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Simultaneous determination of iodate and periodate by capillary zone electrophoresis: Application to carbohydrate analysis
177,62-66
ANALYTICALBIOCHEMISTRY
(1989)
Simultaneous Determination of lodate and Periodate by Capillary Zone Electrophoresis: Application to Carbohydrate Analysis Susumu Honda,’ Kenji Suzuki, and Kazuaki Kakehi Faculty of Pharmaceutical Sciences,Kinki University, 3-4-l Kowakae, Higashi-Osaka,Japan
Received
July
28,1988
MATERIALS Iodate and periodate were rapidly (in 11 min) separated from each other with high column efficiency by capillary zone electrophoresis, using a fused silica tube (50 pm i.d., 80 cm) and 100 mM acetate buffer, pH 4.5, as carrier. On-column uv detection at 222 nm allowed sensitive detection down to the picomole level, and measurement of relative peak area to that of pyromellitic acid (internal standard) enabled reproducible determination of these ions. This method was proved useful for periodate oxidation analysis of various carbohydrates. 0 1999
Academic
Press,
Inc.
Periodate oxidation analysis is one of the chemical methods for elucidation of carbohydrate structure. Although it is less widely used than methylation analysis, it is still important, since periodate consumption, as well as iodate formation, provides useful information on the number of vicinal dihydroxyl groups. Periodate consumption can be easily determined by titrimetric (1,2), photometric (3,4), polarographic (5), or potentiometric (6) methods. Estimation of iodate formation, however, is difficult in the presence of an excess of periodate. The only available method is isotachophoresis (7,8). Recently a more powerful technique, i.e., capillary zone electrophoresis (CZE),2 initiated by Mikkers et al. (9), has become accessible for separation of ions, and the authors also have demonstrated its usefulness in simultaneous determination of some aromatic carboxylic acids (10,ll) and carbohydrate-borate complexes (12). In the present paper we propose the use of CZE for simultaneous analysis of iodate and periodate in periodate oxidation analysis of carbohydrates. 1 To whom all correspondence should be addressed. 2 Abbreviation used: CZE, capillary zone electrophoresis.
AND
METHODS
Chemicals. Sodium iodate and sodium metaperiodate were purchased from Wako Pure Chemicals (Dosho-machi, Higashi-ku, Osaka, Japan) and used without further purification. All other chemicals and carbohydrate samples were of the highest grade commercially available. The buffer solutions used as carrier were prepared by dissolving pellets of sodium hydroxide in various concentrations of acetic acid to adjust the pH to indicated values. Procedure for CZE. This was similar to those in our previous papers (10-12). A Model HER-SOP1 regulated high voltage power supply, capable of generating up to 30 kV in constant voltage mode, was purchased from Matsusada Precision Devices (Kusatsu, Shiga Prefecture, Japan). A JASCO (Hachiohji, Tokyo, Japan) UVIDEC loo-VI uv spectrophotometer was used at 222 nm for monitoring iodate and periodate, as well as the internal standard. A l-cm portion of the polyimine coating of a fused silica capillary tube (50 pm i.d., 80 cm, Scientific Glass Engineering, Melbourne, Australia) was removed by burning at the 20-cm position from the anodic end of the tube, and the transparent portion was fixed on a slit (50 pm X 5 mm), which was placed in the center of the light path. The tube was filled with carrier, and its ends were dipped separately in electrode solutions contained in PTFE reservoirs. Both solutions had the same composition as the carrier, and their levels were maintained identical throughout the work. For sample introduction the cathodic end of the tube was moved to a sample solution, and its level was raised 5 cm higher than those of the electrode solutions. Standing for 5 s in this state allowed introduction of several nanoliters of the sample solution by hydrodynamic pressure. The cathodic end was quickly returned to the electrode solution and CZE was carried out by applying a high voltage in the constant voltage mode.
62 All
0003-2697/89 $3.00 Copyright 0 1989 by Academic Press, Inc. rights of reproduction in any form reserved.
