Activated Ce IIuI ose for Solution-Adsorptio n Chromatography CLETA K. MILLER, DANIEL STEFFENSON, HARLAN D. FRAME, Jr., and HAROLD H. STRAIN Argonne National I aboratory, Argonne, 111.
b Cellulose, when ground in the presence of polar liquids and carefully dried, exhibits increased adsorption capacity for carotene and related pigments. The selectivity of this activated cellulose for the separation of carotenoid pigments is improved, but it is still more like that of the unground, nonactivated cellulose than that of the activated inorganic adsorbents such as magnesia.
C
is usually a weak, surface-active adsorbent. Fibrous cellulose, as employed in paper chromatography, and powdered cellulose, as eniployed in column chromatography, exhibit little or no adsorption affinity for various hydrocarbons dissolved in the least polar liquids, such as lowboiling, aliphatic hydrocarbons. For eramplr, many papers and povidered cellulose do not adsorb the polyuns:tturated hydrocarbon carotenes from their solutions in petroleum et'lier. In this respect,, cellulose resembles other condensed sugars often employed as adsorbents, notably powdered sucrose, inulin, and starch. Because these polysaccharides do not adsorb the carotenes, they do not serve for resolution of mixtures of thesc hydrocarbons (I,@. In the course of our investigations of chromatographic adsorbents, certain preparations of cellulose were found to exhibit pronounced adsorption capacity for carotene. This surprising observation led to an investigation of the condit,ions that enhance the adsorption capacity of cellulose. These studies showed that several conditions, such as grinding with liquids followed by their removal, were essential to the preparation of activated cellulose. Khen these conditions were controlled, reproducible, sorptive preparations 11-ereobtained. ELLCLOSE
EXPERIMENTAL
Materials. All the cellulose employed i3-as powdered cellulose (Whatman powdered cellulose, standard grade. ashless, chemically prepared for chromatography), Liquids employed for wet grinding were reagent grade preparations. Carotene, employed for tests of adsorption capacity, was synthetic @carotene and was chromatographically identical with 8-carotene from leaves. Carotenoid and chloroplast pigments, employed for tests of selectivity, were obtained from natural sources. Lycopene from tomatoes and a-carotene from
carrots mere purified by chromatography on activated magnesium oxide. Chloroplast pigments were extracted from fresh spinach leaves ( 5 ) . Xanthophylls or hydroxycarotenes were portions of preparations described before(7). Determination of Adsorption Capacity and Selectivity. The adsorption capacity of the cellulose preparations was compared by measurement of the R value of a nearly saturated solution of B-carotene (10 mg.) in n-heptane (100 ml.). T o this end, the treated cellulose was packed in chromatographic tubes 1 =t 0.05 cm. i.d. by 24.5 cm., which were filled to a height of 20 cm. by pressing small successive portions of the dry preparations with a dowel. Such columns contained 7.5 5 1.5 grams of adsorbent. The carotene solution was added until the liquid reached the bottom of the column. The column volume mas 10.5 h 1 ml. The observed R values are inversely related to the adsorption capacity of the cellulose ( I ) . The R values for carotene on the activated cellulose decreased slightly over the useful concentration range, which is limited by the solubility (ca. 10 mg. per 100 ml.) and by the visible detectability (ca. 0.1 mg. per 100 ml.) of the carotene. Over this concentration range, the carotene n-as not adsorbed on the untreated cellulose ( R = 1). For the comparison of the various preparations of cellulose, the R values for carotene were determined at but one concentration. For tests of the selectivity of the activated cellulose, mixtures of the pigments were added as a narrow initial zone (about 1 cm. deep), which mas then washed with fresh solvent. The concentrations of the pigments n-ere approximately equal (5 to 10 mg. each per 100 ml.) Grinding Procedures. Three grinding methods were tested: beating with liquid in a blender, vibration of a suspension with ultrasonic radiation (Sonblaster ultrasonic generator, Xarda Ultrasonics Corp., Westbury, S . Y.), and rolling with liquid and marbles in a bottle. The blender generated heat, so that a cooling thimble had t o be suspended from the cover of the container. It could not be operated satisfactorily for long periods. The marble jar Fas a borosilicate glass bottle of about 4-liter capacity (15.5 em. i d . ) . It n a s charged n-ith 200 glass marbles 5 / * inch in diameter, 50 grams of powdered cellulose, and usually 200 ml. of liquid. It n-as rotated on a roller a t 20 r.p.m. This method was studied most extensively. Solvent Stripping. The liquid employed for the grinding was removed in various ways, The slurry was
