(45) Roberts, G., Ibid., 29,911 (1957). (46) Rockwood, S. W., Clark, H. P., Rev. Sci. Instr. 27,877 (1956). (47) Rosenkranta, H., “Analysis of Ster-
oids by Infrared Spectrometry,” “Methods of Biochemical Analysis,” D. Glick, ed., Vol. 11, pp. 15-23, Interscience, Kew York, 1955. (48) Rosenkranta, H., Zablow, L., AXAL. CHEM.25, 1025 (1953). (49) Sadtler & Son, Inc., “Sadtler Catalogue,,’ Philadelphia 3, Pa. 150) Sands. J. D.. Turner. G. S.. ANAL. CHEM.24,791 (i952). ’ (51) Schiedt, C., Rheinwein, H., 2. Naturforsch. 7b, 270 (1952); Instrument Yews (Perkin-Elmer Corp.) 4 , No. 3 (1953). (52) Servoss, R. R., Clark, H. M., J . ~
Chem. Phys. 26, 1175 (1957). (53) Sinclair, R. G., McKay, A. F., Jones, R. N., J . Am. Chem. SOC.74, 2570 (1952). (54) Stewart, J. E., ANAL. CHEM.31, 1287 (1959). (55) Stimson, M. M., O’Donnell, M. J., J . Am. Chem. SOC.74,1805 (1952). (56) Susi, H., Rector, H. E., ANAL.CHEM. 30,1933 (1958). (57) Tai, H., Underwood, A. L., Ibid., 29,1430 (1957). (58) Toribara, T. Y., Di Stefano, V., Ibid., 26,1519 (1954). (59) Walton, W. L., Hughes, R. B., J . Am. Chem. Soe. 79,3985 (1957). (60) Weigl, J. W., ANAL. CHEM. 24, 1483 (1952).
(61) Weir, C. E., Lippincott, E. R., Van Valkenburg, A,, Bunting, E. N., J . Research Natl. Bur. Standards 63A, 55 (1959). (62) White, J. U., Weiner, S., Alpert, N. L., ANAL.CHEM.30, 1694 (1958). (63) Wiberley, S. E,., Sprague, J. W., Campbell, J. E., Ibzd., 29, 210 (1957).
RECEIVEDfor review April 15, 1960. Accepted September 23, 1960. Division of Physical Chemistry, 138th Meeting, ACS, New York, N. Y., September 1960 A portion of a thesis submitted t o the faculty of the Graduate School, University of Maryland, in partial fulfillment of the requirements for the degree of doctor of philosophy.
Dete ct io n a nd Estimatio n of PoIy oxyet hy Ie ne Glycol in Nonionic Surfactants by Ascending Paper Chromatography M. E. GI“,
C. L. CHURCH, Jr., and J. C. HARRIS
Research and Engineering Division, Monsanto Chemical Co., Dayton, Ohio
b Paper chromatographic procedures were developed for detecting polyoxyethylene glycol (PEG) in nonionic surfactants. A semiquantitative, spot area method i s described, permitting estimation of PEG to the nearest per cent.