ELECTROPHORETIC
DETERMINATION
1 4 PH
FIG. 1. pH dependence of the retention times of iodate (0) and periodate (o), as well as their resolution (X). Capillary, fused silica (50 pm i.d., 80 cm); carrier, 100 mM acetate buffer (pH 3.5, 4.0, 4.5, 5.0, 5.5); applied voltage, 20 kV; detection, uv absorption at 222 nm.
Periodate oxidation of carbohydrates. In the standard procedure a sample (2 pmol) of a carbohydrate was dissolved in 20 mM sodium metaperiodate in 100 mM acetate buffer, pH 4.5 (1 ml), containing pyromellitic acid (1,2,4,5-benzenetetracarboxylic acid, internal standard) to a concentration of 2 mM, and the solution was kept at 25°C. After indicated intervaIs of time several nanoliter portions of the solution were introduced to the capillary tube as described above, and iodate and periodate were determined by CZE from their relative peak area to pyromellitic acid. In smaller scale experiments sample amount and volume of the reagent solution were reduced in the same proportion, andperiodate oxidation was performed in a small tapering tube (3 mm i.d., 2 cm). RESULTS
AND
OF
IODATE
AND
63
PERIODATE
tion was so high that a sample introduced to the anodic end of the capillary tube was forced back into the anodic solution in the reservoir, when high voltage was applied. Therefore, in this case samples were introduced from the cathodic end, and the ions were detected near the anode. Electrophoretic migration was toward the anode, and electroosmotic flow opposed to this direction. Hence these ions were moved toward the anode with the velocity of electrophoretic migration subtracted by that of electroosmotic flow. Figure 1 shows the pH dependence of the retention times, T,‘s, of iodate and periodate, together with their resolution, Rs (defined as the difference of retention times divided by average peak width), in 100 mM acetate buffer. It is indicated that the T, values of both ions were increased gradually up to pH 4.5 and rapidly thereafter. Correspondingly Rs was increased at a high rate below pH 4.5 and at a low rate above this pH value. Therefore, selection of pH 4.5 was appropriate. This selection is also reasonable, because periodate oxidation is normally performed at pH 4-5 to avoid non-Malapradian oxidation, which leads to erroneous estimation of the vicinal dihydroxyl groups. We obtained the variation of the electrophoretic mobilities (pep) of both ions and the coefficient of electroosmotic flow (p,,) from the equations 1. L. V-’ (t-’ + to-‘) and 1. L +V-l. t-‘, respectively. In these equations 1, L, and V are the distance between the cathodic end of the capillary tube and the detector (60 cm), tube length (80 cm), and applied voltage (20,000 kV), respectively. The symbol t denotes the retention time of iodate or periodate, and the symbol to designates the time required by methanol to travel distance 1. This could be obtained by inverting the electrodes together with the reservoirs and
DISCUSSION
Separation The principle of CZE has been already reviewed by Jorgenson (13). Our previous papers (10-12) also described it briefly. In most cases of analyzing organic anions, CZE is performed by using a narrow bore capillary tube of fused silica, filled with a neutral or weakly alkaline carrier. Under these conditions rapid electroosmotic flow is generated, which drives anions toward the cathode at a uniform velocity. Each anionic species is pulled back at the same time by electrophoresis depending on charge and size. Since most organic anions have relatively low electrophoretic mobilities, the overall movement is toward the cathode. In CZE of iodate and periodate, however, the velocity of electrophoretic migra-
I 50
I 100 Formality
of
acetic
I 150 acid
(mbl)
FIG. 2. Effect of salt concentration of the retention times, T{s, of iodate (0) and periodate (O), as well as their resolution, Rs (X). Capillary, fused silica (50 pm i.d., 80 cm); carrier, acetate buffer (pH 4.5; concentration 50,75,100,125,150 m&f); applied voltage, 20 kV; detection, uv absorption at 222 nm.
