Anal. Chem. 1986, 58, 1375-1379
here has the great advantage to be more selective without the necessity to separate the fluorescent byproducts from the ALA derivative, combined with the highest sensitivity ever reported for the determination of ALA (detection limits: Ehrlich's reagent, 4 nmol (14); 2-amino-3-naphtho1, 40 pmol (16); OPA/ME, 0.8 pmol). The OPA reagent has the outstanding property of being nonfluorescent until it reacts with the amines (21). A disadvantage may be the still potentially reactive excess of OPA remaining in solution. As indicated by the additional peak at 242 nm in the UV spectrum of the reaction product (Figure 2), the excess OPA reacts with 2-mercaptoethanol to form a 1:l hemimercaptal (22). Nevertheless it is unnecessary to remove this product prior to injection into the HPLC system. In his first publication, Roth ( I ) considered the possibility that the same fluorescent species is produced regardless of the nature of the primary amine. The reaction product has not been isolated up to now, but its possible structure has been extensively discussed by Simons and Johnson (3,22),who identified the structure of the fluorescent adduct as an 1-alkylthio-2-dkyl-substituted-isoindole. In the case of the reaction of OPA/ME with ALA, no information about the structure of the fluorogen is available. In view of the drastically different reaction conditions used in this work (high temperature, long time) and the presence of a reactive carbonyl group, it seems unlikely that a simple alkyl-substituted isoindole has been formed with ALA, OPA, and ME. Further discussion of the product structure should therefore wait on its isolation or characterization. On the other hand, temperatures above 80 OC gave rise to two additional peaks in the elution profile (peaks 1 and 2 in Figure 6). Since isoindoles are quite reactive, these peaks may result from derivatives formed from OPA and ME at elevated temperatures. As Figure 4 indicates, the reaction product of ALA with OPA/ME is very sensitive toward oxygen and light. The adduct has been shown to be stable at room temperature in borate buffer of pH 10.0 for up to 1.5 h, but it decomposed to about 50% within 24 h. Investigations on the structure of the decomposition products suggested that the fluorescent adduct had undergone a spontaneous, albeit slow, intramolecular sulfur-to-oxygen rearrangement (3). This decompo-
1375
sition can easily be prevented by storing the reaction product under nitrogen in the dark. As a fluorometric reagent, OPA is particularly suited for automated use (20) and can be applied for the determination of picomolar amounts of amino compounds (8) like amino acids, primary amines, peptides (8), and recently even as a fluorescence probe of aldolase active site (23). With the new method presented here, the OPA reagent might also be useful as an additional tool for elucidating the biosynthetic pathway to ALA and the chlorophylls in green plants. Registry No. ALA, 106-60-5; OPA, 643-79-8; ME, 60-24-2.
LITERATURE CITED (1) Roth, M. Anal. Chem. 1971, 43, 880-882. (2) Cronin, J. R.; Hare, P. E. Anal. Biochem. 1977, 81, 151-156. (3) Simons, S. S.,Jr.; Johnson, D. F. J. Am. Chem. SOC. 1976, 9 8 , 7098-7099. (4) Slmons, S. S., Jr.; Johnson, D. F. Anal. Biochem. 1977, 8 2 , 250-254. (5) Larsen, B. R.; West, F. 0. J. Chromatogr. Sci. 1981, 19, 259-265. (6) Roth, M. J. Clin. Chem. Clin. Biochen,. 1976, 14, 361-364. (7) Benson, J. R.; Hare. P. E. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 619-622. (8) Lee, L. S.;Drescher, D. G. Int. J . Biochem. 1978, 9 , 457-467. (9) Cronin, J. R.; Pizarello, S.;Gandy, W. Anal. Biochem. 1979, 9 3 , 174-179. 10) Granlck, S.; Sassa, S. I n "Metabollc Pathways"; Vogel, H. J., Ed.; Academic Press: New York, 1971; Vol. 5, pp 77-141. 11) Gorchein, A. Biochem. J. 1984, 219, 883-889. 12) Granick, S.;Beale, S. I . I n "Advances in Enzymology"; Meister, A,, Ed.; Wlley: New York, 1978; Vol. 46, pp 33-204. (13) Castelfranco, P. A.; Beale, S. I.Ann. Rev. Plantfhysiol. 1983, 34, 241-278. (14) Mauzerall, D.; Granick, S . J. Biochem. 1956, 219, 435-446. (15) Dalgliesh, C. E. Biochem. J. 1952, 5 2 , 3-14. (16) Melsch, H.-U.; Reinle, W.; Wolf, U. Anal. Blochem. 1985, 149, 29-34. (17) Meisch, H.-U.; Bielig, H.J. Arch. Microbiol. 1975, 105, 77-82. (18) Lindroth, P.; Mopper, K. Anal. Chem. 1979, 5 1 , 1667-1674. (19) Roth, M.; Hampal, A. J. Chromatogr. 1973, 8 3 , 353-356. (20) Bohlen, P.; Schroeder, R. Anal. Biocbem. 1982, 126, 144-152. (21) Chen, R. F.; Scott, C.; Trepman, E. Biochlm. Biophys. Acta 1979, 576, 440-455. (22) Simons, S. S., Jr.; Johnson, D. F. J. Org. Chem. 1978, 43, 2886-2891. (23) Palczewski, K.; Hargrave, P. A.; Kochman, M. Eur. J. Biochem. 1983, 127, 429-435.
