Examination of Distribution of Substituents in Partially Methylated Cellulose by Gas Liquid Partition Chromatography W. BROCK NEELY, JULIE NOll, and C. B. ROBERTS Biochemical Research laboratory and Analytical laboratory, The Dow Chemical Co
b Gas liquid partition chromatography has been used to identify and examine the disiribution of methoxyl groups in partially methylated cellulose. The method has been successful in identifying the tri-, and di-0-methyl isomers of methyl glucosides. More work will be required to extend the technique to the mono methyl derivatives and to unsubstituted glucose. The results of this technique on methylcellulose ranging in degree of substitution from 0.28 to 3.0 are given.
I
THIS laboratory, we have been interested in applying gas liquid chromatography to identify and examine the various methyl ethers of glucose obtained from the partial methylation of cellulose. I t was felt that GLC analysis of these substituents would be more rapid than the time consuming column paper chromatographic techniques of Croon and Lindberg (3). Several investigators ( I , 7-9) have studied the possibility of using GLC in this regard, and great strides have been made in the identification of the various methyl glucosides. I n such a study, it is difficult to obtain known samples of the glucose ethers of suitable purity for identification purposes, and to obtain a derivative which will be volatile a t the column temperature. The most convenient procedure for preparing this type of compound has been the conversion of the glucose ethers to the methyl glucosides. Unfortunately, this technique converts the methyl ether of glucose to a mixture of alpha- and betaisomers. Because both isomers are usually separated on the chromatogram, they both require identification. This introduction of isomers compounds the difficulty of identification and interpretation. The use of glycose acetates is receiving attention (4, 11) as is also the forniation of glycitol acetates (4). The latter method may become quite important because the formation of glycitol acetate destroys the asymmetry of the anomeric carbon atom, thus giving rise to only one peak in GLC. Another technique which has not received too much attention has been the use of N
, Midland,
trimethyl silyl ethers of sugar derivatives (2, 5, 6). All of these methods are important and will undoubtedly receive more attention in the future. I n this study me have used methyl glucoside formation to obtain material suitable for injection in the GLC unit. EXPERIMENTAL
Apparatus. The gas chromatograph was a n Aerograph Hy-F1 Model 600, Wilkins Instrument a n d Research, Inc., Walnut Creek, Calif. A hydrogen flame ionization detector was used, with nitrogen as the carrier gas. The column packing which provided best results was 25y0 LAC-2-R-446 (polydiethyleneglycolpentaerythritoladipate) and 2% phosphoric acid on 80 to 100 mesh Chromosorb W. The packing was prepared in the manner described by Metcalfe ( I O ) . A %foot, '/*-inch stainless steel column was coiled to fit into the chromatograph and packed by vacuum, with vibrating, to ensure a tightly packed column. I t was allowed to bleed overnight a t 200" C. before use. The sample injection block was heated to 240' C., and the carrier gas was set a t 30 p.s.i.'inlet pressure wirh the oven temperature a t 200" to 210" C. Attenuation was adjusted to provide the desired peak size, and samples ranging from 2 to 4 pl. were injected. A 1-foot LAC column was prepared in the same way and operated a t 220" C. and 23 p.s.i. carrier gas to detect compounds which had longer retention times than methyl-2,B-di-O-methyl-nglucopyranoside. Other columns which were tried included a 6-foot Carbowax 20hI Alkaline on 60 to 80 mesh Chromosorb R and a %foot Apiezon L on 60 to 80 mesh Chromosorb R. Hydrolysis Procedure. Two techniques were investigated for the hydrolysis of the polysaccharide. METHANOLIC HCl. The material was dissolved in equal portions of chloroform and 10% methanolic HC1 and placed in a sealed tube. The tube and contents were reacted a t 100' C. for 6 hours a t which time the solution was removed, evaporated to dryness, and taken up in chloroform for analysis by GLC. The sample was converted in one step to the corresponding methyl glucoside suitable for injection.
Mich.