64
HONDA,
SUZUKI,
1
AND
KAKEHI
whereas the pLeovalue continued to increase at a small rate. Under the optimized conditions (100 mM acetate buffer, pH 4.5) iodate (T,, 10.9 min) and periodate (T,., 7.5 min) gave sharp peaks, well separated from each other, as shown in Fig. 3. The use of pyromellitic acid (T,, 9.0 min) as an internal standard was appropriate, since it was eluted between iodate and periodate, and separation from both peaks was satisfactory. The numbers of theoretical plates (defined as square of retention time divided by variance and calculated as 16. Tz. wp2, where w is peak width) of all the ions involved were quite high (iodate, 190,000 at 5 mM; periodate, 40,000 at 5 mM; pyromellitic acid, 80,000 at 2 mM), one order or more higher than ordinary values in high-performance liquid chromatography. Quantification
3
i t
I
0 Retention
time,
10 T, (min)
FIG. 3. Separation of the authentic specimens of iodate and periodate under the optimized conditions. The analytical conditions were the same as those in Fig. 2, except that the concentration in formality of acetate buffer was 100 mM. Sample concentration, 5 mM (iodate and periodate); 2 mM (pyromellitic acid, internal standard). 1, Periodate; 2, pyromeIIitic acid (internal standard); 3, iodate.
by introducing methanol to the anodic end in the reverse state. Application of the same voltage as given to iodate and/or periodate caused migration of methanol to the cathode to give a to signal. It was observed that he0 decreased up to pH 4, but increased above this pH value. The pH dependence of pLe,,for both ions was similar to that of pea, but variation was in a narrow range. The effect of the salt concentration of carrier was such that the velocities of electrophoretic migration of both ions as well as that of electroosmotic flow were first decreased and subsequently increased, giving minimum points at 100 mM, as shown in Fig. 2. The resolution was increased rapidly up to 100 mM and gradually thereafter, although its variation range was narrow. For this reason 100 mM was adopted in the standard procedure. The pep values of both ions were almost unchanged over the whole range of the acetate concentration examined,
As expected from our basic experiments of quantification using cinnamic acid and its analogs as model compounds (lo), relative peak area showed good correlation to sample concentration. As shown in Fig. 4, the plot of relative peak area to pyromellitic acid vs iodate or periodate concentration was linear at least in a common concentration range l-20 mM. The relative standard deviations (rz = 10) of the relative peak areas of iodate and periodate at 10 mM were 3.2 and 2.9%, respectively. The relative peak height method gave less reproducible data, especially in between-run assay. Thus, iodate and periodate could be quantified simultaneously with high accuracy and precision by the relative peak area method. Although the determinable concentration range was relatively high, the minimum amounts introduced into the tube were as small as several picomoles.
10 lodate
FIG. 4.
or perlodate
20 concentration
(mM)
Calibration curves of iodate and periodate. Data of correlation, 2.5, 5, 7.5, 10, 12.5 mM. 0, Iodate (coefficient l , periodate (coefficient of correlation, 0.9994).
points: 1, 0.9995);
ELECTROPHORETIC
.
DETERMINATION
OF
IODATE
AND
65
PERIODATE
1
React,on
i
0----ztime,
Tr
(min)
FIG. 5. Analysis of the reaction mixture of periodate oxidation of xylitol. Reaction time, 2 h. Other reaction conditions are described under Materials and Methods. The reaction mixture was directly introduced to the capillary tube and analyzed under the same conditions as those in Fig. 3. Peak assignment is also the same as that in Fig. 3.
Periodate
Oxidation
Analysis
[hr)
FIG. 6. Time course of the periodate oxidation of xylitol. Data points: 0.25, 0.5,1,2,5,24 h. The reaction conditions and the analytical conditions were the same as those in Fig. 5, except that reaction time was varied as indicated. 0, Iodate formation; 0, periodate consumption.
