standard deviation was 0.005 and 0.012 absorbance units at, 227 mp and 209 mp or a relative btandard deviation of 0.8% and 1 .0%, respectively. .$bout 30 minutes are required for a series of four determinations. The utmost care should be used in measurement a t the 209 mp wavelength because the reagent blank solution is absorbing and small differences in operational procedure or spectrophotometer settings often result in significant deviations. Composition of the precipitate was determined by relating the final ultraviolet absorbance to the amount of molybdate and comparing this with the amount of thallium taken initially. Standard absorption spectra were prepared using known amounts of molybdate and thallium(1) dissolved in the buffer solution. The absorbance obtained for a solut,ion of a dissolved precipitate of thallium(1) molybdophosphate was corrected for the absorbance due to thallium and then the
corresponding amount of molybdate was obtained by referring to the absorbance us. molybdate concentration graph. Molar ratios of molybdate to thallium of 6.2 to 1 when the absorbance was measured a t 227 mp and of 6.1 to 1 when the absorbance was measured a t 209 mp were obtained. These values correspond closely to a formula of T12 H P ( M O ~ Ofor ~~ the ) ~precipitation. This formula corresponds to that obtained for the ammonium salt of molybdophosphoric acid in other investigations ( 5 , 1 5 ,f7). LITERATURE CITED
(1) Ayres, C. H., ANAL.CHEM.21, 652 (1949). (2) Berg, R., Fahrenkamp, E. S., Roebling, W., Mikrochemie (Molisch Festschrzft) 44, p. 44 (1936). ( 3 ) Boltz, I>. F., Mellon, M. G., IND. ENG.CHEM.,ANAL.En. 19, 873 (1947). (4)'Bnsev, A. I., Tiptsova, 1.. G., 'Yauchn. Doh. Vysshei Shkoly, Khim. i Khim. Technol. 1959, No. 1, p. 105. (5) Clarens, J., C o m p . Rend. 166, 259 (1918).
(6) Efremov, G. V., Syiii, C., Vestnik Leningrad. Cniu. 13, Ser. Fiz i Khim. KO. 3, p. 156 (1958). (7) Foley, W. T., Pottie, R. F., ANAL. CHEM.2 8 , 1101 (1956). ( 8 ) Gladyshev, V. P., Tolstikov, G. A,, Zavodskaya Lab. 22, 1166 (1956). ( 9 ) Iijinia, S., Kamemoto, Y., J . Chem. SOC.Japan, Pure Chem. Sect. 75, 1294 i 1S.i4\ \ - -
- I
(10) Jamrog, D., Piotrowski, J., M e d . Pracy 9, 299 (1958). (11) Merritt, C., Jr., Henhenson, H., Rogers, L. B., ANAL. CHEM. 25, 572 (1953). (12) Onishi, H., Bull. Chem. SOC.Japan 29, 945 (1956). (13) Pavelka, T., Morth, H., Mikrochemze 11, 30 (1932). (14) Ringbom, A., Z. Anal. Chem. 115, 332 (1939). (15) Stockdale, D., Analyst 83, 24 (1958). (16) Voskresenskaya, N. T., Zhur. Anal. Khzm. 11, 585 (1956). (17) Wendlandt, W. W., Anal. Chzm. Acta 20, 267 (19.59). RECEIVEDfor review March 20, 1964. Accepted November 19, 1064. Presented March 3, 1964, at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy.