The major disadvantage was that only those methylcelluloses with a degree of substitution greater than 2 were soluble in the reaction mixture. The technique appeared to be unsuited for those materials with a degree of substitution less than 2. SULFURICACID HYDROLYSIS.This method of Croon and Lindberg (3) appeared to be best for our purposes. We modified the procedure slightly in that we refluxed the sample directly in the diluted sulfuric acid after dissolution in the cold 72% acid. A comparison of yields of the methyl ethers from identical samples subjected to 3-, 6-, and 15-hour hydrolysis showed essentially no difference; consequently a 4- to &hour refluxing was adopted as standard procedure. The variation in hydrolysis time appeared to affect the extent of acid degradation products of the unsubstituted glucose. This will be discussed briefly in the Results. After the acid hydrolysis, the solution was neutralized with barium carbonate, filtered, washed, and concentrated to dryness in vacuo. The residue was
Table
I.
Methylcellulose Samples Used
Material
OCH,, %
1 2 3 4" 5
5 3 9 3 14 3 26 30 3 38 3 45 2
6 r
l
Degree of substitution 0 28 0 0 1 1 3 3
5
78
5 8 4
Material was prepared by using dimethyl sulfate and 307, XaOH as the methylating reagents.
Table II. Possible Glucose Ethers Resulting from Depolymerization of Partially Methylated Cellulose 1. 2,3,4,6-tetra-0-methyl-~-glucose 2. 2,3,6-tri-0-methyl-o-glucose 3. 2,6-di-0-methyl-n-glucose 4. 2,3-di-O-methyl-n-glucose 5. 3,6-di-0-methyl-n-glucose 6. 6-mono-O-methyl-~-glucose
7. 3-mono-O-methy~-~-glucose 8. 2-mono-O-methy~-~-glucose 9. D-glucose
VOL. 34, NO. 1 1 , OCTOBER 1962
1423
2
4
6
8 10 I2 TIME (MINUTES)
14
,6
I8
Figure 1. Chromatogram of hydrolyzed and methanolyzed sample 3, Table 111 Column was a I-foot LAC-446 (25yoJ, H 3 P 0 4 Column temperature was 220' C. with port temperature of 240OC. Nitrogen pressure was 23 p s i .
(2%).
taken up in 27, methanolic HCl and refluxed. The solution was again evaporated to dryness in vacuo and extracted with chloroform. The resulting solution m s filtered, evaporated to dryness, and taken up in a known volume of chloroform for injection. Identical samples were refluxed for I, 2 , 4, and 8 hourq. Results indicated that maximum amount of glucosides were produced with 2 or more hours of refluxing. I n subsequent experiments n e used 4hour reflux as standard procedure. Materials. The methylcelluloses t h a t were examined by these procedures are liqted in Table I. -ill ma-
TIME (MINUTES1
Figure 2. Chromatogram of hydrolyzed and methanolyzed sample 5, Table 111 Column was a 3-foOt LAC-446 (25%), H 3 P 0 4 (2%). Column temperature was 212'C. with port temperature of 24OoC. Nitrogen pressure was 30 p.s.i.
terials except where noted were experimental samples provided by the Cellulose and Plastics Laboratory, The Dow Chemical Co., Midland, hlich. RESULTS
Type of Column. Of the three column packings investigated, the LAC substrate with phosphoric acid on Chromosorb W gave the best results. The peaks obtained from this column were symmetrical and well defined, and resolution of components wa5 good. T h e peaks obtained from both t h e Carbowax 2011 -4lkaline Column and Apiezon L were diffuse and poorly defined; in view of this, no qualitative or quantitative nieasurenients were made with these tn-o columns. Table 111. Retention Times of Various The &foot LAC column provided Methyl Ethers of Methyl-D-glucopyranopeaks which could be determined qualiside, Relative to MethyI-2,3,4,6-tetratatively and quantitatively for com0-methyl-@-D-glucopyranoside ponents eluted before and including 2,6-di-O-methyl-~-glucose. Higher boilMethyl glucoside Rt ing components were not detected 8-2,3,4,6" 1 00 on a &foot column, but some passed ~~-2,3,4,6~ 1 3 7 i o 1 through a 1-foot LAC 446 column as @-2,3,6' ' 9 5 i O l 0-2.3.6 3 6 5 i 0 1 shonx by the chromatogram in Figure 1. The identity of these compounds is not certain although the last peak is believed to be a mono derivative. P e a k Identification. We have been able to identify the first five of the Furnished by 11. L. \\olfrom, Chem. derivatives in Table I1 by GLC Dept., Ohio State University, Columbus, Ohio. using the 3-foot L,IC 446 column. 