3
Retention
t>me
of Carbohydrates
The C-C bonds in 1,2-diol and 1,2,3-trio1 compounds are readily cleaved with periodate in aqueous media to yield two aldehyde compounds. In this Malaprade oxidation the periodate consumed is equimolar to the oxidized C-C bond and also to the iodate formed. Since the present method can determine iodate and periodate simultaneously, it was applied to periodate oxidation of various carbohydrates. Figure 5 shows a typical chromatogram of a periodate oxidation mixture of xylitol. In this analysis the reaction mixture was directly introduced to the capillary tube for CZE without any particular cleanup treatments. The elution profile was quite similar to that of an authentic mixture of iodate and periodate (Fig. 3), showing no interference by accompanying substances. Figure 6 depicts the time course of oxidation of xylitol at pH 4.5. It is observed that this compound was rapidly oxidized, consuming periodate and yielding iodate both in theoretical amounts (commonly 4 mol/mol of xylitol). These observations were consistent with our reported data (7), indi-
eating the reliability of the present method. Based on this result various amounts of xylitol were oxidized by the standard procedure for 1 h. The plot of iodate formation vs xylitol concentration gave a straight line at least in the range of 500 nmol-4 pmol, passing through the origin with a high coefficient of correlation (0.9997). Based on this result accurate quantification of xylitol was possible. Although the volume of the reagent solution in the standard procedure was 1 ml, it could be reduced to 10 ~1 with careful handling of sample and reagent solution by using a miniature tapering tube as a reaction vessel. In this case the minimal determinable amount of xylitol was as small as 5 nmol. Table 1 gives additional data for other carbohydrates. When oxidation was performed for 2 h at 25°C in 100
TABLE
1
Periodate Consumption and Iodate Formation in the Periodate Oxidation of SelectedCarbohydrates Periodate Carbohydrate xylito1 Glucitol Xylose Glucose Rhamnose Trehalose Maltose Dextran
consumption (mol/mol)
3.86 4.85 3.92 4.95 4.05 3.99 4.26 1.25
(4) (5) (3,4”) (3,5”) (3,4”) (4) (4,5”) (2)
Iodate formation (mol/mol) 4.02 5.07 3.96 5.02 4.03 3.98 4.29 1.42
Note. The oxidation was carried out for 2 h at 25°C in 100 tate buffer, pH 4.5. The numbers in parentheses are theoretical y Accompanied by hydrolysis of the formyl ester.
(4) (5) (3,4”) (3,5”) (3,4”) (4) (4,5”) (2) acevalues.
mM
66
HONDA,
SUZUKI,
acetate buffer, pH 4.5, and the reaction mixture was analyzed directly by CZE, all monosaccharide samples gave periodate consumption and iodate formation in good agreement with the theoretical values. Since in the oxidation of reducing monosaccharides the intermediate formyl esters were rapidly hydrolyzed and the resultant diols were further oxidized, they gave the numbers having a superscript for the theoretical values. The theoretical values of periodate consumption, accordingly iodate formation, of the 1 + 1 and 1 + 2 linked glucobioses are 4 and 3 mol/mol, respectively, because no formyl esters are formed. The 1 + 3, 1 + 4, and 1 + 6 linked glucobioses will give values more than 3,4, and 5 mol/mol, respectively, the surplus portion being dependent on the ease of hydrolysis of the formyl esters. The present data for trehalose and maltose were almost exactly 4 mol/mol and between 4 and 5 mol/mol, respectively. Assuming that oxidation was complete under these conditions, these are the evidences that these glucobioses were 1 + 1 and 1 + 4 linked. Both periodate consumption and iodate formation of dextran were somewhat lower than the expected values, possibly due to slow oxidation. The foregoing series of experiments of periodate oxidation demonstrated the usefulness of the present mM
AND
KAKEHI
method. Owing to its simplicity and rapidity this CZE method may be used conveniently in structure elucidation and determination of carbohydrates. REFERENCES 1. Malaprade, L. (1928) C. R. Acad. Sci. 186,382-384. 2. Fleury, P., and Lange, J. (1933) J. Pharm. Clin. 17,107-113. 3. Dixon, J. S., and Lipkin, D. (1954) Anal. Chem. 26,1092-1093. 4. Honda, S., Adachi, K., Kakehi, K., Yuki, H., and Takiura,
5.
(1975) And. Chim. Acta 78,492-494. Takiura, K., and Koizumi, K. (1958) 964.
6. Honda,
S., Sudo, K., Kakehi, Acta 77,274-277.
Chim.
7. Honda,
S., Wakasa,
Oka,
K., and
Zasshi
Takiura,
M., and Kakehi,
78,
K. (1975) K. (1979)
961Anal.
J. Chro-
177,109-115.
matogr.
8.
H., Terao,
Yokugaku
K.
