carbon-hydrogen bonds more freely toward the external environment than does neosamine C. As a result, neomycin B is more strongly retained on the resin column than neomycin C. The Donnan effect explains a t least partially the relatively slow migration of the catenulin constituents. The neamine moiety in neomycin contains a 2,6-diamino-2,6-dideoxyglucosylgroup, whereas pseudoneamine in catenulin contains only a 2-amino-2-deoxyglucosy1 group. Since in aqueous solution the amino groups are protonated to an appreciable degree, the neomycin antibiotics will be excluded from the resin beads more extensively than the catenulins. In fact, under similar experimental conditions the total amount of effluent necessary for a quantitative elution of the catenulins was greater by a factor of about 1.5, in comparison to the total amount of effluent necessary for the elution of the neomg c‘ins. 7
ACKNOWLEDGMENT
The authors express sincere appreciation to William Rieman I11 for a valuable discussion on ion exclusion, to Oskar Wintersteiner for helpful comments, and to Walter Celmer, Chas.
H z e r & Co., Inc., for supplying the sample of catenulin hydrochloride. The authors are also grateful for the able technical assistance of Peter Wintersteiner during the course of these studies. LITERATURE CITED
(1) Arcamone, F., Bertazzoli, C., Ghione, M., Scotti, T., Giom. microbiol. 7, 251 (1959). (2) Brodasky, T. T., ANAL.CHEM.35,343 1196.1). \ ~ _ . _ ,
(3) Davisson, J. W., Solomons, I. A., Lees, T. M., Antibiotics & Chemotherapy 2,460 (1952). (4) Dutcher, J. D., Hosansky, N., Donin, M. N., Wintersteiner, O., J . Am. Chem. SOC.73,1384 (1951). (5) Ford, J. H., Bergy, M. E., Brooks, A. A., Garrett, E. R., Alberti, J., Dyer, J. R., Carter, H. E., Ibid., 77, 5311 (1955). (6) Hagemann, G., NominB, G., PBnasse, L., Ann. Pharm. Fmnc. 16, 585 (1958). (7) Haskell, T. H., French, J. C., Bartz, Q. R., J . Am. Chem. SOC.81, 3482 (1959): (8) Horii, S., J . Antibiotics (Japan) 14A, 249 (1961). (9) Ibid.,.lSA, 187 (1962). (10) Horii, S., et al., Ibid., 16A, 144 (196.1).
(11) Leach, B. E., Teeters, C. M., J . Am. Chem. SOC.73,2794 (1951). (12) Moore. S., Stein, W. H., J . Biol. . Chem. 176, 367 (1948).
(131 Pan. S. C.. Dutcher. .T. D.. ANAT..
, W. A., Sohler, A., Schaffne J . Am. Chem. SOC.82,3938 ( Rlnohart, K. L., Hichens, IVI., Argoudelis, A. D., Chilton, W. S., Carter. H. E.. Georeiadm. M. P.. Schaffner, C. P., Sck&ngk, R. T.; Ibid., 84,3218 (1962). 6) Rothrock, J: W Goegelman, R. T., Wolf, F. J., in “Antibiotics Annual 1958-1959,’’ P. 797, Medical Encvclopedia, New Ybrk, 1959. .7) Saito, A,, Schaffner, C. P., Abst’r. IIIrd Intern. Congr. Biochem., Brussels, 1955, p. 98. .8) Sargent, R. N.,Graham, D. L., Ind. Eng. Chem., Process Design Develop. 1,56 (1962). .9) Schaffner. C. P.. in “Antibiotics ‘ Annual 1954-1955,”’ p. 153, Medical Encyclo edia, Sew York, 1954. (20) Schdngs, R. T., Ph.D. thesis, Rutgers University, Kew Brunswick, N. J., 1960. (21) Schillings, R. T., Schaffner, C. P., in “Antimicrobial Agents and Chemotherapy-1961,” p. 274, American Society for Microbiology, Detroit, 1962. (22) Swart, E. A., Hutchison, D., Waksman, S. A., Arch. Biochem. 24, 92 (1949). (23) .Waksman, S. A., Lechevalier, H. A., Snence 109, 305 (1949). (24) Wheaton, R. M., Bauman, W. C., Ann. X. Y . Acad. Sci. 67, 159 (1953). RECEIVEDfor review May 28, 1963. Accepted September 30,1963. (161
Determination of Hydroxyethyl Group in Hydroxyethyl Starch by Pyrolysis-Gas Chromatography Technique HAN TAl, R. M. POWERS, and T. F. PROTZMAN A , E. Staley Manufacturing Co., Decatur, 111.
b The hydroxyethyl group in the hydroxyethyl starch ether can be quantitatively determined by gas chromatographic analysis of the volatile pyrolytic products obtained under a controlled condition of 400’ C. and 10 minutes. The acetaldehyde peak is selected as the analytical peak, and its peak height correlates quantitatively with the per cent hydroxyethyl values. This technique offers a simple and rapid method for quantitative analysis with reproducible results. The procedures can also be adopted for other starch derivatives.
