lution was used, the slope of calibration curve was 0.045 ppb-l for the digested solution or 0.0101 ppb-' for the original sample; the linear range is up to 7 ppb and 30 ppb for digested solution and original standard, respectively. The precision of this method is very good. For original standards, the RSD was 1.0% a t 2 ppb, 0.5% a t both 10 ppb and 20 ppb when 4 mL of digested solution was used for analysis. The amount of space in the syringe barrel should be as close to the available volume of the absorption cell as possible. If the amount of space in the syringe barrel is greater than the available volume of the absorption cell, some of the mercury vapor which exists in the air phase will not be transferred into the cell for measurement. If the amount of space in the syringe barrel is less than the available volume of the absorption cell, the maximum available amount of mercury will not be distributed in the air phase for measurement. In both cases, the sensitivity will be reduced. The study presented here provides an AAS method for measuring mercury in liquid samples. This method is sensitive, accurate, precise, and easy to perform. Preconcentration, scale expansion, and background correction are not necessary. Because of its sensitivity, the concentration of mercury can be measured at the 0.1 ppb level in an original sample even with a sample size of 1mL. It is believed that this method can be applied to other kinds of samples which are properly digested.
4
-16
Figure 1. Schematic diagram of experimental arrangement: (1)Teflon insert: (2)glass syringe barrel; (3)Teflon plunger; (4)screw; (5) stopper: (6) polyethylene tubing (0.11 cm Ld.); (7)two way valve; (8) rubber septum: (9) needle: (10) drylng tube filled with Mg(CIO,),; (11)two way valve: (12)absorption cell: (13)burnner head: (14)three way valve: (15)to vacuum pump; (16) to laboratory vacuum line.
septum of the drying tube, valve 14 was closed and then valve 7 opened. The plunger was pushed into the barrel by atmospheric pressure and the mercury vapor was driven into the absorption cell. The maximum absorbance which was reached in a few seconds was recorded. After the measurement, the needle was pulled out from the septum and valve 14 was switched to the laboratory vacuum line. Valve 11was opened for a few seconds to let in room air to flush the inside of the absorption cell. Then valve 14 was switched to connect the absorption cell with the vacuum pump again. The plunger was removed from the barrel, and the barrel was rinsed thoroughly with deionized water and shaken dry. With this procedure, 20 determinations can be made in an hour.
ACKNOWLEDGMENT The authors wish to thank William Gasparac for making the syringe, the plunger, and the absorption cell. LITERATURE CITED (1) Hatch, W. Ronald; Ott, Welland
(2) (3)
RESULTS AND DISCUSSION When 4 mL of digested solution was used, the detection limit was 0.013 ppb or 0.05 ng of Hg and the calibration curve was linear to 3.5 ppb with a- slope-of 0.080 ppb-l for the digested solution and linear to 15 ppb with a slope of 0.0177 ppb-l for the original standard. When 2 mL of digested so-
(4) (5) (6)
L. Anal. Chem. 1968, 40, 2085-2087. Hawley, J. E.: Ingle, J. D., Jr. Anal. Chem. 1975, 47, 719-723. Velghe, N.; Campe, A.; Claey, A. A t . Absurpt. News/. 1978, 17, 37-40. Tong, Soo-Loong Anal. Chern. 1978, 50, 412-414. Bourcler, D. R.; Sharma, R. P. J. Anal. Toxicol. 1981, 5, 65-68. Feldman, Cyrus Anal. Chem. 1974, 46, 99-102.
RECEIVEDfor review September 26,1983. Resubmitted March 19, 1984. Accepted March 21, 1984.
Colorirnetrlc Determination of y-Cyclodextrin Takashi Kato* and Koki Horikoshi
The Institute of Physical and Chemical Research, Wako-shi, Saitama 351, Japan Analysis of cyclodextrin (CD) with high-performance liquid chromatography (HPLC) on a silica derivative containing amino groups, with acetonitrile-water mixtures as the eluent, has been described ( I , @ . However, maltooligosaccharides, such as maltotetraose, maltopentaose, maltohexaose, maltoheptaose, and maltooctaose, interfere with the analysis. To avoid this interference, oligosaccharides which were not converted to C D s should be digested with glucoamylase (3),but this method is somewhat complicated. No simple and specific quantitative analysis for -y-CD has yet been reported. The inclusion of a compound in a CD mixture may cause a change in the absorption spectrum. Such a characteristic spectral change has been reported for the inclusion complex of a dye molecule such as Congo red, methyl orange, and crystal violet with a-CD, (3-CD, and y-CD (4). We found that bromocresol green (BCG) also made a inclusion complex with 0003-2700/84/0356-1738$0 1.5010
y C D having a stronger absorption spectrum than those of other CD's. This paper deals with a rapid and simple colorimetric analysis of y C D by using BCG.