S., Hirotsune,
M., and Shigeta,
S. (1979)
Anal.
Biochem.
98,
417-428. 9. Mikkers, F. E. P., Everaerts, F. M., and Verheggen, (1979) J. Chromatogr. 169,11-20. 10. Fujiwara,
S., and Honda,
S. (1986)
And.
Chem.
Th.
P. E. M.
S&1811-1814.
11. Fujiwara, S., and Honda, S. (1987) Anal. Chem. 59,487-490. 12. Honda, S., Iwase, S., Makino, A., and Kakehi, K. (1988) script submitted for publication. 13. Jorgenson, J. W. (1986) Anal. Chem. 58,743A-760A.
manu-
ANALYTICALBIOCHEMISTRY
(1989)
Simultaneous Determination of lodate and Periodate by Capillary Zone Electrophoresis: Application to Carbohydrate Analysis Susumu Honda,’ Kenji Suzuki, and Kazuaki Kakehi Faculty of Pharmaceutical Sciences,Kinki University, 3-4-l Kowakae, Higashi-Osaka,Japan
Received
July
28,1988
MATERIALS Iodate and periodate were rapidly (in 11 min) separated from each other with high column efficiency by capillary zone electrophoresis, using a fused silica tube (50 pm i.d., 80 cm) and 100 mM acetate buffer, pH 4.5, as carrier. On-column uv detection at 222 nm allowed sensitive detection down to the picomole level, and measurement of relative peak area to that of pyromellitic acid (internal standard) enabled reproducible determination of these ions. This method was proved useful for periodate oxidation analysis of various carbohydrates. 0 1999
Academic
Press,
Inc.
Periodate oxidation analysis is one of the chemical methods for elucidation of carbohydrate structure. Although it is less widely used than methylation analysis, it is still important, since periodate consumption, as well as iodate formation, provides useful information on the number of vicinal dihydroxyl groups. Periodate consumption can be easily determined by titrimetric (1,2), photometric (3,4), polarographic (5), or potentiometric (6) methods. Estimation of iodate formation, however, is difficult in the presence of an excess of periodate. The only available method is isotachophoresis (7,8). Recently a more powerful technique, i.e., capillary zone electrophoresis (CZE),2 initiated by Mikkers et al. (9), has become accessible for separation of ions, and the authors also have demonstrated its usefulness in simultaneous determination of some aromatic carboxylic acids (10,ll) and carbohydrate-borate complexes (12). In the present paper we propose the use of CZE for simultaneous analysis of iodate and periodate in periodate oxidation analysis of carbohydrates. 1 To whom all correspondence should be addressed. 2 Abbreviation used: CZE, capillary zone electrophoresis.
AND
METHODS
Chemicals. Sodium iodate and sodium metaperiodate were purchased from Wako Pure Chemicals (Dosho-machi, Higashi-ku, Osaka, Japan) and used without further purification. All other chemicals and carbohydrate samples were of the highest grade commercially available. The buffer solutions used as carrier were prepared by dissolving pellets of sodium hydroxide in various concentrations of acetic acid to adjust the pH to indicated values. Procedure for CZE. This was similar to those in our previous papers (10-12). A Model HER-SOP1 regulated high voltage power supply, capable of generating up to 30 kV in constant voltage mode, was purchased from Matsusada Precision Devices (Kusatsu, Shiga Prefecture, Japan). A JASCO (Hachiohji, Tokyo, Japan) UVIDEC loo-VI uv spectrophotometer was used at 222 nm for monitoring iodate and periodate, as well as the internal standard. A l-cm portion of the polyimine coating of a fused silica capillary tube (50 pm i.d., 80 cm, Scientific Glass Engineering, Melbourne, Australia) was removed by burning at the 20-cm position from the anodic end of the tube, and the transparent portion was fixed on a slit (50 pm X 5 mm), which was placed in the center of the light path. The tube was filled with carrier, and its ends were dipped separately in electrode solutions contained in PTFE reservoirs. Both solutions had the same composition as the carrier, and their levels were maintained identical throughout the work. For sample introduction the cathodic end of the tube was moved to a sample solution, and its level was raised 5 cm higher than those of the electrode solutions. Standing for 5 s in this state allowed introduction of several nanoliters of the sample solution by hydrodynamic pressure. The cathodic end was quickly returned to the electrode solution and CZE was carried out by applying a high voltage in the constant voltage mode.