separated with a Buchner funnel or a filter stick, and the filter cake was dried in a 1-liter, round-bottomed flask using vacuum from a water aspirator and a bath temperature of 100". Alternatively, the slurry itself was dried as just described. (If the dried material had formed a solid cake, it was broken up with a mortar and pestle.) By another procedure the slurry was diluted with about 1 liter of acetone, filtered, and washed with fresh acetone and then with petroleum ether (b.p. 20-40°), which was finally removed by evaporation in dry nitrogen. This last procedure usually yielded a finely powdered preparation even from suspensions that had been ground with water until they mere gelatinous. Variable Conditions. The effects of some five conditions upon the adsorption capacity of the cellulose were investigated: method of grinding, time of grinding, liquid employed in grinding, solvent stripping procedure, and residual solvent retained by the preparation. B y systematic variation of these conditions, it was possible to determine how each one affected the cellulose adsorption capacity. RESULTS
Ultrasonic libration of dry cellulose or of cellulose suspended in various solvents did not alter the R value of carotene ( R = 1). JIicroscopic examination indicated that the cellulose particles had not been broken up. Beating the cellulose suspension with a blender increased the adsorption capacity (decreased the R value). The activity was greatest with polar liquids such as water and methanol ( R value after beating rrith methanol for 2 hours and drying of the slurry Tvith vacuum, 0.38). I t \vas least with nonpolar organic liquids such as 72-heptanr ( R value after beating with n-heptane for 6 hours and desolvation of the slurry with vacuum, 1.00). When the dry cellulose was ground Kith marbles, the initial R value of 1.00 remained unchanged. When the cellulose m-as ground with various liquids, the adsorption affinity for carotene increased nith the polarity of the solvent, as indicated in Table I, ranging from 0.70 with carbon tetrachloride to 0.13 with water. \17ith methanol as the grinding liquid, the activity of the cellulose increaqed with the duration of the grinding up to about 20 hourq. Beyond that time, significant variations were not obserT-ed, a$ indicated iii Table 11. VOL. 35, NO. 1, JANUARY 1963
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Provided all the grinding liquid was removed, the method of solvent stripping was of secondary importance. Material ground under the same conditions provided similar R values when dried by each of the &ripping methods, except vacuum dehydration of the filtered mass, which produced a hard, less active product. For the gelatinous mass produced by grinding in water, preliminary Table 1. Variation of R Value for Carotene Adsorbed in Columns of Cellulose Ground for 20 Hours with Marbles and Various Liquids
Liquids for grinding None Carbon tetrachloride Benzene Acetone Methanol Pyridine Water
R value 1.00 0.70 0.67 0.40 0.33
0.30 0.13
Table II. Variation of R Values for Carotene on Cellulose with Time of Grinding with Marbles (Desolvation by filtration followed by
vacuum drying) Hours of Liquid grinding Methanol 0 5 10 15 20 25 40 60 80 Acetone 0 20
n-Heptane
40 20 40
R value 1.00 0.42 0.38 0.35 0.33 0.37 0.23 0.44 0.40 1.00
0.39 0.22 0.77
0.78
dilution with about 1 liter of acetone followed by filtration and washing with acetone and petroleum ether provided the most filterable mixture and a final product of high activity. Microscopic examination of the ground cellulose indicated that extensive disintegration of the cellulose particles occurred in all the preparations with high adsorption activity. There was also some agglomeration of particles when the preparations were dried, especially when the filter cake was dried in vacuum a t 100' with the water aspirator. Most preparations of the ground cellulose retained a little volatile liquid that could be removed by additional drying a t a pressure of 0.001 mm. of mercury and a temperature of 100'. This residual liquid (usually 1 to 3y0) did not affect the R value, Table 111. The adsorption capacity of the ground cellulose varied with the solvents used to dissolve the carotene. This effect, which is typical of most surface-active adsorbents (1, W), is shovn in Table IV. The untreated cellulose powder employed in these experiments, commercial, microcrystalline cellulose, such as Avicel, and numerous filter papers did not adsorb carotene from solution in n-heptane. The R value for carotene on these preparations was not decreased by washing with various solvents, by dehydration in vacuum, or by reduction of the carotene concentration. For comparison of the selectivity of activated cellulose with that of other adsorbents, the adsorption sequences and the separability of various carotenoid pigments were determined with the activated preparations using various solvents (5, 6). The most significant of these results are presented in Table V. Brackets indicate pigments that were but partially separated.