0 Obtained from methylation of CYThese first five sugars were identified methyl-D-glucopyranoside. by injecting known compounds in a G. Mercer, Cellulose Laboratory, The chloroform solvent into the chromatoDOTVChemical Co., Midland, Mich., graph. The column temperature was supplied a mixture of the CY- and &isomers. The 3,6-di-0-methyl-o-g~ucopyranose 200' to 210' C. The retention times and the methy1-2,3-di-O-methyl-cu-~-gluco- were measured from the point of injecvranoside were supplied by C. T. Bishop, tion to the middle of the peak. All ational Research Council, Ottawa. Free retention times are recorded relative to sugar Tvas converted to methyl glucoside by refluxing a sample in 2 5 methanolic the retention times for methy1-2,3,4,6HCl for 3 hours. tetra - 0 - methyl - p - D - glucopyranoe F. Smith, Dept. of Biochemistry, side. The standard sugars and their University of Minnesota, St. Paul, llinn., furnished this sample. corresponding retention times are listed in Table 111. LL
0
5
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ANALYTICAL CHEMISTRY
Identification of Standards. From the observations of Bishop ( I ) the first peak of the anomeric pair of the tetra- and tri-0-methyl isomers has been given the beta configuration. The 3,6- and the a-4,6-di-O-niethyl isomers were not separated on the &foot LAC column. However: in partially methylated cellulose this presents no problem, as the 4:6-di-O-niethyl isomer could only arise from a partial niethyletion of a terminal end group end would not be present in sufficient amount to be detected. Seither were the a- and @-isomers of meth~-1-3,6-and methyl2,6-di-O-methyl-~-glucoaides separable on this column, This as an adrantage in this investigrtion as it resulted in fewer pesks in the chromatograms of hydrolyzed methylcellulose. Analytical Determinations on Methylcellulose. -1typical chromatogram resulting f r o m the standard procedure for hydrol:, nolysis is shown in Figure 2 . This chromatogram resulted from t'rent'ing sample 5 , Table I. The retention times of t,he various peaks corresponded t o t h e kno1vn derivatil-es and are marked accordingly o n the chromatogram. The peak areas m-ere determined by triangulation. The results are shown graphically in Figures 3 a n d 4. =issuming that the m o u n t of methyl2,3.6-tri-0-methyl-~-ylucoside resulting from the hydrolysis and methanolysis of bhe fully methylated cellulose (Sample 7 , Table I) represents 100% recovery, the right hnnd scale in Figures 3 and 4 may be di\.ided into mole per cent of methyl glucoside. The results so obtained agree with the results of Croon and Lindberg (3) u-ho used column and paper chromatographic techniques for their analyses. Unidentified peaks. There were a number of peaks in the methyl-
treated in a manner similar to the methylcelluloses. Il70rk is in progress to identify the nature of these components. It is felt that some of these low boilers probably result from acid degradation of the unsubstituted glucose in the methylcellulose.
'0
c1
2,J-DI-O.METHYL
i A'\
08
I
44
06
4u\q20 ACKNOWLEDGMENT
"t
The authors express their appreciation t o M. L. Wolfrom, F. Smith, and C. T. Bishop for their generous gifts of the methyl glucosides in Table 111.
/
2
0
1
0
0 1.0
2.0
Figure 3. Plot o f mm.? of methyl glucoside/microgram o f methylcellulose against degree of methoxyl subsritution (ds.)
cellulose samples t h a t appeared before the tri-0-methyl-isomer. They seemed to vary in a random way; however, the peak t h a t emerged immediately after the chloroform appeared to vary inversely with the degree of substitution of the polymer used and directly with the extent of acid hydrolysis. This peak also appeared in samples of glucose, cellobiose, and cellulose that n.ere
E 04
l l
I
0 2
c
/ / .
