely, the uv detector which monitors absorption at 210,254 nm or other wavelengths depends on the ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
657
aromaticity or unsaturation in a molecule which is not specific for carboxylic acids. A variety of compounds present in practical specimens have large extinction coefficients in these ranges and thus cause difficulties when the uv detector is used to monitor carboxylic acids in liquid chromatography except for artificial mixtures. The selectivity of the color reaction is combined with efficient separation by ion-exchange chromatography, and the combination makes it possible to eliminate sample preparation before the analysis of practical samples. This is demonstrated in the analysis of white wine (Figure 4). In this example, 55.2 pl of commercial wine was directly applied on the column. Some of the peaks are tentatively identified by their retention times. Oxalacetic acid was decomposed to pyruvic acid during chromatographic separation under these conditions. Concerning the sample preparation, brief filtration or centrifugation to remove insoluble substances is usually sufficient for juice of fruits and vegetables, fermentation broths, or even various kinds of sauces. In some experiments when biological mixtures like blood plasma are analyzed, deproteinization is preferable.
CONCLUSION Automated liquid chromatography for carboxylic acids has been previously investigated, but there are surprisingly few analyses of biological samples in the literature. The principal reason seems to be lack of a specific color reaction for carboxyl group. On the contrary, the advantages of the present method are 1)little interference by other substances present, 2) wider possibility of selecting chromatographic conditions, 3) minimum sample preparation, and 4)high reproducibility without refined techniques. This method seems to be most suitable for the analysis of practical specimens. The application of the method to various fields including agricultural, clinical, and environmental analyses is in progress in our laboratory.
658
ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
LITERATURE CITED (1) F. A. Isherwood, Biochem. J., 40, 688 (1946). (2) W. A. Bulen, J. E. Varner. and R. C. Burrell, Anal. Chem., 24, 187 (1952). (3) H. G. Wagner and F. A. Isherwood, Analyst (London), 86, 260 (1961). (4) E. Stahl and E. Laub, 2.Lebensm. Unters.-Forsch., 152, 280 (1973). (5) J. K. Palmer and D. M. List, J. Agric. f o o d Chem., 21, 903 (1973). (6) R. A. Henry and J. A. Schmit, Chromatographia, 3, 116 (1970). (7) L. Kesner and E. Muntwyler, Anal. Chem., 38, 1164 (1966). (8) C. D. Scott, J. E. Attrill, and N. G. Anderson, Proc. SOC.Exptl. Biol. Med., 125, 181 (1967). (9) C. A. Burtis and K. S. Warren, Clin. Chem. ( Winston-Salem, N.C.), 14, 290 (1968). (10) C. D. Scott, Clin. Chem. (Winston-Salem, N.C.), 14, 521 (1968). (11) L. A. Barness, G. Morrow Ill, R. E. Nocho, and R. A. Maresca, Clln. Chem. ( Winston-Salem, N.C.), 16, 20 (1970). (12) J. W. Rosevear, K. J. Pfaff, and E.A. Moffitt, Clin. Chem. (Winston-Salem, N.C.), 17, 721 (1971). (13) J. E. Mrochek, W. C. Butts, W. T. Rainey, Jr., and C. A. Burtis, Clin. Chem. ( Winston-Salem, N.C.), 17, 72 (1971). (14) S. Katz and W. W. Pltt, Jr., Anal. Lett., 5, 177 (1972). (15) S. Katz, W. W. Pitt, Jr., and G. Jones, Jr., Clin. Chem. ( Winston-Salem, N.C.), 19, 817 (1973). (16) 0. Forsander and P. Neuenschwander, J. Chromatogr., 5, 515 (1961). (17) H. F. Mueller, T. E. Larson, and M. Ferretti, Anal. Chem., 32, 687 ( 1960). (18) K. Tanaka, Y. Ishihara, H. Sunahara, and E. Mikami, Jpn Anal., 23, 380 (1974). (19) S. Katz and C. A. Burtis, J. Chromatogr., 40, 270 (1969). (20) N. E. Skelly and W. B. Crummett, J. Chromatogr., 55, 309 (1971). (21) J. J. Kirkland, J. Chromatogr. Sci., 7, 361 (1969). (22) K. Shimomura and H. F/ Walton, Anal. Chem., 37, 1012 (1965). (23) R . C. Zerfing and H. Veening, Anal. Chem., 38, 1312 (1966). (24) L. Bengtsson and 0. Samuelson, J. Chromatogr., 61, 101 (1971). (25) C. Yamazaki, N. Nagashima, and T. Takenishi, Anal. Chem., 32, 733 (1960). (26) R. P. W. Scott, D. W. J. Blackburn, and T. Wilkins, J. Gas Chromatogr., 183 (1967). (27) F. Baumann and W. J. Blaedel, Anal. Chem., 28, 2 (1956). (28) Y. Takata and G. Muto, Anal. Chem., 45, 1864 (1973). (29) Y. Kasai, T. Tanimura, and 2. Tamura, Anal. Chem., 47, 34 (1975). (30) P. Jandera and J. Churacek, J. Chromatogr., 86, 351 (1973). (31) R. W. Von Korff, Methods Enzymol., 13, 425 (1969). (32) D. H. Spackman, W. H. Stein, and S. Moore, Anal. Chem., 30, 1190 (1958).