I
polyoxyethylated surfactants, mixtures arise from addition of ethylene oxide to hydrocarbon compounds containing active hydrogen (1, 4, 6, 1.9). Polyoxyethylene glycol (PEG) can form dong with the surfaceactive isomers if water is present during synthesis. The quantity of PEG produced depends on two factors: the amount of water initially present, and the tendency of the surfactant hydrophobe to react with ethylene oxide. In analysis. PEG is rpadily separated from ionic surfactants by well established ion exchange procedures (2, 9, 11). The problem is more difficult in the case of nonionic surfactants because of their similarity to PEG in chemical reactivity. Hence these separations are often based on differences in physical properties, as by selective solvent solubility (5) and preferential adsorption in column chromatography (7). The described method of ascending paper chromatography is particularly useful in qualitative and semiquantitative work where simplicity and convenience are required. Application can be made to a wide variety of polyoxyethylated surfactants. Adducts specifically tested were from long-chain N PREPARIXG
alcohols, CS to CIS, and alkyl phenols, Cp to Clz, with ethylene oxide chain lengths of 5 to 30 units. Samples are applied to the base of a paper cylinder. Development or resolution of PEG and active zones is accomplished with a water- and acetic acid-saturated solution of 1-butanol. Chromatographed spots are detected by spraying with a modified Dragendorff reagent. These reagents were adapted from the work of Nakagawa and Nakata ( 8 ) and Jaminet ( 3 ) . EXPERIMENTAL
Apparatus and Materials. The simple apparatus used for developing paper chromatograms consists of a Petri dish, 90 mm. in inside diameter and 15 mm. deep, as solvent reservoir; and a covering glass tube sealed a t one end, of 95-mm. inside diameter and 12 inches high. The paper is Whatman No. 1, chromatography grade, supplied as 18l/r X 22l/* inch sheets. A 10-pl. micropipet was used for sample transfer. A deVilbiss atomizer was used for indicator spray and a compensating polar planimeter for spot area measurement. Special Solvents and Reagents, Developing solvent was prepared by mixing 1 volume of glacial acetic acid with 5 volumes of distilled water and 4 volumes of 1-butanol. After standing a minimum of 72 hours, the saturated butanol layer was siphoned off and used as developing solvent. The indicating spray reagent was prepared by mixing the following ingredients in the order given :
50 ml. of 0.606y0 solution of bismuth hydroxynitrate in 71% glacial acetic acid 50 ml. of 50y0 KI solution in distilled water 200 ml. of glacial acetic acid 500 ml. of distilled water
Polyoxyethylene glycols studied were Carbowaxes 300, 600, 1000, and 1500 (Union Carbide Chemicals). EO adducts and PEG’S were vacuum dried a t 50’ C. before use. Procedures. The PEG and active fractions used as internal standards in the chromatographic determinations were isolated by multiple hot brine extractions (6) followed by ion exchange purification ( 8 ) . A 25-gram sample of the polyoxyethylated nonionic surfactant was extracted five to six times with 50-ml. portions of NaCl solution (at 90% of saturation). After mixing a t room temperature. the separatory funnels were immersed in boiling water for 15 to 30 minutes and the lower brine phase containing PEG was removed while hot (for each extraction). PEG was recovered from the combined brine extracts by extraction with SOpropyl alcohol. The resulting isopropyl alcohol solution of PEG was completely desalted by batch ion exchange purification. Salt-free PEG was isolated finally by evaporating the aqueous isopropyl alcohol solution to dryness. The active fraction was similarly isolated by putting it directly through ion exchange resins following the brine extractions. Resolution of PEG and active fractions was checked by paper chromatography as described below. Internal standards for paper chromatography were prepared by recombining the PEG with purified active in proportions covering the 0 t o 20% PEG range. VOL. 33, NO. 1 , JANUARY 1961
143
Figure 1. Photogrc PEG blends