Spectrophotometric Determination of High Molecular Weight Quaternary Ammonium Cations with Picric Acid Application to Residual Amounts in Polysaccharides J. H. SLONEKER, J. B. MOOBERRY, P. R. SCHMIDT, J. E. PITTSLEY, P. R. WATSON, and ALLENE JEANES Northern Regional Research laboratory, Peoria, 111.
b Quaternary ammonium compounds serve as precipitants for isolation and purification of acidic polysaccharides. An assay procedure was developed to measure the residual quantities of quaternary ammonium cation remaining in the purified polysaccharide. After the polysaccharide is hydrolyzed with phosphoric acid, the quaternary ammonium cations are precipitated quantitatively with picric acid. The insoluble quaternary ammonium picrate salt is selectively extracted from the aqueous hydrolyzate with chloroform, and the absorbance of the salt is measured spectrophotometrically ( A 365 mg, e 1.5 X 1 04). The procedure is sensitive to about 8 gg. of quaternary ammonium cation in 80 mg. of the microbial polysaccharides and is not affected by the presence of amino acids or amino sugars. The procedure may be adapted also to the determination of certain secondary and tertiary amines.
Q
UATERNARY AMMONIUM COMPOUNDS
have been used frequently in research either to purify polyanionic polysaccharides or to fractionate acidic polysaccharides from neutral polysaccharides by a selective precipitation technique (16, 17). Xt this laboratory an industrially feasible method has been developed by which an acidic microbial polysaccharide is precipitated and recovered from the fermentation broth with Arquad 16-50, a commercial preparation of cetyltrimethylammonium (CTA) chloride (1, 2). Since CTh ions have no visible or ultraviolet absorption spectrum, residual amounts of this base in the polysaccharide cannot be measured direct'ly by spectrophotometry. Previous assay procedures (4, 7 , 2 1 , f2), like our method, took advant'age of the strong cationic properties of quaternary ammonium ions (QK+). i\nionic dyes were reacted with the Q N + and the resultant color complexes were measured sl)ectrol)hotometrically,
However, these methods were unsatisfactory for our purposes. Hydrolysis and neutralization of the polysaccharide solutions produced high concentrations of carbohydrate and salt. In such mistures the polyfunctional dyes developed comples equilibria with the Q N + (f2), which caused poor reproducibility of results. Att'empts to sei)arate the QS+ from carbohydrate and salt on cellulose ion-exchange columns were also unsatisfactory ( 11 ) . The determination of Q N + in the presence of unhydrolyzed polysaccharide and in the absence of salt was prohibited by the extremely viscous nature of t'hese solutions. Picric acid mas chosen as the reagent for assaying QS+because it has a strong ultraviolet absorl)tion spect um and a single strong anionic functional group that reacts railidly and quantitatively with the base. 'This reagent has been used for many years to precipitate and identify organic cations and to determine the molecular weight of organic, VOL. 37, NO. 2, FEBRUARY 1965
243
cations both by spectrophotometric (6, 14, 15) and titrimetric (3, 8 , 19) means. In our assay procedure the QN+ picrate salt is conveniently separated from the excess picric acid by a simple chloroform extraction, and the absorbance of the extract is measured spectrophotometrically a t 365 mp. The assay is simple, rapid, insensitive to carbohydrates (including amino sugars) and amino acids, and is accurate to about 8 pg. of QN+ in 75-80 mg. of polysaccharide. EXPERIMENTAL