RECEIVEDfor review April 1, 1975. Accepted March 2, 1976.
Yoshlnori Orawa Central Research Laboratories, Kikkoman Shoyu Co. Ltd., Noda 399, Noda-shi, Chiba-ken, Japan
The speclfic spectrophotometricdeterminationof a carboxyl group Is adopted for the detection in automated anion-exchange chromatography of water-soluble carboxyllc aclds using diluted hydrochloric acid as mobile phase. The method requires simple clean-up procedures for sample preparation. The ease of operation is similar to that of the usual amino add analyzer. The plot of peak area vs. amount of carboxylic aclds was linear between 0.2 and 4 ymol. The method was evaluated on the separatlon of synthetlc mixtures and was applied to the analysis of white wine.
Despite the recent advancement of chromatographic separation of carboxylic acids, the problem of chromatographic determination of water-soluble aliphatic carboxylic acids in biological mixtures remains to be solved. Gas chromatographic analyses are facile and efficient for those carboxylic acids which are extractable into nonpolar organic solvent from aqueous solution, but they require cumbersome sample preparations for hydroxy acids or polyfunctional acids such as those included in the tricarboxylic acid cycle. Since the laborious extraction and derivatization that is essential for gas chromatographic analysis is not required, it has also been extensively studied and automated for the analyses of carboxylic acids (1-16), environmental waters ( I 7, 18),and artificial mixtures (19-28). Optical, chemical, and electrochemical methods of detection were used in these experiments. The uv detector, which has been most widely used in liquid chromatography, was adopted for the analyses of aromatic and unsaturated acids (4,6,8-10, 13,15,20,21).However, it is insensitive to saturated aliphatic acids. A differential refractometer ( 4 , 5 , 2 2 )is responsive to any compound which has a different refractive index from the eluent, but it is seriously inter€ered with by the minor variations of eluting solvent. Kesner (7) introduced the pH indicator method, and it was applied to the analysis of biological fluid on a column of silicic acid. However, this method is interfered with by either acidic or basic compounds, and requires a refined technique for column preparation and elution. The chromic acid oxidation method was devised by Zerfing e t al. (23) and Samuelson et al. (24)for artificial mixtures of hydroxy acids, keto acids, and di- and tricarboxylic acids. In this case, determination is based on the measurement of amount of dichromate consumed or chromium(II1) produced. The cerate oxidation method (14,15) depends upon the fluorescence measurement of cerium(II1) produced from the reaction of cerium(1V). These oxidation methods, however, are not applicable to the persistent carboxylic acids and interfered with other oxidizable compounds. A variety of other monitoring methods have been explored, e.g., conductometric (4), infrared spectrophotometric ( 2 5 ) ,flame ionization (26), high frequency (27),and coulometric (28)methods. However, these methods of detection belong to either acidity measurement or measurement of bulk properties, and are not specific
for carboxyl group in a molecule. Therefore, they are interfered seriously with the presence of other compounds and have limited usefulness for the analysis of biological mixtures. Our previous paper (29) described a spectrophotometric determination of carboxylic acids based upon the reaction sequence of Figure 1. This method is based on the measurement of hydroxamic acid (IV) which is formed through a direct coupling of carboxylic acid (I) and hydroxylamine by aid of dicyclohexylcarbodiimide (DCC)(11).Hydroxamic acid develops a wine-red color with ferric ion in an acidic medium. The absorption spectrum has ,A,, of 500 to 550 nm. In the present paper, the specific color reaction which we have developed is combined with an efficient separation by ion-exchange chromatography. The resulting method was applied for the determination of hydroxyl or polyfunctional carboxylic acids.