Several significant effects deserve to be pointed out. At a 1%concentration of acetone, the three hydrocarbon pigments, lycopene, @-carotene, and CYcarotene, were readily sorbable and separable. When the concentration of the acetone was increased to 4%, the a- and ,%carotene were nonsorbed and, therefore, inseparable. At 20% acetone (not shown), these three pigments were inseparable. With n-heptane plus benzene, CY- and @-carotene could not be separated from each other, although they were readily separable from lycopene. With n-heptane plus 0.5 and 2% 1-propanol and also plus 50% benzene, the sequences were similar to those shown for n-heptane plus 4% acetone. With 4% 1-propanol, zeaxanthin and lutein m r e together, and the remaining four pigments formed overlapping zones. With no solvent was there detectable separation of zeaxanthin and lutein. The activated cellulose also served for the separation of the chloroplast pigments. With n-heptane plus 4% 1propanol as solvent, the pigments separated in the same sequence observed with powdered sugar when n-heptane plus 0.5% 1-propanol was the solvent. The adsorption capacity of the cellulose for these pigments was much greater than that of powdered sugar (6,6). DISCUSSION
The results summarized herein show that a typical surface-active, activated adsorbent may be prepared from nonadsorptive cellulose. For this preparation, there are two primary requisitesgrinding or beating with a polar liquid and removal or stripping of this liquid without agglomeration of the particles after the grinding. There are important secondary variables, such as the nature of the grinding liquid and the time of grinding. For high activity, ex%ensive grinding with the polar liquid is critical. As residual polar liquids would elute many adsorbed substances, these liquids must be removed after the grinding. The adsorption capacity of the activated cellulose is related, only in part, to the size of the particles. It may also be related to the special surface of the particles, because the grinding of cellulose with polar solvents is reported to separate the cellulose fibers into their component fibrils (3).
Table 111. Effect of Additional Drying (Usually 3 Hours at 100" and 0.001 Mm.) on Activity of Cellulose Ground with Marbles and Various Liquids for 20 Hours (Measured as R values for carotene)
Liquid Methanol Acetone Benzene
RE?
Method of drying Filtration, heat in vac. Filtration, heat in vac. Filtration, heat in vac. Extraction, acetone, pet. et. Extraction, acetone, pet. et,
Table IV. Effect of Wash Liquids on R Value for Carotene Adsorbed on One Preparation of Activated Cellulose
Wash liquid Petroleum ether (b.p. 20-40') *Heptane Petroleum ether (b.p. 65-110') Benzene Acetone
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ANALYTICAL CHEMISTRY
R value 0.27 0.38
0.54 1.00
1.00
% volatiles 0.8
2.7 0.9
2.8 0.9
Table V.
R value Before After drying drying 0.33 0.36 0.40 0.36 0.67 0.72 0.30 0.26 0.13 0.13
Chromatographic Sequences of Carotenoid Pigments Adsorbed in Columns of Activated Cellulose
Solvents Sequences
+
n-Heptane acetone 1% Zeaxanthin Lutein Cryptoxanthin Lycopene 8-Carotene a-Carotene
+
n-Heptane acetone 4% Zeaxanthin [Lutein Cryptoxanthin Lycopene @-Carotene a-Carotene
c
+
n-Heptane benzene 5-10% Zeaxanthin Lutein Cryptoxanthin Lycopene &Carotene a-Carotene
[c
Relative to many other adsorbents, the activated cellulose exhibits intermediate adsorption capacity. It is a much stronger adsorbent than other polysaccharides, but it is much weaker than activated alumina or magnesia. Only by measurement of the adsorptive properties of particular preparations can cellulose be ranked in a precise position with other adsorbents arranged in the order of their adsorption capacities (4). Earlier studies of chromatographic sequences indicated that polysaccharides had little or no affinity for carotene molecules or for the polyene portions of the xanthophylls (6). The results with the activated cellulose nom show that this material does, in fact, hare significant adsorption capacity for the polyene hydrocarbons. Moreover, this affinity is selective because it permits the separation of lycopene, p-carotene, and a-carotene. But with the isomeric zeaxanthin and lutein, the difference between the adsorbability of their dif-
ferent but similar polyene systems is not sufficient to produce a complete chromatographic separation like that accomplished with activated magnesium oxide. Moreover, the position of the lycopene below the xanthophylls in a cellulose column contrasts with the position of the lycopene uppermost with the zeaxanthin in a magnesia column. From these relationships one may conclude that activation of the cellulose has increased the adsorption affinity not only for the conjugated polyene aspect of the carotenoid molecules, but also for the hydroxy aspects of the xanthophylls as well. From this standpoint, the activated cellulose is analogous to the nonactive cellulose with relatively much stronger affinity for hydroxyl groups than for the polyene skeleton. LITERATURE CITED
(1) HeftFann,
raphy,
E., ed., “ChromatogReinhold, New York, 1961.
