814
Anal. Chem. I@83,55, 814-816
_____
Table I. Comparison of Siloxane Membrane Probe to Hollow Fiber Robe Responses (50 ppm (v/v)) peak height, mm acrylonitrile methylene chloride m/z 53 m/z 84 (1) membranr probe prepared from 10% polymer solution ( 2 ) membrane probe prepared from 15% polymer solution (3) siloxane hollow fibrr probe a
Background pressure for water blanks was 0.4
X
22
105
14 6
71 60
ion sowce pressure,' torr 0.6 X 0.45 X 0.55 X
torr for all three cases. em2 which was very close to the 1.06 cm2 for the 15-element siloxane hollow fiber probe. Organwsiloxaneblock copolymers with higher siloxane content, such as X4-2541 (75% poly(dimethylsiloxane), 25% a-methylstyrene) provided good permeability for many compounds hut lower enrichment factors. The enrichment factor of chloroform in aqueous solution with this membrane prohe was determined to be 0.6 X 10' as compared to 1.1 X 10' reported by Westover et al. (4) with the use of the siloxane hollow fiber probe. Other polymer membrane probes, including poly(viny1 alcohol), poly(cyanoacrylate),and poly(styrenea-methyLstyrene) were found to he much less permeable than the organo-siloxane block copolymer. This new membrane probe possesses the following advantages over other sampling devices: (1)It is easy to construct. (2) There is no need for gasket seals or adhesives which could lesd to vacuum leaks or memory effects. (3) A large number of polymers can be tested for applications of tailor-made separations for mass spectrometer or other instrument probes. (4) The membrane probe can he heated through the metal hy heat conduction to study Permeability of polymers a t higher temperatures. (5) It is easy to evaluate permeation properties of polymers that cannot he fabricated as hollow fihers. Registry No. Water, 1732-18-5.
LITERATURE CITED Figure 1. Diagram of membrane probe.
to he more widely applicable for a variety of organic components. T o demonstrate the usefulness of the membrane probe device as compared to the siloxane hollow fiber probe made from the same block copolymer, we prepared two membrane probes of siloxane block copolymer (X4-2517,40% poly(dimethylsiloxane), 60% a-methylsytrene) using two solutions of 10% and 15% copolymer in toluene. Fifty part-per-million (v/v) standards of acrylonitrile and the common industrial solvent, methylene chloride, were made up in water. As shown in Table I equal or better response was obtained with the membrane probe at comparable source pressures. The effective membrane probe surface area was calculated to be 1.14
(1) Klain, E.: Elcheiberger. J.: Eyer. C.; Smith. J. Water Res. 1975. 9 . 807. (2) Relszner. K. D.; West. P. W. Enwon. Scl. T e M . 1973. 7 . 526. I31 Hardv. J. K.! DBsou~tB.P. K.: Reismer. K. D.: West. P. W. Endron.
.
(8) Saam, J. C.; Ward. A.~H.:Fearon. F. W. G.'"Advances in Chemistry Series"; American Chemical Society: Washington, DC 1973: Adv. Chem. Ser. No. 129. p 239. (9) Monroe. C. M.: Ward. A. H.: Abstracts. R u b k Divisbn 01 the AmerC can Chemical Society. Toronto. Canada. MY7. 1974. No. 15. (10) Ward, A. H : Kendrick, T. C.: S a m . J. C. ."Advances in Chemistry Series". No. 142. American Chemical Society: Washington, DC. 1975. Adv. Chem. Ser. NO. 142. p 300.
RECEIVED for review July 21,1982. Accepted December 14, 1982.
Determination of Carbon Isotope Ratios in Plant Starch via Selective Enzymatic Hydrolysis Arndt Schlmmelmann' and Mlchael J. DeNlro' Department of Earth and Space Sciences. Unlversny of California. Los Aflgeles. California 90024
Previous determinations of the stable carbon isotopic compasition of biopolymers involved isolation and purification of the intact polymers from complex organic mixtures, followed by their combustion and isotopic analysis of the re'Also Program in Archaeology.
sulting carbon dioxide (e.g., ref 1-3). We present here a method based on the selective enzymatic hydrolysis of a biopolymer. The resulting monomer is collected after passage through a dialysis membrane which serves to separate it from other components present in the original biological material. 'The monomer is then combusted prior to isotopic analysis.
0 1983 American Chemical Society 0003-2101)/(1~~0955-0814$01.50~0
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
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Table I. s 13CValues and Yields for Starch Prepared by the Chemical Purification Method and for Glucose Obtained from Starch by the Enzymatic Methoda enzymatic method chemical purification sample 13C,0 1 0 0 yield,b % 6 13C,0 1 0 0 yield,b % ' potato starch corn starch bean powder
-25.8 * 0.1 ( n 4 ) -10.0 f 0.1 ( n = 4 ) -23.9 f 0.3 (n = 4 ) E
a The number of replicates is indicated by n. applicable.