EXPERIMENTAL SECTION Enzymes. The cyclomaltcdextringlucanotransferase (CGTase) (5)of alkalophilic Bacillus sp. No. 38-2 (ATCC 21783) was kindly supplied by Meito Sangyo Co., Ltd. The CGTase of Bacillus macerans (IAM 1243) was prepared according to the method of Kitahata et al. (6). Reagent. a-, p-, and y-CD and CH-30, which is a mixture of a-,p-, 7-CD and maltooligosaccharides, were kindly supplied by Nihon Shokuhin Kako Co., Ltd. (Tokyo). BCG was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo). Analytical Method. A Hitachi spectrophotometer, Model 200-10, was used. The analysis by HPLC was done with a Shi0 1984 American Chemlcai Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 9, AUGUST 1984
1739
a ' 4 I ' 400 500 600 700 800 900 1'
1
Wavelength (nm)
Flgure 1. Absorption spectra of BCG complexes of CDs: (---) aCD (- -) 0-CD (-) y-CD. One milliliter of 1% ( w h ) CD solution was mixed with 0.1 mL of 5 mM BCG and 2 mL of 0.2 M citrate buffer (pH 4.2), and absorbance spectra were measured.
-
L
rng o f T-CD
Flgure 3. Linearity between absorbance and concentration of y-CD: (0)1 mM BCG; (0)2 rnM BCG; (A)4 mM BCG (a)5 mM BCG; (A) 6 mM BCG (O),8 rnM BCG (W) 10 mM BCG. One milliliter of various concentrations of y-CD was mixed with 0.1 mL of various concentrations of BCG in 2 mL of 0.2 M citrate buffer (pH 4.2), and absorbances at 630 nm were measured. Table I. Effect of Various Carbohydrates on the BCG Method amt of carbohydrate, pg 7-CD
(u-CD pCD
440 440 440 440 440 440 440 44 0 440 440 440 440 440 440 440 440 440 440 440 440
0 200 400 4000 0
Wavelength ( n m )
Figure 2. Effect of pH on absorption spectrum of BCG complex of CD: (A) pH 3.0; (6)pH 3.5; (C) pH 4.0; (D) pH 4.5; (E) pH 5.0; (F) pH 5.5. One rnillillter of 1% (w/v) CD solution was mixed with 0.1 mL of 5 rnM BCG in 2 mL of 0.2 M citrate buffer (various pH values), and absorption spectra were measured. Symbols are the same as those in Figure 1. madzu Model LC-3A liquid chromatograph equipped with a s a detector. The Shodex Model SE-11 differential refractometer i column (4.6 X 250 mm) was packed with Nucleosil 5NHz (Macherey-Nagel Co., Germany). The analysis was carried out as follows: eluent, acetonitrile/water (65/35); flow rate, 1.0 mL/min; pressure, 30 kg/cm2; sample size, 5-25 pL; column temperature, 50 "C.
RESULTS AND DISCUSSION Absorption Spectra of the Inclusion Complexes of BCG in CD's. One milliliter of 1% (w/v) CD solution was mixed with 0.1 mL of 5 mM BCG (in 20% ethanol solution) and 2 mL of 0.2 M citrate buffer (pH 4.2). Then the differential absorption spectrum was measured. The results are shown in Figure 1; the addition of a-CD and p-CD did not cause any change in their absorption spectra. However, the inclusion complex of the BCG molecule with y-CD exhibited significant change in the absorption spectrum showing maximum absorbance a t 630 nm. Effect of pH on Absorption Spectrum of BCG-CD Complex. BCG is a pH indicator; therefore, the pH values of the B C G C D complex should be critical in measuring the absorption spectra. One milliliter of 1% (w/v) CD solution was mixed with 0.1 mL of BCG (5 mM) and 2 mL of 0.2 M citrate buffer (pH 3-6), and the absorption spectra were measured. As shown in Figure 2, the absorption maximum of the mixture was 630 nm in the pH range 3.0-4.5, higher pH
a
0 0 0 0 0 0 0 0
200 400 4000 400 200 400 4000
0
0 0 0
200 400 4000 0 0 0 0 0 0
200 400 4000 400 200 400 4000
amt soluble found, % glucose starch pg error a 0 0 0 0 0 0 0 200 400 4000 0 0 0 0 0 0 400 200 400 2000
0 0 0 0 0 0 0 0 0 0 200 400 4000 0 0 0 0 200 400 2000
440 430 470 460 440 460 450 450 440 450 440 450 460 440 440 410 460 430 460 440
-2.3 t 6.8
t4.5 0 t4.5 t 2.3 t 2.3 0 t 2.3
0 t 2.3 t 4.5
0 0
-6.8 t 4.5
-2.3 t 4.5 0
Error ( W ) = (found/(r-CD - 1))x 100.