RECEIVED for review November 8, 1985. Accepted January 13, 1986.
High-Performance Liquid Chromatographic Determination of Electrically Neutral Carbohydrates with Conductivity Detection Tetsuo Okada* and Tooru Kuwamoto Department of Chemistry, Faculty of Science, Kyoto University, Sakyo-ku, Kyoto 606, Japan Hlgh-performance liquid chromatographic determination of electrically neutral carbohydrates was carrled out with conductometrlc detectlon. Although these compounds are not detected directly by a conductlvlty detector because of thelr low dissociation, most of thelr borate complexes are strong enough acids to be detected with a conductlvlty detector. Therefore, use of a boric acid solutlon as an eluent permltted these compounds to be determined wlth conductometric detection. Moreover, this method can be applied to simultaneous determination of carbohydrates with organic aclds. However, only a ilmlted number of organic acids can be measured together wlth carbohydrates because of the llmltatlon of using a low-acidity eluent. The detectlon limits for carbohydrates typically ranged In 1 X lo-' M levels, and this method can be used for determlning carbohydrates In food samples.
High-performance liquid chromatographic analysis of carbohydrates has been investigated by many workers from the viewpoints of both the separation and detection (1-16). Carbohydrates can be separated with silica gel (1-4), an amino-bonded silica gel (5-9),a cation-exchange resin (7,10-13), and an anion-exchangeresin (14,15). Every stationary phase has particular advantages. For example, silica-based stationary phases are superior in the separation of the structural analogues of carbohydrates, and a cation-exchangeresin permits the use water as a mobile phase for separating many watersoluble carbohydrates. There remain, thus, few problems concerning the separation. On the contrary, there are many problems in the detection as follows: refractive index detection (2, 7-11, 16), which is used generally for detection of carbohydrates, is less sensitive; most of the postcolumn reaction ( I , 12,14,15) and precolumn
0003-2700/88/0358-1375$01.50/00 1986 American Chemical Society
1376
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
derivatization techniques (3-5) need very high reaction temperatures (90-100 "C) and long reaction times (1-10 min). Therefore, electrochemical detection (6,13) or postcolumn detection with mild reaction conditions have been investigated (12). Thus, many problems concerning the detection remain to be solved. On the other hand, a conductivity detector has been developed recently for ion chromatography (17-19) and has become widely used. It can theoretically detect ions in water with detection limits of 1 X M but cannot directly sense electrically neutral compounds. We noticed that poly01 compounds including carbohydrates form ionized complexes with boric acid (20). If a borate complex of a carbohydrate is dissociated to a high degree in an effluent containing boric acid of low conductance, it should be detectable by a conductivity detector with high sensitivity. In this paper, the conductometric determination of carbohydrates (D-xylose, D-ribose, D-glucose, D-galaCtOSe, D-fructose, lactose, sucroge, maltose, raffinose, ethylene glycol, glycerol, D-mannitol, and D-sorbitol) after the high-performance liquid chromatographic separation with a cation-exchange resin is investigated. Moreover, possibility of their simultaneous determination with organic acids being retained on a cation-exchange resin similarly to neutral carbohydrates is also described.