-26.0 * 0.1 ( n = 2) -10.1 i- 0.4 ( n = 4 ) -24.1 * 0.0 ( n = 3 )
79 ( n = 1) 62 k c 7 ( n = 4 ) n.a. Expressed as percentage of amount of starch present initially. Not 98 ( n = 1 ) 96 k 2 ( n = 5) n.a.c
Although we chose to develop the method to determine the carbon isotope ratios of starch, this approach should find general applicability to biopolymers that can be degraded quantitatively to monomers and/or oligomers by using specific hydrolytic enzymes.
of C02 formed during the combustion of an aliquot of the starch/NaCl mixture. The yield for the enzymatic method was calculated from the glucose content of an aliquot of the solution against which the starch and enzyme mixture had been dialyzed. The glucose content of this solution was determined gravimetrically.
EXPERIMENTAL SECTION
RESULTS AND DISCUSSION
Corn starch, potato starch, and amyloglucosidase (exo-1,4-aglucosidase, E.C. 3.2.1.3) from Aspergillus niger (4) were obtained from Sigma Chemical Co. Dried beans (Phaseolus vulgaris) were purchased from a grocer. Dialysis tubing Spectrapor No. 6, with a 2000 molecular weight cutoff, was obtained from Spectrum Medical Industries, Inc. Starch was isolated from powdered beans and from the commerical starch samples by a standard chemical purifmtion method (5). One of the last steps in this procedure involves removal of iodine from a precipitated starch-iodine complex with alcoholic NaOH solution which has a pH of 12. At this pH, the wet precipitated starch may trap atmospheric carbon dioxide. To avoid contaminating the starch carbon with inorganic carbon in the form of carbonates, we used either freshly precipitated starch or starch that had been dialyzed one day against running distilled water for the isotopic measurements on chemically purified starch. In the enzymatic method, starch in the beans and the commerical starch samples were hydrolyzed with amyloglucosidase and the resulting glucose separated from the other components present in the starting mixture by passage through a dialysis membrane, according to the following procedure. First, the commercial starches were dissolved in quantities of 100 mg in 10 mL of distilled water by boiling the mixture for 10 min. The solutions were transferred quantitatively into dialysis bags. Next, 200-mg aliquots of finely pulverized bean were heated with 10 mL of distilled water to 100 OC for 10 min; after being cooled, the supernatant solutions were transferred into dialysis bags. A few drops of toluene (approximately 0.1 mL) were added to all solutions undergoing dialysis to prevent microbial degradation. The starch solutions were dialyzed one day against running distilled water at room temperature to remove all water-soluble components with molecular weights below 2000. Next, 1mL of amyloglucosidase solution (approximately 100 units of enzyme) that had been predialyzed for 1day against running distilled water was added to the dialysis bags containing the starch solutions and toluene. The dialysis bags were then incubated at 40 OC for 2 days in flasks containing 40 mL of digtilled water. The completeness of the enzymatic hydrolysis of starch at the end of 2 days was verified by the absence of an iodine-starch reaction (5) on small samples from the inside of the bags. The presence of glucose in the 40-mL solution was verified by the positive reaction with Fehling's solution (6). Freeze-dried starch and glucose samples were combusted by a modified version of the Stump and Frazer (7)method and the 13C/12Cratios of the resulting carbon dioqde measured as described in ref 8. The results are expressed in the usual 613C notation, where
The standard is the Peedee belemnite carbonate (PDB). Chemically purified starch contains some sodium chloride from the alcoholic NaCl washing solution. Thus the yield for the chemical purification of starch was calculated from the amount
The results of the yield and isotope ratio measurements are given in Table I. The 613C values for both the chemically purified commercial potato and corn starches and the glucose produced enzymatically from them are in good agreement with each other. Comparisons of yield determinations on both starches show that the enzymatic approach has the advantage of an almost complete conversion of the starch to glucose, whereas the chemical purification causes 21% and 38% losses of starch for the commercial potato and corn starches, respectively. These losses probably occur during the numerous washings and centrifugations using alcoholic-aqueous solutions in which starch has a noticeable solubility. The results for the whole bean powder show the same good agreement between the 613C values of the chemically purified starch and the enzymatically obtained glucose. Some enzymatically produced oligomers with molecular weights less than 2000 may have passed through the dialysis membrane and escaped complete conversion to glucose. This possibility does not affect the results or interpretations presented here.