EXPERIMENTAL Reagents. Hydroxylamineperchlorate and benzohydroxamic acid were prepared in our laboratory (29).Other compounds were obtained from Kanto Chemical Co. Ltd., Tokyo. A 0.02 M hydroxylamine perchlorate solution, a 0.06 M triethylamine solution and a 0.25 M DCC solution were prepared by dissolving each reagent in anhydrous ethanol or isopropanol. A 0.01 M ferric perchlorate solution was prepared by dissolvingFe(C10&.6H20 in 0.5 M HClOh solution which was prepared by diluting commercial 70% perchloric acid with ethanol or isopropanol. Reagent solutions are stable for at least one month at room temperature when kept in a brown bottle. Materials. Strongly basic anion-exchange resin, Diaion CA 08 (16-20 p ) was obtained from Mitsubishi Kasei Kogyo Co. Ltd., Tokyo. A pump for eluent, a sampling device,flow switchingvalve, connection fittings, thick wall chromatographic tubing (made from Pyrex glass, 0.3-cm i.d. and 100 cm in length, which could contain pressure up to 100 kg/cm2)with a jacket for temperature control and a reaction bath with a thermoregulator were obtained from Kyowa Seimitsu Co. Ltd., Tokyo. Pumps for reagents, a photometric detector with a 10-mmflow cell, and a recorder with multipoint pen which prints one point every 6 s, were obtained from JOEL Ltd., Tokyo. Three ranges of absorbance, that is 0-100%, 70-100%, and 90-100% transmittance full scale, are measurable with the recorder, and two of them were chosen depending on the sample concentration. Column Preparation and Sample Application. The column was slurry-packed with Diaion CA 08 in chloride form. Diluted HCI, 0.15 or 0.2 N, was employed as the mobile phase. While conditioning the column, the effluent was diverted to waste, keeping the detection system unaffected.The inlet pressure was monitored during the operation and maintained below 70 kg/cm2. This limitation permitted use of three columns connected in series (total length, 300 cm) with a flowrate of 0.2 ml/min. The pressure fluctuation due to the pulsation of the pump was within 5 kg/cm2. The column temperature was maintained at 50 f 0.1 "C.Sample solution was applied on the column by a sampling device, which had two loops of Teflon tubing. The volumes of these loops were determined as 55.2 and 73 pl by titration, respectively.
RESULTS AND DISCUSSION Silicic acid column chromatography is most widely used for the separation of carboxylic acids, but it requires a freshly prepared chromatographic column in every analysis. Since ANALYTICAL CHEMISTRY, VOL.
49, NO. 4,
APRIL 1977
655
NR'
6
RCOOH
WR' ( 1 )
IV
+
-
NR' RCOO; NHR'
NHR'
HZNOH
RCONHOH +
O=$ NHR'
(IVI
(I!)
(111)
Fe"
e (RCONHOFe)2'
t
( V )
H' R ' = COHI,
Flgure 1. Reaction sequence of ferrlc hydroxamate formation
easy conditioning and reproducibility are preferable for analysis in automated liquid chromatography, strongly basic anion-exchange polystyrene resin was used. Formate or acetate buffer, which has previously been used for the elution of carboxylic acids from anion-exchange columns (30),could not be used, because the presence of such salts leads to the formation of hydroxamic acids, thus interfering with the detection method reported here. As the consequence, hydrochloric acid is used as eluent in this experiment (31). Illustration of Flow Diagram. The flow scheme (Figure 2) of the method was designed to produce reaction conditions similar to the manual procedure (29). The mixture of carboxylic acids was separated by anion-exchange chromatography with diluted hydrochloric acid as eluent. Hydrochloric acid in the effluent was neutralized by triethylamine which did not interfere with the determination. Triethylamine from Pump 2 was mixed with hydroxylamine perchlorate from Pump 3, and the mixture was fed to the effluent stream through Coil I. The pH value of the resultant solution was between 4.0 and 5.0 which was checked with both bromophenol blue and bromocresol green prior to introducing DCC by Pump 4. The reaction bath temperature was maintained at 50 f 0.1 "C and the length of reaction coil (Coil 111) was 50 m (0.5-mm i.d.). The ferric perchlorate was delivered by Pump 5 to form ferric hydroxamate, and this final mixture cooled in the cooling bath. The absorbance was measured a t 520 nm or 536 nm with a filter photometer. The outlet of the detector was attached to Coil V (0.5-mm i.d. X 10 m) which provided enough back pressure to prevent bubble formation in the flow cell and maintain pulseless flow throughout the system.