EXPERIMENTAL SECTION Apparatus. The chromatographic system was composed of a computer-controlled pump, an injection valve equipped with a 45-pL sample loop, a conductivity detector, a Toyo Soda UV detector (UV-8 Model 11,at 200 nm), and two separation columns TSK-gel SCX (packed with H+-form cation-exchange resin 7.8 mm i.d. X 300 mm). The pump, the injection valve, and the conductivity detector are parts of a Toyo Soda nonsuppressed ion chromatograph, Model HLC-601. The columns and the conductivity detector were kept at 35 OC. The flow rate was 0.8 mL/min with a pressure of 40 kg/cm2. Reagents. An eluent was prepared by dissolving analytical grade boric acid in distilled deionized water and deaerating. Standard solutions of carbohydrates(D-XylOSe,D-ribose,D-glUCOSe, D-galaCtOSe, D-fructose, lactose, sucrose, maltose, D-mannitOl, D-sorbitol,glycerol, ethylene glycol, methanol, and ethanol) and organic acids (formic, acetic, glycolic, lactic, pyruvic, oxalic, malonic, maleic, fumaric, succinic, malic, tartaric, citric, gluconic, and D-a-galacturonic acids) were prepared by dissolving their analytical grade reagents in the eluent. Pretreatment columns for food samples were prepared by packing a Dowex 50WX2 cation-exchangeresin (100-200 mesh; H+form) and an Amberlite IRA-400 anion-ekchange resin (particle size, 0.4-0.53 mm; C1form) in glass columns (7 mm i.d. X 80 mm) and rinsing them adequately with 0.1 M HCl and water. Food Samples. Wine was diluted to a 10-fold volume with a boric acid solution so as to adjust the concentration of boric acid to that in the eluent. A milk sample was centrifuged and filtered with a Millipore filter (Type HA, pore size 0.45 Wm) after the same dilution as a wine sample. These samples were used after pretreatments with the anion-exchange column and cation-exchange column mentioned above. RESULTS AND DISCUSSION Response of a Conductivity Detector for Carbohydrates. Poly01 compounds (P) including carbohydrates react with boric acid (B) as shown KP
P + B S P B PB
+ H 2 0 & PBOH- + H+
(1)
where K and K , are the formation constant of the complex (PB) and its dissociation constant, respectively. The dissociation constant of P B is generally larger than that of boric acid (pK, = 9.2), e.g., the value is pK, = 4.5 in the presence of 0.5 M fructose and pK, = 5.3 in the presence of 4 M glycerol
(17). This complexation has been utilized for anion-exchange chromatographic separation of sugars (14,15), titration of boric acid (20), and pH-gradient elution of peptides and amino acids
(21).
If it is assumed that polymerization of boric acid can be ignored, background conductance ( K B ) of the eluent, the concentration of which is C B (mol/L), is given by KB
=
(AH+
+ ~B-)CBKB,/[H+]~OOO
(2)
where AH+ and AB- are limiting ion equivalent conductance of hydrogen and boric acid ions and KBais a dissociation constant of boric acid. Since many carbohydrates are very weak acids (e.g., pK, = 12 for fructose), they are not dissolved in an acidic solution. When C, (mol/L) of a carbohydrate is injected into the stream of a boric acid eluent, conductance in a sample band ( K B ) is given by the following equation: K,
+
= [(AH+ -k AB-) (CB - [PB] - [PBOH-])KB,/[H+] (AH+ Ap~-)[PBoH-]]/1000 (3)
where XPB- is limiting ion equivalent conductance of the dissociated form of the polyol-borate complex. If CB >> C,, eq 3 can be rewritten to K,
=
[(AH+
+ AB-)C&B~/[H+]+ (AH++ APB-) [PBOH-]]/lOOO (4)
Therefore, response of a conductivity detector is proportional to a difference (AK)between KB and K, AK =