(5.0-6.0) shifted the spectra to longer wavelength. Absorbance a t 630 nm was relatively low at pH 3.0 and pH 3.5. No significant effect due to the addition of a,P-CD's was observed. Therefore, the analysis of y C D was carried out at pH 4.0. Linearity of Absorbance and Concentration of BCG. One-milliliter portions of various concentrations of 7-CD (up to 1000 pg of -y-CD) were mixed with 0.1 mL of various concentrations of BCG (1mM to 10 mM) and 2 mL of 0.2 M citrate buffer (pH 4.2). Then the absorbance a t 630 nm was measured. As shown in Figure 3, linear relationships were observed up to 600 pg of y-CD under various concentrations of BCG. At low concentration of BCG (1 mM and 2 mM), the increase of absorbance a t 630 nm was relatively low. In contrast at a high concentration of BCG (8 mM and 10 mM), the absorbance of reference solution was too high to determine a t 630 nm. Therefore, the concentration of BCG used for analysis of y C D was from 4 mM to 6 mM. The recommended method for analysis of y C D is as follows: 1mL of sample solution (up to 700 pg as yCD) is mixed with 0.1 mL of BCG (5 mM) and 2 mL of 0.2 M citrate buffer (pH 4.2), and the absorbance is measured a t 630 nm. Effect of Other Carbohydrates on the 7-CD Analysis. In the presence of various carbohydrates, such as a-CD, p-CD, glucose, and soluble starch, the 7-CD analysis using our BCG
Anal. Chem. 1984, 56, 1740-1741
1740
Table 11. Application of the BCG Method for the Determination of ?CD in the Enzymatic Products from Starch for r-CD
Alkalophilic Bacillus sp. NO. 38-2 Bacillus macerans CH-30 a
by HPLC, pg for for a-CD pCD
HPLC,
by
by BCG,
%
kg
pg
errora
1210
1290
t6.6
656
9640
320
350
t9.4
7440
1670
6380
5900
-7.5
6330
12300
Error (%) = (BCG/(HPLC - 1))x 100.
method was tested. Addition of a-CD, 0-CD, glucose, or soluble starch did not cause significant error as shown in Table I. Our new method was not strongly disturbed by the presence of a-CD, 8-CD, and glucose. Although the mixture of high concentrations of a-CD, @-CD,glucose, and soluble starch yielded approximately a 5% error in determining y-CD for this method, it is concluded that the presence of other carbohydrates does not strongly influence the determination of y-CD. Analysis of y-CD in several enzymatic products from starch. Tests were carried out on the enzymatic products of CGTases of alkalophilic Bacillus sp. No. 38-2 and from Bacillus macerans. The enzyme reaction mixture containing 5 mL of 2% (w/v) of soluble starch in an incubation system
containing 10 mM CaClz and 20 pL (190 units) of CGTase was incubated at 50 "C for 120 min. The incubation systems used were as follows: acetate buffer (80 mM, pH 5.0) for Bacillus macerans CGTase and Tris-HC1 buffer (80 mM, pH 8.0) for the CGTase of alkalophilic Bacillus sp. No. 38-2. The CH-30 which is a mixture of CD's and linear maltooligosaccharides was also tested. The amounts of y-CD in the samples were determined with the HPLC method and our BCG method. The results are shown in Table 11. The errors of the amount of y-CD were less than 8%. Although, the analysis of Bacillus macerans CGTase product gave 10% error, this may be due to the lower concentration of y-CD in the mixture. Anyhow, our BCG method for y-CD analysis can directly measure y-CD in the enzymatic products without glucoamylase treatment. This study shows that the BCG method is rapid and quantitative in the determination of y-CD in mixtures of cyclodextrins and maltooligosaccharides. Registry No. CD, 17465-86-0;BCG, 76-60-8;CD-BCGinclusion complex, 90106-88-0.
