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observed stable carbon isotope depletion for NBS 22 that occurs with prolonged storage is a consequence of the interaction of the gaseous products of combustion with the copper oxide wire, it is not surprising that an increase in the size (34 cm) of the combustion tubes diminished the isotope depletion effect that was observed for the shorter (22 cm) combustion tubes (Table 11). The synthesis of copper carbonate minerals such as malachite results in an isotope fractionation such that the carbonate carbon becomes enriched in carbon-13 relative to the COz starting material (7). Also, the fact that copper carbonate minerals such as malachite and azurite decompose at 200 and 220 "C, respectively, well below the combustion temperature employed in this study (i.e., 550 "C), lends credence to the hypothesis that (1) the formation of a copper carbonate phase is responsible for the observed isotope depletion of C02 with prolonged storage of the NBS 22 samples subsequent to combustion and (2) reheating of older NBS 22 samples prior to stable isotope analysis results in the destruction of this copper carbonate mineral phase and the release of this I3Cenriched component back into the COz reservoir. X-ray diffraction analysis of the preheated (850 "C) copper oxide wire indicated that the dominant mineral present was, as expected, tenorite (CuO). X-ray analysis of the wire subsequent to the combustion and storage of NBS 22 samples was apparently not sensitive enough to detect the copper carbonate phase. It was observed, however, that a significant percentage of the tenorite had been converted to cuprite (Cu,O), which is a t an appropriate oxidation state for the formation of copper carbonate (Cu2C03). In summary, the sealed tube combustion method is rapidly becoming the method of choice for the conversion of organic
matter to C02 for stable carbon isotope analysis. Unlike those who use dynamic combustion, scientists who employ static (sealed tube) combustion are typically combusting 30 or more individual samples at a time. As indicated above, the storage of samples for prolonged time intervals (Le., several weeks) subsequent to combustion will result in 613C values that are depleted by up to 1-3%0 relative to what would have been observed had the samples been analyzed within several days subsequent to combustion. For situations that arise (such as instrument down time) when combusted samples must be stored prior to analysis, it is possible to avoid this problem by isolating the COz gas from the copper oxide wire. A more practical solution, however, is to simply reheat the tubes immediately prior to analysis.
ACKNOWLEDGMENT We thank D. Powell (OU) for performing the X-ray diffraction analyses and Z. Sofer (Amoco Production Co.) for his critical review of this manuscript.
LITERATURE CITED Stuermer, D. H.; Peters, K. E.; Kaplan, I. R. Geochim. Cosmchim. Acta 1978, 4 2 , 989-997. Sofer, 2. Anal. Chem. 1980, 5 2 , 1389-1391. Schoell, M.; Faber, E.; Coleman, M. L. Org. Geochem. 1983, 5 , 3-6. DesMarais, D. J.; Hayes, J. M. Anal. Chem. 1976, 4 8 , 1651-1652. ( 5 ) Bonilla, J. V.; Engel, M. H. Org. Geochem. 1986, 10, 181-190. (6) Hut,G. Report to Director General, International Atomic Energy Agency: Vienna, 1987, 42 pp. (7) Smith, A. W. Archammetry 1978, 20, 123-133.
RECEIVED for review January 24,1989. Accepted June 1,1989. We acknowledge the National Science Foundation, Division of Earth Sciences (Grant No. EAR-8352055) for partial support of this research.