CONCLUSIONS The enzymatic method described here is especially useful for the preparation of purified glucose from small amounts of starch in a complex organic mixture because the method has a quantitative yield. The enzymatic approach should be generally applicable to biopolymers for which a hydrolytic enzyme converts the polymer quantitatively to monomers or oligomers small enough to permeate through dialysis tubing or filtration membranes. Quantitative conversion of polymer to monomer and/or oligomer is required to avoid introduction of artifacts relating to isotope effects during enzymatic reactions (9). Hydrolases such as chitinases, proteases, and cellulases are capable of hydrolysis of their respective substrates to monomers or oligomers (10-12) and thus appear to be good candidates for use in the enzymatic method described here. The enzymatic approach could be adapted for use in the study of isotopic differences among biopolymers of the same kind but with different molecular weights by using a series of dialysis tubings with increasing molecular weight exclusion limits. Finally, we note that many enzymes require special pH ranges to develop their maximum activity. For example, the enzyme used in this study shows maximum activity in the pH range of 4-6. A carbon-free buffer system with pH 6 (saturated solution of molybdic acid adjusted with dilute sodium molybdate solution) was developed and did not inhibit the enzymatic activity. While amyloglucosidase showed sufficient activity without the buffer system to hydrolyze starch quantitatively, other enzymes might require buffered conditions.
818
ANALYTICAL
CHEMISTRY,
VOL. 55, NO. 4, APRIL
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In such cases, carbon-free buffer systems such as the one described here should be employed to avoid the added step of having to separate the monomer and/or oligomer from a carbon-containing buffering component prior to combustion and isotopic analysis.
Purcher, G. W.; Leavenworth, C. S.;Vickery, H. 8. Anal. Chem. 1948, 9 , 850-853. Stanek, J.; Cherng, M.; Kocourek, J.; PacBk, J. "The Monosaccharides"; Academic Press: New York, 1963; p 868. Stump, R. K.; Frazer, J. W. Nucl. Sci. Abstr. 1973, 28, 746. Northfelt, D. W.; DeNlro, M. J.; Epstein, S. Geochlm. Cosmochim. Acta 1981, 45, 1895-1898. O'Leary, M. H. I n "Isopote Effects on Enzyme-Catalyzed Reactions"; Cleiand, W. W., O'Leary, M. H., Northrop, D. B., Eds.; University Park Press: Baltimore, MD, 1976; pp 233-251. Stirling, J. L.; Cook, G. A.; Pope, A. M. S.I n "Fungal Wails and Hyphal Growth"; Burnett, J. H., Trinci, A. P., Eds.; Cambrldge: London, 1979; pp 169-188. Cowling, E. 6.; Kirk, T. K. Blotechnol. Bioeng. Symp. 1976, 6 , 95-123. Fruton, J. S. I n "The Proteases and Blological Control"; Relch, E., Rifkin, D. B., Shaw, E., Eds., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1975; pp 33-50.
ACKNOWLEDGMENT We thank Leo Sternberg for his valuable advice and criticism. Dave Winter performed the stable isotope ratio determinations. Registry No. Carbon-12, 7440-44-0; carbon-13, 14762-74-4; starch, 9005-25-8; E.C. 3.2.1.3, 9032-08-0; hydrolase, 9027-41-2. LITERATURE CITED Epstein, S. Geochlm. Cosmochim. Acta
(1) DeNlro, M. J.; 1978, 42, 495-506. (2) Hsu, J. D.; Smlth, B. N. Plant Cell Physlol. 1972, 13, 689-694. (3) Deleens, E.; Garnier-Dardart, J. Planta 1977, 135, 241-248. (4) Marshall, J. J.; Whelan, W. J. FEBS Lett. 1970, 9, 85-88.
RECEIVED for review October 4, 1982. Accepted November 22,1982. The research was funded by a grant from the Academic Senate Committee on Research at UCLA and NSF Grant ATM 79-24591.
CORRECTIONS Determination of Diffusion Coefficients by Flow Injection Analysis Greg Gerhardt and Ralph N. Adams (Anal. Chem. 1982, 54, 2618-2620). There is an unfortunate error in eq 1, appearing on page 2618. The terms a2f were omitted from this equation of Vanderslice and the definition of a is also missing. The correct equation should read
-( L;) 35.4a2f
=
064
and the sentence following should read: L is the distance between the injection port and the detector in centimeters, q is the flow rate in milliliters per minute, D is the diffusion coefficient, a is the radius of the flow path tubing in centimeters, f is a concentration and detector sensitivity factor, and AtB is the peak width in seconds. Circuit for Constant Current Operation of a n Electron Capture Detector
W. B. Knighton and E. P. Grimsrud (Anal. Chem. 1982, 54, 1892-1893). There is an error in Figure 1appearing on page 1893. The 2.7 kQ resistor at the bottom of Figure 1 A was inadvertently shown as being connected between pin 3 of the CD 4011B IC (the output of the f i s t NAND gate) and ground. This resistor should be placed on the other side of the 470-pF capacitor so that it is connected to pins 5 and 6 of the IC (the inputs of the second NAND gate) and ground. The resulting configuration is then consistent with the pulse width control shown in Figure 2.