LITERATURE CITED (1) Zsadon, B.; Otta, K. H.; Tudos, F. J . Chromatogr. 1979, 172, 490-492. (2) Hokse, H. J . Chromatogr. 1980, 789, 98-100. (3) Kitahata, S.; Yoshikawa, S.; Okada, S. J . Jpn. SOC. Starch Sci. 1978, 25, 19-23. (4) Hirai, H.; Toshima, N.; Uenoyama, S. Polym. J . 1981, 73, 607-610. (5) Nakamura, N.; Horikoshi, K. Agric. Bioi. Chem. 1978, 4 0 , 753-757. (6) Okada, S.; Kitahata, S. Proc. Symp. Amylase. (Osaka) 1973, 8, 21-30.
RECEIVED for review January 18,1984. Accepted March 21, 1984.
Determination of Rhenium in Acid Solution by Anodic Stripping Voltammetry with the Amalgamated Rotating Gold Disk Electrode Heinz Ruf* and Margarete Friedrich Kernforschungszentrum Karlsruhe, Institut fur Radiochemie, 7500 Karlsruhe, Postfach 3640, Federal Republic of Germany Polarographic methods of quantitative rhenium determination have been developed by Heyrovsky ( I ) and Lingane (2). Recently, reports have been published about voltammetric methods of rhenium determination in acid and alkaline solutions. The electrode materials used for this application have been glassy carbon and also mercury as a thin film on a glassy carbon support ( 3 ) . In anodic stripping voltammetric determination of Re(V), platinum and paraffin soaked spectral carbon have been used (4). The contents of metallic rhenium in powder materials have likewise been determined by anodic stripping voltammetry using a carbon paste electrode. The sample to be assayed is homogeneously mixed with the carbon paste (5). Also the studied anodic behavior of rhenium electrodeposited from perrhenate solutions on a mercury cathode is of interest in this context (6). According to the study the amalgamated metal is dissolved again while the polarization of the electrode gets more and more positive and the potentials are still negative (-0.4 to -0.3 V vs. SCE) so that anodic stripping voltammetric determinations of rhenium seem to be possible in principle on the mercury electrode as well. By use of a thin film mercury electrode (TFME) which to our knowledge has previously been applied only in connection with a voltammetric (not a stripping voltammetric) method, a substantial increase in the detection sensitivity might rightly be expected for this element ( 3 ) . However, experiments we performed with the hanging mercury drop electrode (HMDE) on the basis of these findings 0003-2700/84/0356-1740$01.50/0
did not lead to success. Similar experiments with the TFME and with glassy carbon as the supporting material failed also. On the other hand, the amalgamated rotating gold disk electrode (HgAuRDE) proved to be extraordinarily well suited for this purpose. On this electrode rhenium present as perrhenate in dilute sulfuric acid (0.01 N H,SO,) can be enriched at -800 mV (Ag/AgCl) through electroreduction. In a subsequent anodic DP mode polarization of the electrode in the same electrolyte, a distinct oxidation peak appears at -280 mV (Ag/AgCl) whose height is proportional to the perrhenate concentration. A correlation coefficient of r = 0.9954 has been calculated from the results of calibration. It is possible via the current signal to record quantitatively rhenium even in low concentration solutions with sufficient accuracy. After 2 min of enrichment 5 X 10" g of Re/mL still furnish a signal which can be very well utilized. The stripping voltammograms recorded are shown in Figure 1. As the stripping voltammetric peak of lead (Pb2+)occurs close to the rhenium peak, it interferes with the determination. Manganese and technetium do not interfere. EXPERIMENTAL SECTION Coating of the gold electrode is performed by electrodeposition of mercury from a stirred solution of 20 mL of 0.05 M KNOBwhich contains approximately 300 pg of Hg2+.For this purpose a voltage of -1.0 V is applied to the electrode for 1 min. When coating is terminated, the electrode is rinsed with deionized water and subsequently immersed into the working 0 1984 American Chemical Soclety