Foam Countercurrent Chromatography of Bacitracin with Nitrogen and Additive-Free Water Hisao Oka,' Ken-ichi Harada? Makoto Suzuki? Hiroyuki N a k a ~ a w aand , ~ Yoichiro Ito* Laboratory of Technical Development, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 Foam separation has long been used for separation of a broad spectrum of samples ranging from metal ions to mineral particles (1). The separation is based on a unique parameter of foaming capacity and/or foam affinity of samples in an aqueous solution and bears a great potential for application to biological samples. Utility of the method in research laboratories, however, has been extremely limited mainly due to a lack of efficient instruments to fully exploit versatility of the method. Recently, an innovation of the foam separation technology has been achieved by development of foam countercurrent chromatography (CCC), which utilizes a true countercurrent movement between foam and its mother liquid through a long, narrow coiled tube by the aid of a particular mode of planetary motion generated by a coil planet centrifuge (2). Introduction of a sample mixture into the coil results in separation of the Visiting Scientist from the Aichi Prefectural Institute of Public Health Nagoya, Ja an. Permanent adgesa: Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468, Japan. 3Permanentaddress: The Institute of Public Health, Tokyo 108, Japan. 0003-2700/89/0361-1998$01.50/0
sample components: Foam-active materials quickly move with the foaming stream and are collected through the foam outlet while the rest are carried with the liquid stream in the liquid outlet. The method has been applied to various test samples including ionic compounds collected with suitable surfactants and surface-active proteins separated in a phosphate buffer solution to prevent denaturation of the molecule (2-4). This paper describes a successful application of the foam CCC technology to chromatographic fractionation of bacitracin complex using nitrogen gas and distilled water entirely free of surfactant or other additives. Bacitracin (BC) is a basic, cyclic peptide antibiotic commonly used as a feed additive of livestock all over the world. It consists of more than 20 components, each with different antimicrobial activities, and chemical structures of these components other than BCs-A and -F are still unknown. Because of its strong foaming activity, BC is an ideal test sample to demonstrate capability and usefulness of the present method.
EXPERIMENTAL SECTION The apparatus used for the present study is a multilayer coil planet centrifuge,which produces a synchronousplanetary motion of the gear-driven column holder and has been described pre0 1989 American Chemical Society
ANAL.YTICAL CHEMISTRY, VOL. 61, NO. 17, SEPTEMBER 1, 1989
1999
14lBC-FI
1 1 IBC-Ai
~~
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Figure 1. HPLC analysis of bacitracin in the original sample. The experimental conditions are given in the text.
viously (2, 3). Although our prototype was originally designed and fabricated at NIH, the apparatus may be duplicated in a reasonable time period by the following two companies: Pharma-Tech Research Corp., Baltimore, MD, and P.C., Inc., Potomac, MD. Also, interested readers may try to use their commercial high-speed CCC models with a 10 cm revolutional radius by modifying the column holder and the separation column. Separation was initiated by simultaneous introduction of distilled water at a flow rate of 3.2 mL/min from the tail and nitrogen gas pressured at 80 psi from the head into the rotating coil at 500 rpm while the needle valve on the liquid collection line was fully open (13.5 turns). After a steady-state hydrodynamic equilibrium was reached, the pump was stopped and 0.5 mL of a sample solution containing bacitracin (Sigma Chemical Co., St. Louis, MO), 1% (w/v) in distilled water, was injected through the sample port. After a standing time of 5 min, the opening of the needle valve was adjusted (0.8 turn) to bring the volume ratio of foam and liquid fractions to 15-6, and the pumping resumed at 3.2 mL/min. Effluents through the foam and liquid outlets were each manually fractionated at 30-s intervals. An aliquot of each fraction was analyzed by reversed-phase high-performance liquid chromatography (HPLC). The HPLC analysis was performed with a Shimadzu HPLC set consisting of a Model LC-6A pump, a manual injector kit, a Model SPD-6A detector, and a Model C-R5A recording data processor (Shimadzu Corporation, Kyoto, Japan) using a Capcell Pak CI8column, 0.46 cm i.d. X 15 cm (Shiseido, Tokyo, Japan). The mobile phase, composed of methanol and 0.04 M Na2HP04(pH 9.4) at a volume ratio of 62:38, was isocratically eluted at a flow rate of 1 mL/min, and the effluent was monitored at 234 nm.
I
I . I
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Figure 2. HPLC analysis of the first foam fraction. 1 1 IBC-Ai
- A - J L ) i
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Figure 3. HPLC analysis of the 1 lth foam fraction.