KB
- K,
= (AH+
+ APB-)
[PBOH-]/1OOO
(5)
From this equation, we can see that a poly01 compound is observed by a conductivity detector with sensitivity corresponding to its reactivity with boric acid and the degree of the dissociation of the borate complex. Thus, poly01 compounds having large Kp and K, are detected with high sensitivity and those having small Kpand/or K, are detected only with low sensitivity. When the eluent is very acidic, dissociation of a borate complex of a poly01 compound is reduced. Equation 5 is accordingly rewritten to the following form: AK =
-(AH+
-k AB-)[PB]KB,/[H+]~OOO
(6)
A negative peak is observed in such a case. A change in conductance is generally larger in an aqueous solution than in a nonaqueous solution. When a cation-exchange resin is used for separating carbohydrates, water containing only boric acid can be used as an eluent. However, a mobile phase should contain 70-80% acetonitrile when a silica-based column is used (1-9). Table I shows changes in conductance obtained by adding glucose or fructose to 5 mM boric acid in water and to 10 mM boric acid in 80% acetonitrile water (v/v). Conductivity greatly increases in an aqueous solution compared with in a nonaqueous solution. From this result, sugars can be detected with high sensitivity in an aqueous system. Therefore, a cation-excahnge column, which permits the use of an aqueous eluent, was selected for the present purpose. Elution of Carbohydrates with Boric Acid as an Eluent. Variation of retention behavior of carbohydrates on a cation-exchange resin with the change in the eluent concentration was investigated. Figure 1 shows the retention times of glucose, fructose, lactose, glycerol, and some organic acids retained on a cation exchanger similar to electrically neutral carbohydrates. In this investigation, the retention times were measured by using a UV detector and 0.1-0.8 M boric acid solution as eluents. The retention times of these four carbohydrates are typical of those of the other carboh-
1377
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
Table I. Change in Conductance by Adding Glucose o r Fructose to Boric Acid Solutions concn of sugar,
solution
mM
5 mM H3B03in water
+ fructose
+ fructose + glucose + glucose
10 mM
conduc- increase in tance, conductance, S/cm S/cm
0 5.0 9.9 5.0 9.9
1.20 2.26 3.40 1.25 1.28
1.06 2.20 0.05 0.08
0 5.0 9.9 5.0 9.9
0.62 0.90 1.18 0.65 0.68
0.28 0.54 0.03 0.06
in 80% (v/v)
acetonitrile-water + fructose + fructose + glucose + glucose
Table 11. Variation of Peak Heights of Carbohydrates with the Concentration of Boric Acid carbohydratesa xylose
ribose glucose galactose fructose lactose sucrose maltose raffinose glycerol mannitol sorbitol a
peak height (rS/cm) at the following boric acid concns
Table 111. Detection Limits of Carbohydrates Obtained by the Present Method carbohydrates xylose
ribose glucose galactose fructose lactose glycerol
mannitol sorbitol
detection limita (pg/mL) at the following boric acid concns 0.02 M 0.05 M 0.1 M 0.2 M 0.8 M 5.6 1.1 26 13 0.88 175 43 1.4 0.67
0.05 M
0.1 M
0.2 M
0.4 M
0.8 M
0.124 0.652 0.042 0.056 0.80 0.004 b b b 0.016 0.52 1.04
0.172 0.648 0.051 0.096 1.42 0.010 b b b 0.033 0.76 1.76
0.252 0.920 0.075 0.134 1.92 0.015 b b b 0.046 1.12 2.16
0.292 1.110 0.090 0.136 2.08 0.016 b b b 0.056 1.16 2.44
0.314 0.980 0.091 0.124 2.00 0.013 b b b 0.061 1.14 2.08
0.320 0.830 0.090 0.064 1.92 -0.006 b b b 0.070 0.96 1.60
Concentration is 1 mg/mL in all cases. Not detected.