In analyzing an unknown nonionic surfactant sample, 5% solutions were prepared in acetone. Acetone solutions of internal standards were also prepared to cover the range of expected PEG content. For unfamiliar or exploratory runs, internal standards.of 2.5 and 20% PEG were useful, while for familiar samples, internal standards differing by 2.5 or 5.0% PEG gave more accurate results. In cases of high PEG content, the sample was diluted with a known quantity of PEG-free active before analysis to increase sensitivity. Greatest sensitivity is ohtained between the 2.5 and 10% PEG range for a 500!4. A-,~~... . solutions were pipetted onto~igepaper (9'/8 jnches high X 10% inches wide) a t points 1 inch from the base and 1 to l'/P inches apart. Five samples (duplicates for unknown and three internal standards) were generally run on each sheet. The paper was carefully rolled into a cylinder along the short axis and stapled flat with no overlap. The cylinder was placed in a Petri dish containing 35 ml. of developing solvent, and after it was covered with the glass tube, the solvent was allowed to ascend the paper for 5 hours a t room temperature (tolerance 74' to 84" F.). After development, the solvent front was marked for R , values and the paper was air-dried. The seam was cut away and the unrolled paper was sprayed with indicating reagent to define active (reddish orange) and PEG (red) zones. Spot areas were measured by planimeter immediately after the paper was dry. ~~
~~
~~~~
~~
~
~~
The assignment of PEG content for the unknown may be qualitative from visual comparison with the PEG spots of the internal standards. A semiquantitative value is calculated by interpolation from the measured areas of the unknown and known PEG zones. For runs with internal standards a t 2.5 144
ANALYTICAL CHEMISTRY
or 5.0% PEG increments, interpolations were made between the two nearest per cent PEG contents; while with 2.5 and 20% PEG internal standards, interpolation was made between log per cent PEG. PEG contents were also determined from measurement of color density (spot reflectance using a Photovolt scanner with a blue filter on photographed and unphotographed sheets) and by an elution-colorimetric procedure in which excised PEG zones were eluted with water and the PEG was determined colorimetrically after precipitation as the phosphomolybdic complex according to Stevenson (10). Neither of these procedures yielded higher accuracy or precision than was obtained with the spot area method. In addition, the spot area method was found most sensitive of the methods studied to variations in PEG content and molecular weight. RESULTS AND DISCUSSION
Detection of PEG. The typical .nm>l+ I-UUI"
.-.h+-inarl fin nhmmn+nnmnhinn " Y Y a l l l l Y "11 "LL'"'Ya""~'ay111116
nonionic surfactants with varying P E G contents is shown in Figure 1. Two discrete zones or fractions are apparent for each sample: a rapidly moving upper fraction (R, of 0.9) near the solvent front, identified as active material; and a more slowly moving lower fraction (R, of 0.6) identified as PEG. The active ingredient zones are usually colored reddish orange and PEG spots red by the indicating reagent. This results from the tendency of the Dragendorff reagent to form a more intense red color complex with increasing ethylene oxide content in the sample. With increasing PEG content, the PEG
Figure 2. ERect of time on separation of PEG (P) from active (A) froctions Schematic diogram for 10% PEG level, small scale apparatur
internal standards of known PEG content, the PEG level of a given unknown may be easily estimated visually from color intensity and area to within a 5% range. Sensitivity commences a t approximately 10 pg. (2% of a 500-pg. sample) for PEG in the 1000 molecular weight range and a t 75 pg. of PEG (15% of a 50O-fig. sample) for a molecular weight of about 200. The separation is shown schema6 ically in Figure 2 as a function of developing time. The slower motion of PEG apparently results from its greater sorption by the cellulose relative to active material. Active zones are little sorbed by the paper and move rapidly near the solvent front. Separation was accomplished within 4 hours, though a 5-hour development period is recommended for more satisfactory resolution. R , values generally varied between 0.8 and 0.95 for active zones and between 0.4 and 0.7 for PEG fractions. Nonionic surfactant actives showed little variation in RJ values even for samples differing widely in hydrophobe chain length. However, trends of decreasing R, with increasing EO chain lengths were found for the various hydrophohes tested. Spreads between RJ values for 5 and 30 EO adducts were usually within 0.1; therefore, i t is possible to distinguish only he-
data for Carboaaxes, it was observed that spot areas also increase logarithmically with PEG molecular weight (the increase rapid at first, then diminishing). PEG used in preparing internal standards should, therefore, be in the same molecular weight range as the PEG contained in the sample being analyzed. The precision and accuracy of results obtainable with the spot area method are given in Table I. Determined PEG contents have a standard deviation of 4 to 6y0>permitting assignment of PEG to the nearest per cent. Absolute accuracies or recoveries were 94 to 98% of actual values.