T
degradation of polymeric materials, if made a t a controlled temperature, often yields volatile products of d e m t e compositions. Direct determinations of these products by gas chromatography have been utilized for analyzing various polymers (1, 3,6). Greenwood, Knox, and Milne (2) have
illustrated characteristic gas chromatograms of the pyrolysis products of various carbohydrates. In this laboratory, we have extended this technique to investigate starch and its hydroxyalkyl derivatives. For hydroxyethyl starch ether it has been shown that one of the gas chromatographic peaks is due to acetaldehyde. Furthermore, the intensity of the peak correlates quite satisfactorily with the amount of hydroxyethyl group. Simplicity of method, speed in obtaining results, and reproducibility recommend this procedure for quantitative analysis. EXPERIMENTAL
HE THERMAL
108
ANALYTICAL CHEMISTRY
Apparatus. Pyrolysis Oven. A pyrolysis oven (Figure 1) was constructed for carrying out the pyrolysis a t a definite temperature with a sample size of about 1 mg. T h e body of the oven is a cylindrical stainless block of 3-inch diameter and 4l/Z-inch length. A l/&nch diameter hole is drilled axially through the center as a
chamber for the sample tube. The block is heated by three cartridge heaters inserted in three w e b s p metrically drilled parallel to the axis of the block. A thermocouple well of l/&ch diameter is drilled in the center plane of the block, reaching to within inch of the sample chamber. An Iron-Constantan protected thermocouple (Type 5, Thermo-Electric Co.) is used for registering the temperature. The whole assembly is insulated by a 1inch thick asbestos cover. The cartridge heaters are connected in parallel to a variable transformer. A constant temperature of 400’ C. is maintained by applying about 30 volts. Gas Chromatograph. The gas chromatograph used for the present study consists of F&M Model 1609 flame ionization detector attachment, F&M Model 240 temperature programmer, F&M Model SI-4 solid sample injector and Sargent Model SR recorder. The column is an 8-foot coiled copper tube inch i.d.) packed with Burrell adsorbant 341-133, with polyethylene glycol as the stationary phase. Helium is used as the carrier gas.
?
+ Figure 2. Gas chromatogram of pyrolysis products of unmodified corn starch Figure 1. Upper:
Pyrolysis oven
Front view.
lower:
Side view
Weight of sample pyrolyzed: 1.2 mg. Peak identification: 1. Light gases. 2. Acetaldehyde. 3. Furan. 4. Propionaldehyde. 5. Acetone and 2-methylfuron. 6. 2-Butanone. 7. 2,3-Butadione. 8. 2 Methyl-1-buten-3-one
I
c
c(
-e
I
6
m
e
Figure 3. Gas chromatogram of pyrolysis products of hydroxyethyl starch, 5.68% hydroxyethyl
Figure 4. Gas chromatogram of the pyrolysis products of hydroxyethyl starch, 17.63% hydroxyethyl
Weight of sample pyrolyzed: 1.1 mg. Peak identification; 1. Ligh gases. 2. Acetaldehyde. 3. Furan. 4. Propionaldehyde. 5. Acetone and 2-methylfuran. 6. 2-Butanone. 7. 2,3-Butadione. 8. Unidentified
Weight of sample pyrolyzed: 1.0 mg. Peak identification: 1. light gases. 2. Acetaldehyde. 3. Furan. 4. Propionaldehyde. 5. Acetone and 2-methylfuran. 6. 2-Butanone. 7. unidentified. 8. 2,3-Butad i o n e l 9 . unidentified
VOL. 36, NO. 1, JANUARY 1964
109
Table 1.
Peak Heights of Acetaldehyde Peak
% HE" Peak heights (mm./mg.)* Mean Std. dev. 27 25 4 2.1 26 22 27 25 5.68 60.6 5.6 66 58 66 53 60 10.06 95.0 4.1 101 91 94 97 92 17.63 6.9 110 105 . 2 113 104 95 18.54 104 131.6 3.2 129 132 137 130 130 23.08 a Weight er cent of the hydroxyethyl group. b The peal heights are the differences between the measured values and that of the unmodified starch.