ydrates. The change in the eluent concentrationhardly affects the elution of carbohydrates, while it strongly affects the retention of organic acids because of increases in degrees of their dissociation accompanied with decreasing the eluent concentration (or increasing eluent pH). These results show that borate complexes of carbohydratesare weakly dissociated. A dissociation constant of a borate complex in the presence of 0.8 M boric acid is roughly estimated as pK = 5.6 for fructose and pK = 7.1 for glucose from comparing their conductometric detection data with that of lactic acid (a dissociation constant means the product of K,, and K,). Sensitivity in Conductometric Determination of Carbohydrates. Table I1 shows the peak heights of carbohydrates studied in the present work. Ethylene glycol, methanol, ethanol, and oligosaccharides except for lactose cannot be detected because they do not form borate complexes or their borate complexes are not dissociated under this condition if they form the complexes. However, the peaks of monosaccharides, sugar alcohols, and lactose can be observed with a conductivity detector. The peaks of the carbohydrates increase in size with increasing eluent concentration and become constant in the concentration range 0.1-0.2 M of the eluent. An increase in the peak size results from an increase in [PBOH-1. This result can be understood by eq 1 and 4. Since the dissociation of the borate complexes of carbohydrates is reduced by decreasing the eluent pH, their peaks decrease a t the eluent concentration of >0.4 M. The most remarkable example can be seen in the case of lactose; a negative peak is observed at the eluent concentration of 0.8 M as predicted by eq 6. It was indicated that lactose had less reactivity with boric acid because of its weak retention on an anion-exchange resin (15). However, these phenomena observed in lactose can be explained by the low degree of dis-
6.8 1.8 22 15 0.96 125 36 1.7 0.82
20 7.7 71 100 3.3 1070 91 6.7 4.0
aDetection limit was defined as a signal-to-noiseratio of 2. Table IV. Comparison of Detection Limits Obtained with Various Detection Methods detection method
0.02 M
5.5 1.5 19 10 0.73 93 30 1.3 0.65
5.8 1.5 20 10 0.70 100 30 1.3 0.57
refractive index UV (precolumn derivertization) UV (postcolumn reaction) electrochemical conductivity
detection limit, M 1x 1X 1x 1X 1x
ref
104-1x 10-3 7 10-8-1 X lo4 4, 5 10-6-1 x 10-4 i,12,14
10-'-1 X 10+-1 x
6, 13
this method
sociation of its borate complex rather than by its low reactivity with boric acid. This consideration may be confirmed by the fact that the other carbohydrates, which have high reactivity with boric acid, also gave negative peaks when eluents acidified by adding phosphoric or sulfuric acid were used. Therefore, under a very low pH condition, the response of a conductivity detector can be described not by eq 5 but by eq 6. Difference in sensitivity between carbohydrates is due to their structures, especially sequence of hydroxyl groups. Carbohydrates easily form the ionized borate complexes when two adjacent hydroxyl groups are closely situated (cis formation) and undissociated complexes when they are distantly situated (trans formation). However, it is not obvious how the sequence of hydroxyl groups and the elution order in anion-exchange chromatography can be related to the order of sensitivity (sorbitol > fructose > mannitol > ribose > xylose > galactose > glucose > glycerol > lactose). An increase in background conductance with increasing eluent concentration cannot be ignored, although boric acid is a very weak acid. Since an increase in background conductance accompanies an increase in base line noise, use of high eluent concentrations increases detection limits of carbohydrates. Table I11 shows changes in the detection limits and Table IV shows a comparison of detection limits obtained with various detection modes. The detection limits typically range from sub-part-per-millionto part-per-million levels (1 X to 1 X M) in the present method. This method corresponds to precolumn derivatization, postcolumn reaction, or electrochemical detection in sensitivity and is superior to these methods in simplicity. Most carbohydrates, especially fructose, ribose, mannitol, and sorbitol, can be detected conductometrically with higher sensitivity than with direct UV or refractive index detection, while conductometric detection of lactose is less sensitive than with direct UV detection. Linear ranges of calibration curves are 0-0.5 mg/mL for fructose and 0-10 mg/mL for glucose, glycerol, and lactose. Peak broadening is observed at more than these concentrations. Relative standard deviations for 2 mg/mL glycerol and 0.2 mg/mL fructose were 1.6% and 3.6%, respectively. Figure 2 shows a typical chromatogram of glucose, lactose, fructose, and glycerol with conductometric detection. If the concentration of boric acid in a sample solution is different
1378
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
2
1
I
{-ace.
--ace.
--ace.
_ _ _ _ _ _ glyceml
__..._ glycerol
_.....fructose
..I.- fructose
-----sue.
____.. gIuc ose --SUC.
__,... glucose
--
tor.
lactose
___ lactose __.
!1i-
_-
--tor
--
L
U
91Y.
glY
SUC.
’
--lac. --fum.