Reagents and Materials. All solutions were prepared from reagentgrade chemicals. X phosphoric acid solution (3M) was prepared by diluting 85y0 phosphoric acid with distilled wafer (1 : 5). A sodium hydroxide solution (3M) was prepared and adjusted so that a volume of it neutralized an equal volume of 3J1 phosphoric acid to give a salt solution of p H 4.3. A one-half saturated picric acid solution (picric acid solubility: 1.2 grams per 100 ml. of water) was prepared and stored in a brown bottle. Cetavlon (CTA bromide, Eastman Kodak Co.), the purity of which was established by elemental analysis, was used as a standard. Solutions of Cetavlon were prepared that had a CTA cation concentration of 0.01 to 0.05%. Crystalline CTA picrate was prepared by mixing 100 mg. of Cetavlon dissolved in 10 mi. of 1% potassium chloride with 150 mg. of picric acid dissolved in 20 ml. of distilled water. The crystalline precipitate was recovered by filtration, washed with distilled water, and dissolved in a minimal quantity of absolute ethyl alcohol. Vpon addition of water to the alcohol, crystalline platelets of CTA picrate formed. Three recrystallizations from alcohol and water afforded 108 mg. of a pure product. The crystalline CTX picrate has used to make standard curves by serial dilution in chloroform. The samples of acidic polysaccharides were produced by the bacterium Xanthomonas campestris XRRL B-1459 ( 9 , 13) and by yeast Cryptococcus laztrentii var. jlavescens S R R L Y-1401 (5, IO). Apparatus. Spectrophotometric measurements were made with a Beckman Model I3 spectrophotometer using selected and matched 18- X 150-mm. borosilicate glass test tubes. Procedure. STANDARD CURVEI N ABSENCEO F POLYSACCHARIDE. A11 determinations including reagent blanks, were run in duplicate. Into 12-ml. conical centrifuge tubes was pipetted 0.1 t o 1 ml. of 0.01% Cetavlon solution, which was equivalent to 10 to 100 p g . of CTA cation. Distilled water was added to make the final volume to 1.5 mi. Phosphoric acid (1.0 ml. of 351 solution) was mixed into the contents of each tube. The phosphoric acid solution wab neutralized to pH 4.3 with 1.0 ml. of 3.11 sodium hydroxide solution. Picric acid (0.1 nil. of the one-half saturated solution) was added to each tube, and upon
244
ANALYTICAL CHEMISTRY
ZL
E
Salt Concentration in Aqueous Layer, X
2
Figure 1 . Distribution of cetyltrimethylammonium picrate between chloroform and aqueous salt solution; absorbance of aqueous layer A.
Sodium chloride, p H 4.7
B. Sodium chloride, p H 7.0 C.
Sodium phosphate; X, p H 4.3 and 0 , p H
7.0
mixing the contents of the tubes, a precipitate of CTA picrate formed. The precipitate was extracted from the aqueous picric acid solution with three 1.5 ml. portions of chloroform. A Vortex J r . test-tube mixer was used to obtain intimate dispersion of the immiscible liquids. After each extraction, the chloroform layer was allowed to clarify and then vas transferred to a IO-ml. volumetric flask with the aid of a capillary medicine dropper. The chloroform solution was made to volume and transferred to a colorimetric tube. The absorbance of the CTA picrate in the chloroform was measured at 365 mF. Care was taken to minimize the amount of aqueous picric acid mechanically carried over to the 10-ml. volumetric flask during extraction. However, the picric acid remained in the aqueous phase, and the error caused by minute changes in the volume of chloroform was canceled by use of reagent blanks. STANDARD CURVE IN PRESENCE OF POLYSACCHARIDE. Varying amounts of polysaccharide, ranging up to 80 mg., were weighed into 12-ml. centrifuge tubes and allowed to dissolve overnight in 1.5 ml. of distilled water containing appropriate amounts of CTA cation. One milliliter of 3M phosphoric acid was added and mixed into the extremely viscous solutions of polysaccharide. T o destroy viscosity, the acidified solutions of polysaccharide were autoclaved (120’ C., 15 p.s.i.) for 20 to 25 minutes. After the tubes and their contents were cooled, 1 ml. of 3M sodium hydroxide solution was added to neutralize the acid. The rest of the procedure is the same as already stated, except that during extraction with chloroform an emulsion sometimes forms owing to the high concentration of carbohydrate material and traces of partially hydrolyzed protein in the aqueous layer. This emulsion is readily broken by centrifugation of the assay tubes to obtain efficient separation of the two phases. Identical standard curves were obtained in either the presence or absence