RESULTS AND DISCUSSIONS HPLC analysis of the original BC sample revealed major component BC-A, its oxidation product BC-F, and over 20 UV-absorbing minor components as shown in Figure 1. In the present foam CCC method, all these components were divided into two groups based on their foam activity: BC-A and several components with higher hydrophobicity (or longer retention time in Figure 1) were eluted through the foam collection line while the rest of the components were eluted through the liquid collection line. In the foam fraction, hydrophobic components corresponding to peaks 10 through 15 (Figure 1)were eluted in such a manner that the most hydrophobic component (peak 15) emerged first near the foam front followed by the rest of the components exactly in decreasing order of hydrophobicity or retention time. Furthermore, each component in the foam fractions showed substantial enrichment relative to that in the original sample solution. In the first foam fraction, most hydrophobic components corresponding to peaks 15 and 14 (BC-F) were enriched 2.8 and 2.2 times, respectively (Figure 2). Peak 15 is hardly visible in Figure 1due to the low solute concentration in the original sample solution, whereas the same peak is clearly observed in Figure 2 after near 3-fold enrichment in the foam fraction. In the 11th fraction, BC-A (peak 11)was enriched 1.8 times and almost entirely isolated
, 0
* 5
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Figure 4. HPLC analysis of the second liquid fraction.
from other components (Figure 3). It should be noted that these separations were achieved within 6 min of elution or 11 min after sample injection. On the other hand, much less efficient separation was observed in liquid fractions as shown in the HPLC analysis of the second fraction (Figure 4). Nevertheless, the components corresponding to peaks 1 through 9 appear to be eluted in decreasing order of their polarity, and more hydrophobic components (peaks 11-15) are almost entirely eliminated from all liquid fractions. The above results were obtained by optimizing various operational parameters such as liquid feed rate, needle valve opening on the liquid collection line, standing time after sample injection, etc., while N, feed pressure and revolution
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speed of the centrifuge were kept constant. Liquid feed rates lower than 3.2 mL/min failed to elute foam, while higher feed rates yielded less efficient foam separation. The opening of the needle valve determined the relative volume between the foam and liquid fractions. Opening the valve over 1.2 turns gave no foam fraction, while less than 0.5 turn lowered separation efficiency in foam fraction. By manipulation of the needle valve opening between the above critical range, the components with intermediate hydrophobicity can be eluted from either the foam or liquid outlet. Standing time after sample injection was also an important factor affecting both the foam elution pattern and the separation efficiency. Long standing time improved separations between peaks 10 and 15 (Figure l),but when it exceeded 5 min, foam elution became intermittent. Pumping was stopped during the standing time because even low flow rates of 0.2-0.5 mL/min delayed foam elution resulting in longer separation times required. As described above, we were able to separate BC components in the order of hydrophobicity by using foam CCC without any surfactant or buffer solution. The combined use of nitrogen gas and distilled water in an open column provides a number of advantages over other chromatographic methods such as (1) minimum decomposition or deactivation of bio-
logical samples, (2) nonadsorptive sample loss onto the solid support matrix, (3) no risk of contamination, (4) easy recovery of samples after fractionation, (5) low cost in operation, etc. In addition, the method is capable of enrichment of foam active samples so that a minute amount of compounds can be effectively concentrated and detected in the foam fraction. The system also permits continuous operation by continuous sample feeding. Efficiency of the method may be increased in many folds by the use of a longer coil and the sample loading capacity by the use of a larger-bore coil. We believe that the present method has a great potential in enrichment, stripping, and isolation of various natural and synthetic products in research laboratories. Registry No. BC, 1405-87-4; BC-A, 22601-59-8; BC-F, 22601-63-4; nitrogen, 7727-37-9;water, 7732-18-5.
LITERATURE CITED (1) (2) (3) (4)
Somasundaran. P. S e p . Purlf. Methods 1972, 1 , 117. Ito, Y. J . L i q . Chromtogr. 1985, 8 , 2131. Bhatnagar. M.:Ito. Y. J . L i q . Chromafogr. 1988, 1 1 , 21 Ito, Y. J . Chromatogr. 1987, 403, 77.
RECEIVEDfor review December 29, 1988. Accepted May 24, 1989.