-gly tor’
-L o
30
20
10
min.
Time,
Figure 3. An attempt tp simultaneously detect carbohydrates and organic acids with conductivity detection: (A) conductivity detection, (B) UV detection (full scale: 0.16 AU); eluent, 0.8 M boric acid: peak identiflcation, (1) tartaric acid (25 pg/mL), (2) malic acid (25 pg/mL), (3) lactic acid (25 pg/mL), (4) lactose (20 mglmL), (5) glucose (5 mglmL), (0) fructose (0.2 mg/mL), (7) succinic acid (10 pg/mL), (8) glycerol (5 mg/mL), (9) acetlc acid (25 pg/mL). Other conditions are given in the text. 2
/e /“all.
-
:/n-alo.,tar. i
OM.
., - \ Tale gu.,pyr. ‘cit.
Flgure 1. Retention times of carbohydrates (glucose, glycerol, lactose, and fructose) and organic acids obtained with use of boric acid solutions as eluents: eluent, (1) 0.1 M boric acid (pH 4.92), (2) 0.4 M boric acid (pH 3.954, (3) 0.6 M boric acid (pH 3.59), (4) 0.8 M boric acM (pH 3.44); identification for organic acids, ace., acetic acid; cit., citric acid; for., formic acid; fum., fumaric acid; gal., a-D-galacturonic acid; glu., gluconic acid; lac., lactic acid; male., maleic acid; mall., malic acid; malo., malonic acid oxa., oxalic acid; pyr., pyruvic acid; SUC., succinic acid. I
w u)
c l
t
:I
A I - _.......__. ....k......R.k ._.. k ... B
1 -L
o
10 20 Time, min.
Figure 2. Chromatogram of carbohydrates with conductivity detection: (A) mnducthrity detectbn, (B) UV detection (full scale; 0.16 AU); eluent, 0.1 M boric acid: peak identification, (1) unknown peaks, (2) lactose (2.5 mg/mL), (3) glucose (1.25 mg/mL), (4) fructose (0.05 mg/mL), (5) glycerol (1.25 mg/mL). Other condltlons are given in the text.
from that in an eluent, a vacant peak of boric acid is observed in the eluting position between glucose and fructose. Moreover, unknown peaks appear in the retention time range 12-15 min. These peaks are caused by neutral impurities because they are not removed by pretreatments with cation- or an-
0
10
20
30
Time, min.
Flgure 4. Detection of carbohydrates in wine: (A) conductivity detection, (B) UV detection (full scale: 0.16 AU); eluent, 0.05 M boric acid; peak identification, (1) glucose, (2) fructose, (3) glycerol. Other chromatographic conditions and preparation of the sample are given in the text.
ion-exchange resins. However, their origin is not clear. Simultaneous Analysis of Carbohydrates with Organic Acids. Organic acids are also retained on a cationexchange resin similar to carbohydrates. If an organic acid is dissociated with a high degree in an effluent, it is detected by a conductivity detector. Accordingly, organic acids interfere with determination of carbohydrates using the present method. As shown in Figure 1,carbohydrates can be detected by use of 0.1 M eluent without interferences from organic acids. However, glycolic, formic, acetic, and succinic acids elute in the region of the carbohydrate peaks upon use of eluents of higher concentrations. These interferences can be reduced by treating a sample solution with an anion-exchange resin without loss of carbohydrates. However, results illustrated in Figure 1demonstrate the possibility of simultaneous determination of carbohydrates with organic acids. Many important organic acids in biochemical, medical, and food re-
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
such as milk and wine. Samples were treated with an anion-exchange resin to reduce the interference from organic acids and with a cation-exchange resin to remove cations, except for H+, degrading the separation columns. Carbohydrates were not removed with these pretreatments. The results are shown in Figures 4 and 5. The first large peak is the peak of chloride being replaced by organic acids in the sample during the pretreatment step with the anion exchanger. Glucose, fructose, and glycerol are detected in a wine sample, and lactose and glucose and/or galactose are detected in a milk sample. A few other peaks are also observed, but it is not obvious whether the peaks are caused by either residual organic acids or the other compounds. In conclusion, this method is fairly effective for determination of some electrically neutral carbohydrates.