62 All
0003-2697/89 $3.00 Copyright 0 1989 by Academic Press, Inc. rights of reproduction in any form reserved.
ELECTROPHORETIC
DETERMINATION
1 4 PH
FIG. 1. pH dependence of the retention times of iodate (0) and periodate (o), as well as their resolution (X). Capillary, fused silica (50 pm i.d., 80 cm); carrier, 100 mM acetate buffer (pH 3.5, 4.0, 4.5, 5.0, 5.5); applied voltage, 20 kV; detection, uv absorption at 222 nm.
Periodate oxidation of carbohydrates. In the standard procedure a sample (2 pmol) of a carbohydrate was dissolved in 20 mM sodium metaperiodate in 100 mM acetate buffer, pH 4.5 (1 ml), containing pyromellitic acid (1,2,4,5-benzenetetracarboxylic acid, internal standard) to a concentration of 2 mM, and the solution was kept at 25°C. After indicated intervaIs of time several nanoliter portions of the solution were introduced to the capillary tube as described above, and iodate and periodate were determined by CZE from their relative peak area to pyromellitic acid. In smaller scale experiments sample amount and volume of the reagent solution were reduced in the same proportion, andperiodate oxidation was performed in a small tapering tube (3 mm i.d., 2 cm). RESULTS
AND
OF
IODATE
AND
63
PERIODATE
tion was so high that a sample introduced to the anodic end of the capillary tube was forced back into the anodic solution in the reservoir, when high voltage was applied. Therefore, in this case samples were introduced from the cathodic end, and the ions were detected near the anode. Electrophoretic migration was toward the anode, and electroosmotic flow opposed to this direction. Hence these ions were moved toward the anode with the velocity of electrophoretic migration subtracted by that of electroosmotic flow. Figure 1 shows the pH dependence of the retention times, T,‘s, of iodate and periodate, together with their resolution, Rs (defined as the difference of retention times divided by average peak width), in 100 mM acetate buffer. It is indicated that the T, values of both ions were increased gradually up to pH 4.5 and rapidly thereafter. Correspondingly Rs was increased at a high rate below pH 4.5 and at a low rate above this pH value. Therefore, selection of pH 4.5 was appropriate. This selection is also reasonable, because periodate oxidation is normally performed at pH 4-5 to avoid non-Malapradian oxidation, which leads to erroneous estimation of the vicinal dihydroxyl groups. We obtained the variation of the electrophoretic mobilities (pep) of both ions and the coefficient of electroosmotic flow (p,,) from the equations 1. L. V-’ (t-’ + to-‘) and 1. L +V-l. t-‘, respectively. In these equations 1, L, and V are the distance between the cathodic end of the capillary tube and the detector (60 cm), tube length (80 cm), and applied voltage (20,000 kV), respectively. The symbol t denotes the retention time of iodate or periodate, and the symbol to designates the time required by methanol to travel distance 1. This could be obtained by inverting the electrodes together with the reservoirs and
DISCUSSION
Separation The principle of CZE has been already reviewed by Jorgenson (13). Our previous papers (10-12) also described it briefly. In most cases of analyzing organic anions, CZE is performed by using a narrow bore capillary tube of fused silica, filled with a neutral or weakly alkaline carrier. Under these conditions rapid electroosmotic flow is generated, which drives anions toward the cathode at a uniform velocity. Each anionic species is pulled back at the same time by electrophoresis depending on charge and size. Since most organic anions have relatively low electrophoretic mobilities, the overall movement is toward the cathode. In CZE of iodate and periodate, however, the velocity of electrophoretic migra-
I 50
I 100 Formality
of
acetic
I 150 acid
(mbl)
FIG. 2. Effect of salt concentration of the retention times, T{s, of iodate (0) and periodate (O), as well as their resolution, Rs (X). Capillary, fused silica (50 pm i.d., 80 cm); carrier, acetate buffer (pH 4.5; concentration 50,75,100,125,150 m&f); applied voltage, 20 kV; detection, uv absorption at 222 nm.