Chromatographic Conditions. Elution was made with 0.15 N or 0.2 N hydrochloric acid and the column temperature was maintained a t 50 or 60 "C.The separation of lactic and acetic acid which was difficult at other conditions was accomplished with these concentrations of hydrochloric acid, and temperatures. A typical chromatogram of a synthetic mixture of carboxylic acids is shown in Figure 3. The separation of a mixture of usual polyfunctional acids and simple aliphatic acids is facile with this procedure. Reaction Conditions. In the coupling reaction, the presence of water reduced the yield of hydroxamic acid (29),an obvious disadvantage since the effluent from the column was aqueous hydrochloric acid. To reduce the water content in the reaction mixture, therefore, triethylamine, hydroxylamine, and DCC were dissolved in anhydrous ethanol or isopropanol. By adjusting the flow rates of these reagents, the water content was reduced to less than 10%. Ethanol or isopropanol also acted as good solvent for dicyclohexylurea (V in Figure 1) which tends to precipitate from aqueous solution and plug the narrow Teflon tubing in the flow system. No difference was observed in the yield of hydroxamate, whether the coupling reaction was made in ethanol or isopropanol. The concentrations of the hydroxylamine and DCC were adjusted to reproduce the same conditions as the coupling reaction in manual procedure. The adjustment of pH value was not difficult, because 0.2 M hydroxylamine had buffering action in this region. In the actual analysis, the same concentration and flow rate of triethylamine could be used with either 0.2 or 0.15 N hydrochloric acid. Chloride ion is known to suppress the coloration (29),but this effect was negligible a t the concentration of chloride ion in the present experiment. The length of the reaction coil and the temperature of the reaction was adjusted to produce the highest yield of hydroxamate from the same quantity of benzoic acid. The yield of benzohydroxamic acid was determined by injecting a definite amount of benzoic acid at the bottom of the column, and the per cent yield was calculated from peak area (32) vs. the authentic benzohydroxamic acid treated with same conditions (Table I). Since additional peak broadening in the 50-m reaction coil was not significant and the yield was slightly better, the length of reaction coil was chosen as 50 m. The reaction time is much
Pressure gauge
-?
Ch r om a t o gr aph ic
column
1y-y Coil 11
Waste
pH Check
Flgure 2. Flow diagram of Carboxylic acid analysis
856
1
Coil V Reaction bath
ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
Cooling bath
detector
Waste
Table I. Rate of Benzohydroxamic Acid Formation at Different Reaction Temperature and Reaction Time Tube length (0.5-mm i.d.), m Reaction time, min" I
.
.
o
20
110
Flgure 3.
60
80
100
120
iiio
160
~NI
zoo
220
240
260
zm ntn
Elution curve of a synthetic mixture of water-soluble carboxylic
acids
+
+
Column dimensions: 0.3-cm i.d. X (100 100 100) cm; eluent, 0.2 N HCI; wavelength, 536 nm; sample size, 73 pl; other conditions are the same as in Figure 2. Sample amount, @mol:(1) aspartic, 2.02; (2) gluconic, 3.02; (3)glucuronic, 1.0; (4) pyroglvtamic, 1.0; (5) lactic, 1.0; (6) acetic, 1.0 (7) tartaric, 1.57; (8) malic, 1.6; (9) citric, 2.1; (IO)succinic, 1.3; (11) isocitric, 1.8: (12) +butyric, 1.0; (13) a-ketoglutaric, 1.5
Reaction temp., 50 "C 60 "C 70 "C W112 at 50 O C
10
25
50
1.0
2.6
5.2
46%b 54
65% 66 63 20.1
70% 70 65
57
19.0
21.0
Calculated from tube volume and flow rate (1.86 ml/min). b Yield calculated from peak are (H X Wllz)compared with that
of authentic benzohydroxamic acid treated with the same conditions.
Table 11. Relative Peak Areas of Water-Soluble Carboxylic Acids
2
Acid Acetic Aspartic n -Butyric Citric Formic G1uconic Glucuronic Glutamic G1y co1ic Isocitric
Relative peak area" 1.00
0.43 0.90 0.56
1.19 0.23 0.57 1.01 1.43 0.73
Acid a-Ketoglutaric Lactic Malic Mesotartaric Oxalacetic n-Propionic Pyroglutamic Pyruvic Succinic Tartaric
Relative peak area 0.92 1.24 1.25 1.43
* . .b
0.94 1.08 0.72 1.29 0.97
a Relative peak area = Peak area of one Kmol of acidpeak area of one pmol of acetic acid. Peak area was calculated the same as
0
10
20
Figure 4.