ll
-L
o
10 Time,
1379
20 rnin
Figure 5. Detection of carbohydrates in milk: (A) conductivlty detection, (6)UV detection (full scale; 0.16 AU); eluent, 0.05 M boric a c e peak identification, (1) lactose, (2) glucose and/or galactose. Other chromatographic conditions and preparation of the sample are given in the text.
search have only weak absorption in UV region. Therefore, an appropriate detection mode has to be used to chromatographically measure organic acids. Since a conductivity detector is sensitive to organic acids, simultaneous conductometric detection of carbohydrates with organic acids will be useful in a number of fields. Organic acids are not separated adequately from one another because there are only small differences in degrees of dissociation between them at pH range 3.4-4. Although use of eluents of lower pH permits better separation of organic acids, organic acids are detected with lower sensitivity and carbohydrates are not detected even if an eluent contains a sufficient amount of boric acid. Figure 3 shows an attempt to simultaneously detect carbohydrates and some organic acids. In this figure, tartaric acid can be distinguished from the other dibasic acids. However, it cannot be identified in an unknown sample containing oxalic, citric, and maleic acids. Since retention times of the unknown peaks mentioned above also lie in the range 12-15 min, organic acids except for acetic, formic, glycolic, fumalic, lactic, and D-a-galacturonic acids cannot be measured by the present method. Application to Food Samples. The present method was applied to determination of carbohydrates in food samples,
ACKNOWLEDGMENT We wish to thank Toyo Soda Manufacturing Co., Ltd., for providing separation columns. LITERATURE CITED (1) Grimble, 0. K.; Barker, H. M.; Taylor, R. H. Anal. Blochem. 1983, 128,422-428. (2) Baust, J. G.; Lee, R. E., Jr.; Rojas, R. R.; Hendrix, D. L.; Friday, D.; James, H. J. Chromafogr. 1983, 261, 65-75. (3) Schwarzenbach, R. J. Chromatogr. 1977, 740, 304-309. (4) Takada, M.; Maeda, M.; TsujI, A. J. Chromatogr. 1982, 244, 347-355. (5) Chen, C.-C.; McGinnis. G. D. Carbohydr. Res. 1983, 722, 322-326. (6) Buchberger, W.; Winsauer, K.; Breltwieser, Ch. Fresenius' 2. Anal. Chem. 1983, 315, 518-520. (7) Brons, C.; Olleman, C. J. Chromatogr. 1983, 259, 79-86. (8) Lochmuller, C. H.; Hill, W. B., Jr. J. Chromatogr. 1983, 264,215-222. (9) Nikolov, 2. L.; Meagher, M. M.; Reiliy, P. J. J. Chromatogr. 1985, 319, 51-51.
(IO) Gouldlng, R. W. J. Chromafogr. 1975, 103, 229-239. (11) McBee, 0. G.; Maness, N. 0. J. Chromatogr. 1983, 264, 474-478. (12) VrBtnq, P.; Brinkman, U. A. Th.; Frel, R. W. Anal. Chem. 1985, 5 7 , 224-229. (13) Watanabe, N.; Inoue, M. Anal. Chem. 1983, 5 5 , 1016-1019. (14) Nordln, P. Anal. Blochem. 1983, 737, 492-498. (15) Ersser, R. S.; Mitchell, J. D. J. Chromatogr. 1984, 307, 393-398. (16) Shaw, P. E.; Wilson, C. W., I11 J. Chromatogr. Sci. 1982, 2 0 , 209-212. (17) Small, H.; Stevens, T. S.; Bauman, W. C. Anal. Chem. 1975, 47, I 801-I 809. (18) Gjerde, D. T.; Frltz, J. S.; Schmuckler, G. J. Chromatogr. 1979, 186, 509-519. (19) Okada, T.; Kuwamoto, T. Anal. Chem. 1985, 5 7 , 829-833. (20) Braman, R. S. Treatise on Analytical Chemlstry; Koithoff, I. M., Elving, P. J., Eds.; Wiley: New York, 1978;Part 11, Vol. IO, pp 3-88. (21) Troitsky, G. V.; Azhitsky, G. Yu. J. Chromatogr. 1985, 324, 285-297.
RECEIVED for review December 4,1985. Accepted February 10, 1986.