of polysaccharide or by a serial dilution of a known quantity of crystalline CTA picrate dissolved in dry chloroform. At pH 4.3 negligible quantities of picric acid were extracted from the aqueous layer. Thus the chloroform blanks had desirably low absorbance values. DETERMINATION OF & N + IN POLYSACCHARIDES. Polysaccharides (up to 80 mg., dry weight basis) containing unknown quantities of QN+ were weighed into 12-ml. centrifuge tubes and were dissolved in 1.5 ml. of water. If the concentration of Q S + in the polysaccharide is high, an aliquot of an appropriate dilution of the polysaccharide may be transferred to the centrifuge tubes. The samples were treated as previously described for the standard curve containing polysaccharide. Chloroform extracts too concentrated for determining the absorbance values of Q N + picrate can be diluted with suitable amounts of chloroform. DETERMINATION OF Q N + IX PROCESS LIQUORSAFTER RECOVERY OF POLYSACCHARIDES. Hydrolysis of the process liquors ( I , 2 ) is not necessary. .iliquots of the solutions were transferred to assay tubes, buffered with monosodium phosphate (pH 4.3), and assayed as described above for &S+. The small quantities of methanol and salts transferred to the assay solutions from the process liquors did not affect the results of the analysis. RESULTS A N D DISCUSSION
Effects of pH. Increasing amounts of picric acid are extracted by thc
chloroform as the p H of the aqueous solution decreases below 3. The resultant absorbance by the picric acid a t 365 mp produces high-assay blanks, which reduce the accuracy and sensitivity of the method. Between pH 3 and 9 a negligible amount of picric acid is extracted into the chloroform. Because certain quaternary ammonium compounds are unstable a t alkaline pH (17’) no investigations were made above pH 9. Acid hydrolysates of the polysaccharides were neutralized to pH 4.3 before the addition of picric acid and extraction with chloroform. Effects of Salts. T h e standard curves for CTA picrate vary when t h e picrate is extracted with chloroform from aqueous solutions containing certain salts. When the aqueous solutions contain sodium chloride, the standard curves pass through the abscissa rather than the origin. Such behavior indicates a partial solubility of CTA picrate in aqueous solution and incomplete extraction by chloroform. The quantitative role ,of sodium chloride was established by measuring the distribution of C T h picrate between chloroform and aqueous solutions of sodium chloride a t pH 4.7 and 7 . Figure 1 shows the absorbance of the aqueous layer a t 365 mp us. the concentration of salt in the aqueous layer.
An appreciable increase in absorbance is observed in the aqueous layer as the sodium chloride concentration is increased and the p H is lowered from 7 to 4.7. The high ionic strength of the solution of sodium chloride does not salt out the CTA picrate as expected but apparently shifts the equilibrium of the precipitation reaction to produce small quantities of CTA cations and picrate anions in the aqueous layer. -1sa result, C T h picrate is not extracted quantitatively from the aqueous layer by chloroform. Sodium salts of phosphoric acid do not interfere with the precipitation of CT.l picrate as does sodium chloride (Figure 1 ) . The distribution of CTA picrate between chloroform and solutions of sodium phosphate a t p H 4.3 and 7 differed little from that between chloroform and distilled water. Standard curves made with monosodium phosphate in the aqueous layer were linear and passed through the origin. Also, the aqueous solutions of sodium phosphate have good buffering action over a wide range of pH values.