(2) Lederer, E., Lederer, hl., “Chromatography, a Review of Principles
and Applications,” 2nd ed., Elsevier, New York, 1957. (3) Ott, E., Spurlin, H. M., Grafflin, M. W.. eda.. “Cellulose and Cellulose Dehvatiies,” 2nd ed., Interscience, New York, 1954. (4) . . Strain, H. H., ANAL. CHEM.30, 620 (1958): ’ (5) Strain, H. H., “Chloroplast Pigments and Chromatographic Analysis,” 32nd Annual Priestley Lectures, Pennsylvania State University, University Park, Pa., 1958. (6) Strain, H. H., J . Am. Chem. SOL 70, 588 (1948). (7) Strain, H. H.., “Leaf Xanthophylls,” Carnegie Institution of Washington, Washington, D. C., Publ. 490, 1938. RECEIVED for review August 27, 1962. Accepted November 16, 1962. Based upon investigations supported by the U. S. Atomic Energy Commission. Cleta K. Miller was Beloit College participant and Daniel Steff enson was Cornell College participant in the Argonne Semester Program cosponsored by Argonne National Laboratory and Associated Colleges of the Midwest.
Determination of the True Chloride Content of Biological Fluids and Tissues. I. Analysis by Chlorine-36 Isotope Dilution ERNEST COTLOVE’ laboratory o f Kidney and Electrolyte Metabolism, National Heart Institute, National Institutes of Health, Bethesda, Md.
b An isotope dilution method using C P is described by which the true value of the total chloride content of a sample of biological fluid or tissue can be measured with a precision of &Oh% (relative standard deviation) for amounts of 150 to 250 peq. of chloride. The method involves complete isotopic exchange of stable with added radioactive chloride in a solution formed by hot alkaline digestion of the sample, and determination of the correct value of the diluted specific activity in purified solutions obtained by successive stages of alkaline dry ashing, oxidation-reduction, and distillation. Radioassay was performed by infinite thickness liquid counting, and chemical assay was by automatic coulometric-amperometric titration with silver ion. The true chloride contents of tissues of normal adult rats, dogs, and frogs are lower than most values previously reported, particularly for muscle and liver. The isotope dilution method described can serve as a standard of reference for the evaluation and validation of simpler methods of chloride measurement in biological materials.
T
HE analysis of chloride in biological materials and particularly in tissues has presented a difficult problem
which has remained unsolved in spite of repeated efforts over many years. The continued interest in chloride analysis is part of a broader interest in the mechanisms of distribution and transport of chloride and of other substances in tissues. The accurate analysis of chloride also offers a possible means of determining cellular volume and composition from the ratio of chloride concentrations in tissue and plasma (11). The analysis of chloride in a tissue involves two main operations: first, preparation of a solution which contains all the chloride of the tissue sample but no constituents that interfere with the final measurement; and second, measurement of chloride in the solution by a colorimetric or titrimetric method (the latter employing a colorimetric or electrometric end point). The method most commonly used to accomplish these operations has been wet ashing with concentrated nitric acid (open Carius digestion), followed by Volhard titration (19). Because this wet ashing method has given erratic or questionable results, it has been modified repeatedly (11). Particular modifications have been compared directly with other types of methods on replicates of tissue samples, but the findings have been divergent and inconclusive.
Thus, chloride values obtained by 8 dry alkaline ashing method were similar to those obtained by a wet ashing method in one comparison ( I ) , but were 40% higher in another ( 2 ) . Methods employing extraction of fresh muscle with hot water or dilute sodium nitrate solution gave chloride values which were 60 to 70% higher than those by a wet ashing method (16, 21). Closed digestion of tissue by oxidation in a Parr bomb gave chloride results which were higher than those by a wet ashing method by as much as 180% (I7,18). The discrepancies in chloride results obtained by different methods have been largest in analysis of liver and muscle, which have a low content of chloride relative to protein and other organic substances, and have been least in analysis of serum and other biological fluids, which have a high chloride content relative to organic substances. When discrepancies between methods were found, it was usually assumed that the method yielding the higher result was correct, and lower results were ascribed to inadequate extraction of chloride, t o loss of chloride during wet or dry ashing, or to inadequate removal of constituents that interfere with the 1 Present address, Clinical Pathology Department, ’National Institutes of Health, Betheeda 14, Md. VOL. 35, NO. 1, JANUARY 1963
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