64
HONDA,
SUZUKI,
1
AND
KAKEHI
whereas the pLeovalue continued to increase at a small rate. Under the optimized conditions (100 mM acetate buffer, pH 4.5) iodate (T,, 10.9 min) and periodate (T,., 7.5 min) gave sharp peaks, well separated from each other, as shown in Fig. 3. The use of pyromellitic acid (T,, 9.0 min) as an internal standard was appropriate, since it was eluted between iodate and periodate, and separation from both peaks was satisfactory. The numbers of theoretical plates (defined as square of retention time divided by variance and calculated as 16. Tz. wp2, where w is peak width) of all the ions involved were quite high (iodate, 190,000 at 5 mM; periodate, 40,000 at 5 mM; pyromellitic acid, 80,000 at 2 mM), one order or more higher than ordinary values in high-performance liquid chromatography. Quantification
3
i t
I
0 Retention
time,
10 T, (min)
FIG. 3. Separation of the authentic specimens of iodate and periodate under the optimized conditions. The analytical conditions were the same as those in Fig. 2, except that the concentration in formality of acetate buffer was 100 mM. Sample concentration, 5 mM (iodate and periodate); 2 mM (pyromellitic acid, internal standard). 1, Periodate; 2, pyromeIIitic acid (internal standard); 3, iodate.
by introducing methanol to the anodic end in the reverse state. Application of the same voltage as given to iodate and/or periodate caused migration of methanol to the cathode to give a to signal. It was observed that he0 decreased up to pH 4, but increased above this pH value. The pH dependence of pLe,,for both ions was similar to that of pea, but variation was in a narrow range. The effect of the salt concentration of carrier was such that the velocities of electrophoretic migration of both ions as well as that of electroosmotic flow were first decreased and subsequently increased, giving minimum points at 100 mM, as shown in Fig. 2. The resolution was increased rapidly up to 100 mM and gradually thereafter, although its variation range was narrow. For this reason 100 mM was adopted in the standard procedure. The pep values of both ions were almost unchanged over the whole range of the acetate concentration examined,
As expected from our basic experiments of quantification using cinnamic acid and its analogs as model compounds (lo), relative peak area showed good correlation to sample concentration. As shown in Fig. 4, the plot of relative peak area to pyromellitic acid vs iodate or periodate concentration was linear at least in a common concentration range l-20 mM. The relative standard deviations (rz = 10) of the relative peak areas of iodate and periodate at 10 mM were 3.2 and 2.9%, respectively. The relative peak height method gave less reproducible data, especially in between-run assay. Thus, iodate and periodate could be quantified simultaneously with high accuracy and precision by the relative peak area method. Although the determinable concentration range was relatively high, the minimum amounts introduced into the tube were as small as several picomoles.
10 lodate
FIG. 4.
or perlodate
20 concentration
(mM)
Calibration curves of iodate and periodate. Data of correlation, 2.5, 5, 7.5, 10, 12.5 mM. 0, Iodate (coefficient l , periodate (coefficient of correlation, 0.9994).
points: 1, 0.9995);
ELECTROPHORETIC
.
DETERMINATION
OF
IODATE
AND
65
PERIODATE
1
React,on
i
0----ztime,
Tr
(min)
FIG. 5. Analysis of the reaction mixture of periodate oxidation of xylitol. Reaction time, 2 h. Other reaction conditions are described under Materials and Methods. The reaction mixture was directly introduced to the capillary tube and analyzed under the same conditions as those in Fig. 3. Peak assignment is also the same as that in Fig. 3.
Periodate
Oxidation
Analysis
[hr)
FIG. 6. Time course of the periodate oxidation of xylitol. Data points: 0.25, 0.5,1,2,5,24 h. The reaction conditions and the analytical conditions were the same as those in Fig. 5, except that reaction time was varied as indicated. 0, Iodate formation; 0, periodate consumption.