Effects of Amino Acids and Amino Sugars. Amino acids and amino sugars do not interfere with the determination of Q N + in solutions of polysaccharide a t concentrations as high a5 10 to 20% of that of the polysaccharide (Table I). The assay method, therefore, would seem applicable to measuring Q N + content of either acidic polysaccharides that also contain amino sugar residues or crude samples of polysaccharide that contains proteinaceous impurities. Acid Hydrolysis. Aqueous solutions of our microbial polysaccharides have high viscosity which must be eliminated by acid hydrolysis before an analysis for Q N + can be made. The polysaccharide produced by X . campestris, which is rather stable to hydrolysis with hydrochloric acid and sulfuric acid ( I @ , is very stable to phosphoric acid under normal hydrolysis conditions. However, adequate hydrolysis with phosphoric acid was achieved by autoclaving the solutions for 20 minutes a t 120" C. and 15 p s i . For the same degree of hydrolysis at 100" C., 6 to 8 hours were required. Addition of a n equimolar quantity of sodium hydroxide converts the autoclaved hydrolyzate to a solution of 0.8M monosodium phosphate buffered a t pH 4.3. The QK+ is readily recovered without loss from such a buffered solution. Also a t this pH, negligible amounts of picric acid are extracted into the chloroform and the assay blanks have low absorbance values. Q N + Content of Polysaccharides. Quantitative determination of QN+ was made on several preparations of polysaccharide precipitated by Cetavlon
or Arquad 16-50 and purified to different degrees (Table 11). When the polysaccharide samples produced by X. campestris are merely precipitated from cell-free culture broth and dehydrated with methanol, the precipitates contain 9.4% and 13.6y0 Q K + on a dry weight basis (preparations 37 and 38). If the precipitated polysaccharide is washed four times with methyl alcohol containing 0.2% potassium chloride, one-third to onehalf of the Q N + is removed (pilotplant preparations 37-4 and 37-6). Washing the precipitated polysaccharide nine times with alcoholic potassium chloride removes essentially all of the QN (pilot-plant preparations 35 and 110) (1, 2). Laboratory-scale purification of QN +-precipitated polysaccharide by dissolving the product in aqueous potassium chloride and precipitating it with alcohol several times resulted in complete removal of QY+ from the polysaccharide produced by C. laurentii and almost complete removal from duplicate samples of polysaccharide produced by X. campestris (preparations 34h and 34B). Sensitivity of the & N + assay is determined by the viscosity of the microbial polysaccharides, which limits the amount used in each assay tube. For polysaccharide samples having weights above 80 mg., the gel-like solution could not be mixed adequately with the phosphoric acid before hydrolysis. Increased sensitivity may be achieved, however, by hydrolyzing a dilute solution of polysaccharide and by evaporating the hydrolyzate to an appropriate concentration before continuing with the analysis. Adaptability of Assay. T h e assay is applicable to other quaternary ammonium compounds, as well as t o certain secondary amines. Linear standard curves were obtained for cetyltrimethylammonium chloride, +
Table II.
cetylpyridinium chloride, D,L-coniine, dicyclohexylamine, dodecyltrimethylammonium chloride, and trimethylarachidyl - behenylammonium chloride (Adogen 401). The measure of cetylpyridinium chloride is four times more sensitive by the picric acid procedure than by the direct spectrophotometric procedure. The molar absorptivity of cetylpyridinium chloride is 4 x lo3 at 260 mp (171, whereas the molar absorptivity of the cetylpyridinium picrate is approximately 1.5 X lo4 at 365 mp. Our observations show that for cetyldimethylbenzylammonium chloride the picric acid procedure is 20 times more sensitive than the direct spectrophotometric assay procedure. The secondary and tertiary amines of low molecular weight, pyrrolidine and N-methylpyrrolidine, could not be assayed by this procedure. The N methylpyrrolidine reacted with picric acid, but the product could not be extracted quantitatively with chloroform. Application of the assay to compounds containing secondary amines or QN+ functional groups depends on the hydrophobic nature of the cation and the solubility or dissociation of the picrate salt in water. However, the procedure should be applicable to certain tertiary amines since application to secondary amines was demonstrated.
Table I. Effects of Amino Acids and Amino Sugars on Determination of Cetyltrimethylammonium Cation
Compound added L-Proline D,L-ASartic
CetCetavlon avlon Amount, added, found,
Acicf
L-Isoleucine D-Glucosamine
mg. 10 0
fig.
erg.
100
99 6
10 0 10.0 1.0
100 100 100
99 5 101.0 100.5
Quaternary Ammonium Cation Content in Polysaccharides Precipitated with Cetavlona and Arquad 16-5Ob
Polysaccharide source Cry tococcus laurentii d R R L Y-1401 Xanthommas campestris NRRL B-1459
Cation Polysaccharide content, PP". Yo Laboratory-Purified 231 0
Std. dev., ycc 0