30
40
50
60
70
80
90
100
110
120
1%
140 MIN
Elution curve of white wine
+
Column dimensions, 0.3-cm i.d. X (100 100) cm: eluent, 0.2 N HCI; column temp., 60°C; reaction bath temp., 6OoC; sample size, 55.2 @I; other conditions are the same as in Figure 2. (1) amino acids; (2) lactic acid; (3) acetic acid: (4) tartaric acid: (5)malic acid; (6) citric acid; (7) succinic acid
shorter here than in the manual procedure, but the yield obtained is sufficient for analysis. A difference of yield was not observed a t 50 and 60 OC, thus the reaction temperature was set a t 50 O C , the same as the column temperature. Since the coloration of ferric hydroxamate is reduced at elevated temperature, the reaction mixture was cooled to ambient temperature after the addition of ferric perchlorate. Final concentration of perchloric acid and ethanol or isopropanol became 0.05 N and 90%, respectively, and the conditions were suitable for the determination of ferric hydroxamate. Sensitivity. The limit of determination for various carboxylic acids is approximately 0.2 pmol which is comparable with the sensitive detectors used by Kesner (7) or Tanaka (18). The peak area obtained by multiplying peak height times its width at half height was proportional to the amount of carboxylic acids. One of the factors which determines limits of detection is the molecular extinction coefficient of ferric hydroxamate which is about one thousand. This value was one twentieth of that of Ruhemann purple in ninhydrin reaction. The intensity of coloration for various acids as expressed in relative peak area (Table 11) demonstrated that the absorbance was not proportional to the number of carboxyl groups in a molecule. The difference in the absorbances for various acids is due to the unequal extinction coefficients of individual carboxylic acids and the difference in the yield of the coupling
in Table I. Oxalacetic acid was unstable in the present conditions. Conditions: Eluent, 0.2 N HCl; column temp., 50 "C;reaction temp., 50 "C; column dimensions, 0.3-cm i.d. X (100 + 100) cm; wavelength, 520 nm; other conditions were the same a8 in Figure 2.
reaction. However, the reason for low absorbances of glucuronic acid and gluconic acid seems to be different. The relative absorbances of glucuronic and gluconic acid to acetic acid in manual procedure were 0.825 and 0.355, respectively. However, these ratios further decreased to approximately 65% in the automated analysis. This reduction of relative absorbance in the present system seems to depend on the partial conversion of the acids into lactones in acidic media during chromatographic separation, for esters were inert to the reaction (29).For monobasic acids, the spectra obtained on the reaction products were qualitatively identical with those of authentic hydroxamic acids. But comparison of the spectra could not be made for di- or tribasic acids, since their authentic hydroxamates were not available. The reason for the differences in relative absorbances of various carboxylic acids was not investigated, but the differences were not so serious as to give difficulties for the analytical purpose. Selectivity. As described in the previous paper, all the carboxylic acids, except oxalic acid, studied developed color under the reaction conditions used. The wavelength of maximum absorption of ferric hydroxamate is in the visible region and few compounds which chromatograph similarly on ionexchange columns have an absorption in this region. Consequently, little interference is expected by the presence of colored compounds in the reaction mixture except ferric hydroxamate. Conversely, the uv detector which monitors absorption at 210,254 nm or other wavelengths depends on the ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
657
aromaticity or unsaturation in a molecule which is not specific for carboxylic acids. A variety of compounds present in practical specimens have large extinction coefficients in these ranges and thus cause difficulties when the uv detector is used to monitor carboxylic acids in liquid chromatography except for artificial mixtures. The selectivity of the color reaction is combined with efficient separation by ion-exchange chromatography, and the combination makes it possible to eliminate sample preparation before the analysis of practical samples. This is demonstrated in the analysis of white wine (Figure 4). In this example, 55.2 pl of commercial wine was directly applied on the column. Some of the peaks are tentatively identified by their retention times. Oxalacetic acid was decomposed to pyruvic acid during chromatographic separation under these conditions. Concerning the sample preparation, brief filtration or centrifugation to remove insoluble substances is usually sufficient for juice of fruits and vegetables, fermentation broths, or even various kinds of sauces. In some experiments when biological mixtures like blood plasma are analyzed, deproteinization is preferable.
CONCLUSION Automated liquid chromatography for carboxylic acids has been previously investigated, but there are surprisingly few analyses of biological samples in the literature. The principal reason seems to be lack of a specific color reaction for carboxyl group. On the contrary, the advantages of the present method are 1)little interference by other substances present, 2) wider possibility of selecting chromatographic conditions, 3) minimum sample preparation, and 4)high reproducibility without refined techniques. This method seems to be most suitable for the analysis of practical specimens. The application of the method to various fields including agricultural, clinical, and environmental analyses is in progress in our laboratory.
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RECEIVEDfor review April 1, 1975. Accepted March 2, 1976.