/0.3
5 1.0
25
12.5 2.5
5.0
31.5 7.5
Pm
Figure 3.
2%
%:;
>:lo
ii:;
Pli.”tltIr
Relation of PEG spot areas to
PEG
23
quantity ACKNOWLEDGMENT
tween extremely high and low EO adducts with the developing solvent employed. This explains the occurrence of only one zone for each surfactant active tested. PEG components showed greater variation in R/ values than was found for actives; these values also diminished with increasing molecular weight or EO mole ratio of the parent surfactant sample. From Rf data, it appears possible to distinguish PEG’S differing by about 300 units (6.8 EO) in molecular weight. PEG-300 ( R , = 0.6) and PEG-1540 (R, = 0.4) components of Carbowax 1500 were completely resolved by this procedure. Comparison of aqueous solubilities with resulting R/ values for Carbowaxes indicates that differences in travel rates stem from adsorption differences
and not from solvent partitioning. Adsorption of PEG by cellulose increases with EO chain length. Estimation of PEG from Spot Areas. The spot area method was examined most thoroughly and was adapted for routine use. Figure 3 shows the increase in P E G spot areas with P E G quantity between 12.5 and 100 pg, The variation of spot areas decreases substantially above a 50pg. PEG level (10% of a 500-pg. sample). Best sensitivity is obtained between 12.5 pg. (2.5%) and 50 pg. (10%) of PEG, the area a linear function of PEG content in this region. Below 12 pg., PEG areas cannot be measured reliably. Over the entire 12.5- to 100-pg. range, the spot area varies linearly with log pg. (or log per cent) PEG. From
The authors gratefully acknowledge the samples and assistance provided by R. L. Evers, W. B. Satkowski, and associates of the Inorganic Research Division, Monsanto Chemical Co. LITERATURE CITED
(1) Flory, P. J., J . Am. Chem. SOC.62, 1561 (1940). (2) Ginn. M. E.. Church. C. I,.. ANAL. CHEM.’31, 551 (1959). ’ (3) Jaminet, Fr., J . pharm. Belg. 9, 318-24 (1954). (4) Kelly, J., Greenwald, H. L., J. Phys. Chem. 62, 1096 (1958). (5) Malkemus, J. D., Swan, J. D., J. Am. Oil Chemists’ SOC.34, 342 (1957). (6) Mayhew, R. L., Hyatt, R. C., Ibid., 29, 357 (1952). (7) Kakagawa, T., Muneyuki, R., Shinogi & Co., Amagasaki, Japan, “Convenient
.,
Method to Remove Polyethyleneglycol Included in Nonionic Surfactant,” Rept.
7 (1957). (8) Kakagawa, T., Kakata, I., J. Chem. SOC.Japan, Ind. Chem. Sec. 59, 1154-6 (1956); J . Am. Oil Chemists’ SOC.34, 64 (1958). (9) Rosen, M. J., ANAL.CHEM.29, 1675-6
flW571. \---
Table I.
Samples
Accuracy and Precision Obtained by Spot Area Method
Actual % PEG
Detd. RIean
Std. Dev.
%
Std. Dev.
Acciiracv
as % oi Actual Value
A B C A, B, and C prepared by reconstituting active and PEG fractions isolated from a polyoxyethylated alcohol. Eight replicates involved for each sample. 5
I
(10) Stevenson, D. J., Analyst 79, S o . 941, 504-7 (1954). (11) Weeks, L. E., Ginn, M. E., Baker, C. E.. Soaz, Chem. Svecialties 33. No. 8. 47-50, 113; 115 (195f). (12) Wrigley, A. N., Stirton, A. J., Howard, E., J . Org. Chem. 25, 439 (1960).
RECEIVEDfor review May 20, 1960. Accepted August 10, 1960. Division of Analytical Chemistry, 138th Meeting, ACS, New York, N. Y., September 1960.
VOL. 33, NO. 1, JANUARY 1961
145