3
Retention
t>me
of Carbohydrates
The C-C bonds in 1,2-diol and 1,2,3-trio1 compounds are readily cleaved with periodate in aqueous media to yield two aldehyde compounds. In this Malaprade oxidation the periodate consumed is equimolar to the oxidized C-C bond and also to the iodate formed. Since the present method can determine iodate and periodate simultaneously, it was applied to periodate oxidation of various carbohydrates. Figure 5 shows a typical chromatogram of a periodate oxidation mixture of xylitol. In this analysis the reaction mixture was directly introduced to the capillary tube for CZE without any particular cleanup treatments. The elution profile was quite similar to that of an authentic mixture of iodate and periodate (Fig. 3), showing no interference by accompanying substances. Figure 6 depicts the time course of oxidation of xylitol at pH 4.5. It is observed that this compound was rapidly oxidized, consuming periodate and yielding iodate both in theoretical amounts (commonly 4 mol/mol of xylitol). These observations were consistent with our reported data (7), indi-
eating the reliability of the present method. Based on this result various amounts of xylitol were oxidized by the standard procedure for 1 h. The plot of iodate formation vs xylitol concentration gave a straight line at least in the range of 500 nmol-4 pmol, passing through the origin with a high coefficient of correlation (0.9997). Based on this result accurate quantification of xylitol was possible. Although the volume of the reagent solution in the standard procedure was 1 ml, it could be reduced to 10 ~1 with careful handling of sample and reagent solution by using a miniature tapering tube as a reaction vessel. In this case the minimal determinable amount of xylitol was as small as 5 nmol. Table 1 gives additional data for other carbohydrates. When oxidation was performed for 2 h at 25°C in 100
TABLE
1
Periodate Consumption and Iodate Formation in the Periodate Oxidation of SelectedCarbohydrates Periodate Carbohydrate xylito1 Glucitol Xylose Glucose Rhamnose Trehalose Maltose Dextran
consumption (mol/mol)
3.86 4.85 3.92 4.95 4.05 3.99 4.26 1.25
(4) (5) (3,4”) (3,5”) (3,4”) (4) (4,5”) (2)
Iodate formation (mol/mol) 4.02 5.07 3.96 5.02 4.03 3.98 4.29 1.42
Note. The oxidation was carried out for 2 h at 25°C in 100 tate buffer, pH 4.5. The numbers in parentheses are theoretical y Accompanied by hydrolysis of the formyl ester.
(4) (5) (3,4”) (3,5”) (3,4”) (4) (4,5”) (2) acevalues.
mM
66
HONDA,
SUZUKI,
acetate buffer, pH 4.5, and the reaction mixture was analyzed directly by CZE, all monosaccharide samples gave periodate consumption and iodate formation in good agreement with the theoretical values. Since in the oxidation of reducing monosaccharides the intermediate formyl esters were rapidly hydrolyzed and the resultant diols were further oxidized, they gave the numbers having a superscript for the theoretical values. The theoretical values of periodate consumption, accordingly iodate formation, of the 1 + 1 and 1 + 2 linked glucobioses are 4 and 3 mol/mol, respectively, because no formyl esters are formed. The 1 + 3, 1 + 4, and 1 + 6 linked glucobioses will give values more than 3,4, and 5 mol/mol, respectively, the surplus portion being dependent on the ease of hydrolysis of the formyl esters. The present data for trehalose and maltose were almost exactly 4 mol/mol and between 4 and 5 mol/mol, respectively. Assuming that oxidation was complete under these conditions, these are the evidences that these glucobioses were 1 + 1 and 1 + 4 linked. Both periodate consumption and iodate formation of dextran were somewhat lower than the expected values, possibly due to slow oxidation. The foregoing series of experiments of periodate oxidation demonstrated the usefulness of the present mM
AND
KAKEHI
method. Owing to its simplicity and rapidity this CZE method may be used conveniently in structure elucidation and determination of carbohydrates. REFERENCES 1. Malaprade, L. (1928) C. R. Acad. Sci. 186,382-384. 2. Fleury, P., and Lange, J. (1933) J. Pharm. Clin. 17,107-113. 3. Dixon, J. S., and Lipkin, D. (1954) Anal. Chem. 26,1092-1093. 4. Honda, S., Adachi, K., Kakehi, K., Yuki, H., and Takiura,
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