The operating conditions are: (a) column: constant temperature a t 90" C., helium flow rate 7 5 ml. per minute (head pressure 60 p.s.i.g.); (6) flame ionization detector: air flow rate 600 ml. per minute (head pressure 40 p.s.i.g.) ; hydrogen flow rate 50 ml. per minute (head pressure 30 p.s.i.g.) ; range setting, 100; attenuation setting, 4 or 8; injection port temperature 150" C.; (c) recorder: full scale range 1.25 mv.; scanning speed, inch per minute. Procedure. About 1 mg. of t h e sample is weighed into a glass melting point tube, 9 em. long, 1 mm. i.d., with one end sealed. T h e tube is evacuated, vacuum sealed (about 3 cm. from t h e b u t t end), a n d placed in the pyrolysis oven at 400' C. for 10 minutes. It is then inserted in the solid sample injector and allowed to remain in the injection port for about 3 minutes to equilibrate the temperature. The sample tube is then broken by pushing the plunger of the solid injector t o release the volatile pyrolysis products into the carrier gas stream. RESULTS
,4gas chromatogram of the pyrolysis products of unmodified corn starch is shown in Figure 2. The pyrolysis was done at 400" C. for 10 minutes. Studies have been made on the effect of time and temperature during No appreciable amount pyrolysis. of volatile products is formed within 10 minutes for a pyrolytic temperature below 350" C. Between 380" C. and 450' C., the gas chromatograms exhibit well defined patterns with only slight changes a t different temperatures within this range. The products start t o show further decomposition, as indicated by the changing intensities of the peaks, when the temperature reaches above 500' C. At 400" C., the pattern of peaks remains almost unchanged between 8 and 15 minutes for tests made at one-minute increments. Therefore, we chose a pyrolytic temperature of 400' C. and a time of 10 minutes as the optimum conditions for this study.
110
ANALYTICAL CHEMISTRY
Figures 3 and 4 are the representative gas chromatograms obtained from two hydroxyethyl starch ethers containing 5 68% and 17.63% hydroxyethyl, respectively. The increase of intensity of the acetaldehyde peak indicates that acetaldehyde is one of the thermal decomposition products from the hydroxyethyl group. Therefore, this peak is selected as the analytical peak for the quantitative determinations. Since the shape and the half peak width are the same for all the samples studied, the peak intensities are expressed in peak height per mg. of the sample. The peak heights are the distances from the apex to the base line drawn across the peak and expressed in a n arbitrary unit of mm. The unmodified starch also shows an acetaldehyde peak. Therefore, the actual peak heights due to hydroxyethyl group are obtained from the differences between the measured value and the corresponding value from the unmodified starch. Five samples of the hydroxyethyl starch ether of different hydrosyethyl levels were prepared and pyrolyzed. Five repetitive runs mere made on these samples and the unmodified starch on different days. Table I lists the values of peak heights per milligram of sample of the acetaldehyde peak as obtained by the difference of the measured peak height and that of the unmodified starch. Under the present conditions, the unmodified starches gaye a peak height value between 42 and 45 mm. per mg. with a mean value of 43.6 and a standard deviation of mean 0.6 mm. The per cent hydroxyethyl values of the samples are also listed in Table I. The data are the average of five determinations. The analyses were made following a procedure using hydriodic acid as reported by Lorts (4). The results reveal that the peak height increases m'ith increasing per cent hydroxyethyl value, showing a linear relationship. The regression equation is Y = 5.87 X - 4.46, where Y is peak height and X is per cent hydroxyethyl.
The 95% confidence limits for means at three typical hydroxyethyl levels are: X = 7%, 32.8 < Y 6 40.5; X = 15%, 81.2 6 Y 86.0; X = 23%, 126.6 Y 6 134.4. Peak height values can also be calibrated to the weight of acetaldehyde by introducing a known amount of acetaldehyde into the gas chromatograph under the identical operational conditions as that for the pyrolysis products. The present instrument setting gives a peak height of 58 mm. for 0.010 mg. of acetaldehyde. Therefore, 1% hydroxyethyl is equivalent to 6-mm. peak height or 0.001 mg. acetaldehyde in the pyrolysis products. The data indicate that, under the carefully controlled conditions, the pyrolysis of starch and hydroxyethyl starch ether yield reproducible results with good precision. The calibration in the present study is rather empirical because its accuracy would have been influenced by the chemical method which is used as the standard. The measurements can be made on an absolute basis only if the mechanism and the stoichiometry for the pyrolysis reaction are fully understood, but these have not been explored. However, compared with the conventional chemical analysis using hydriodic acid, the method is simple and rapid with equal, if not better. precision. Although the present investigation deals specifically with the hydroxyalkyl ether, with a proper selection of the characteristic peak, the procedures can be readily adapted to the other starch derivatives.