LITERATURE CITED
30
DS
m
k
(1) Bishop, C. T., Cooper, F. P., Can. J . Chem. 38,388 (1960). ( 2 ) Calvin, M. L., Ferrier, R. J., Univ. of
Calif., Berkeley, Calif., personal communication from R. J. Ferrier July 28, 1961. (3) Croon, I., Lindberg, B., Svensk Papperstidn. 60, 843 (1957). (4) Gunner, S. W., Jones, J. K. N., Perry, M. B., Can. J . Chem. 39, 1892 (1961). (5) Hedgley, E. J., Meresz, O., Overend, W. G., Rennie, R., Chem. and I n d . (London), 938 (1960). (6) Hedgley, E. J., Overend, IT. G., Ibzd., 378.
( i jKircher, H. R., ANAL. CHEM. 32, 1103 (1960).
(8) Klein, E., Barter, C. J. Jr., Textile Res. J . 31, 486 (1961). (9) McInnes, A . G., Ball, D. H., Cooper,
3
2 0
I O
30
OS
Figure 4. Plot of m m 2 o f methyl gluof methyl-cellucoside/microgram lose against degree of methoxyl substitution
F P., Bishop, C. T., J . Chromatog. 1 , 556 (1958). (10) Metcalfe, L. D., Facts and 'Methods 2 , No. 1, (1961). (11) Vanden Heuvel, W. J. A,, Homing, E. C., Baochem. and Biophys. Res. Comm. 4, 399 (1961). RECEIVED for review March 15, 1962. Accepted June 4, 1962. Division of Cellulose Chemistry, 142nd Meeting, ACS, Atlantic City. September 1962.
A Complete Separation of a Mixture of Iron(M), Co b a It(II), Molybdenum(VI), AI uminum(Ill), and Nickel(l1) by Ion Exchange Chromatography CARL MICHAELIS, NANCY SPIRES TARLANO,' JULIANNA CLUNEI2 and ROBERT YOLLES3 Chemistry Department, University o f Dayton, Dayton, Ohio
b A detailed method is reported for separation of a mixture o f Fe(lll), Co(ll), Mo(VI), Ni(ll), and AI(III) on Dowex cation and anion exchange resins using hydrochloric acid elution. The nickel and aluminum which d o not form stable anionic chloro complexes in strong hydrochloric acid solution were not retained by the anion exchange resin, while the iron, cobalt, and molybdenum which d o form the anionic chloro complexes were retained b y the resin. After the nickel and aluminum were eluted from the anion exchange column with strong hydrochloric acid, they were separated on the cation exchange column. The cobalt, iron, and molybdenum were eluted successively from the anion exchange column b y selecting the proper concentration of eluting acid. Elution curves were prepared for iron, cobalt, molybdenum, nickel, and aluminum.
T
PROBLEMS of quantitatively separating the metals which occur in very high strength, corrosion resistant, and high temperature resistant alloys are well known. Complex precipitation methods are usually used. Ion exchange affords a convenient method of separating many of these metals. Some metals form stable anionic chloro complexes in hydrochloric acid solutions and therefore can be separated on anion exchange resins from those which do not (5, 6). Those which do not form the complexes may upon dilution be added to a cation exchange column. Then if the proper concentrations of hydrochloric acid can be determined experimentally, the ions can all be eluted successively. Hague, Maczkowske, and Bright (2) separated molybdenum, iron, and cobalt by first precipitating the molybdenum as the sulfide and then separating cobalt HE
and iron on the anion exchange column. Wilkins (8) separated molybdenum from iron and cobalt among other metals by making an HF-HC1 solution and passing the mixture through a n anion exchange column. This requires special apparatus and care in working with toxic HF fumes. 15Tilkins and Hibbs (9) separated aluminum and nickel from a number of other metals and then determined them in presence of each other. The procedure here described is a simple method of separating all f i ~ eof the metals consecutively using only hydrochloric acid. Elution curves were prepared by adPresent address, Chemical Atxtracts, Ohio State University, Columbus, Ohio. Present address, Stanford Research Institute, Palo Alto, Calif. Present address, Chemistry Dept., Univ. of California, Berkeley, Calif'. VOL. 34, NO. 1 1 , OCTOSER 1962
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