than in spheres of the same mass, as the time necessary for diffusion to their centers would be governed by the smallest of the three dimensions. Columns prepared with irregular particles gave higher operating pressures than those filled with spherical particles of the same diameter, presumably because of closer packing of the irregular shapes. The general relationships among column length, particle size, rate of flow, operating pressure, and column performance merit consideration. If column length is increased-e.g., from 100 to 150 cm.-to obtain higher resolving power, the operating pressure is increased proportionately, and this may be undesirable. Because resistance t o flow a t constant rate, and therefore pressure, bears a n inverse relationship to particle size, the operating pressure can be lowered by employing larger particles. Increase in particle size will, however, partially nullify the gain in resolving power. I n turn, this diminution in resolving power may be offset by a reduction in flow rate. Application of these general principles allow resolving power to be manipulated within limits, by the selection of appropriate values for these related parameters. The effects of temperature, pH, and ionic strength of developing solutions on resolving power are beyond the scope of the present discussion. Some minor differences b e b e e n resins, probably owing to factors other than particle size, were noted. The resolution of threonine and serine was
better on Amberlite CG-120 Type 111 and Dowex 50-X8 (bead form) than on Dowex 50M7-X8 (irregular particles). On the Amberlite columns, the separation of threonine and serine a t 50" C. was marginal; a t 40" C., complete. They were not separated a t either 40" or 50" C. on Dowex 50W-X8. The resolution of tyrosine from phenylalanine on Dowex 50\T7-X8 !vas good, but overlapping of p-alanine with tyrosine was sometimes encountered. On the Amberlite resin, the resolution of tyrosine from phenylalanine was not as complete, but @-alanine always emerged Tyell after phenylalanine. Resolution of the basic amino acids was equally satisfactory on both resins. Ammonia usually overlapped hydroxylysine on Dowex 50W. On the Amberlite resin, ammonia usually emerged with ornithine or with lysine, or in some intermediate position between these two amino acids. At the commencement of this work, pulverized resins were available only in limited experimental quantities. NOW, pulverized resins for analytical chromatography are available from commercial sources. ACKNOWLEDGMENT
The author wishes to express his appreciation of cooperative exchange of information with W.H. Stein, S. Moore, and D. H. Spackman, Rockefeller Institute, New York, S. Y. Thanks are due to the Resinous Sales Development Department, Rohm & Haas Co., Philadelphia, Pa., for samples
of the Amberlite CG-120 Type 111, technical data, and helpful cooperation. Thanks are due to the Organic Sales Department, Dow Chemical Co., Midland, Nich., for samples of Dowex 50-X4, -X8, -X12, Dowex 50W-X8, -X12, and for technical data. The Bio-Rad Laboratories, Berkeley, Calif., supplied pulverized Dowex 50W-X8 and the Permutit Co., Birmingham, N. J., donated the Permutit Q-ZP. Assistance in the amino acid chromatography and the preparation of illustrations was rendered by Roberta A. Anderson. LITERATURE CITED
(1) Chamot, E. M.,
Mason, C. W., "Handbook of Chemical Microscopy," 2nd ed., Vol. I, pp. 41215, Wiley, New York, 1948. (2) Martin, G., Blythe, C. E., Tongue, H., Trans. Ceram. SOC. (Eng.) 23, 61-118 (1924). (3) Mayer, S. W., Tompkins, E. R., J. Am. Chem. SOC.69, 2866 (1947). (4) Moore, S., Spackman, D. H., Stein, W. H., ANAL.CHEM.,in press. (5) Moore, S., Stein, W. H., J. Bid. Chem. 176, 367 (1948). (6) Ibid., 192, 663 (1951). ( 7 ) Ibid., 211,893 (1954). (8) Spackman, D. H., Moore, S., Stein, W. H., ~ Z N A L . CHEM.,in press. (9) Spackman, D. H., Stein, W. H., Moore, S., Federation Proc. 15, 358 (1956). RECEIVEDfor review August 5, 1957. Accepted January 27, 1958.
Chromatographic Analysis of Pulps Utilizing Direct Densitometry D. F. DURSO and J. C. PAULSON Buckeye Cellulose Corp., Memphis, Tenn.
b A method is presented which permits fairly rapid estimation of monomeric sugars in wood pulps. The pulp is dissolved and hydrolyzed with 72% sulfuric acid, being carried through a series of dilutions as the temperature is increased. An aliquot of the hydrolyzate is neutralized with sodium carbonate. After crystallization of sodium sulfate, the supernatant is separated by paper partition chromatography using ethyl acetate, acetic acid, and water. The sugar spots are developed with silver nitrate, and the sugar concentration is estimated by direct transmission densitometry. The
method is applicable to fully bleached and unbleached pulps.
T
HE present method was developed as a routine procedure for the determination of the monomeric sugars glucose, mannose, and xylose occurring in wood pulp hgdrolyzates. The procedure, as compared to existing methods, is simplified in that after chromatographic separation the sugars are estimated directly on the paper without eluting them. The paper is sprayed with silver nitrate and the transmission density of the developed sugar spots is measured with a Photovolt densi-
tonieter; the relationship between the maximum transmission density of the sugar spot and the logarithm of the sugar concentration is linear. hlcFarren, Brand, and Rutkowski (3) used a similar technique in measuring lactose, glucose, and galactose. The error was reported to be no greater than 5%. Another unique feature is the manner in which the hydrolyzing acid is neutralized prior to the paper chromatographic separation of the sugars. I n other techniques, the neutralization is usually done with ion exchange resins or by precipitation of the acid as barium sulfate or silver chloride. I n the present VOL. 30,
NO. 5, MAY 1958
919
procedure, the sulfuric acid is neutralized with solid sodium bicarbonate and, as a result, the sugar solution is saturated with sodium sulfate at 4" C. The presence of salt does not interfere with the separation or estimation of the mgars. Moreover, this technique reduces the labor demand of the method. The hydrolysis procedure, although rather lengthy, has been purposely kept as mild as possible, t o minimize degradation of sugars. As a low temperature primary stage results in complete solution before depolymerization begins, the substrate entering the secondary stage undergoes homogeneous reaction.
H
PROCEDURE
Hydrolysis. T h e hydrolysis procedure is described for 2-gram pulp samples, b u t m a y he scaled down t o I-gram samples. T h e pulp is fluffed in a blender-type disintegrator, dissolved in 50 ml. of 72% (weight basis) sulfuric acid, and held overnight at 0" C. in a refrigerated glycol bath. On the following day the hydrolyzate is diluted with 40 ml. of water and kept at room temperature for 3 to 4 hours. An additional 40 ml. of water is added and the hydrolyzate is maintained at room temperature overnight. Water is again added t o bring the volume up t o 200 ml. and, after standing at room temperature for 2 hours, the hydrolysis is completed by heating at 75" C. for 8 hours. This last step is carried out with a device (Figure 1) which automatically lowers the flasks into the 75" bath, maintains them at this temperature for 8 hours, and then withdraws the flasks from the bath. Neutralization. A 50-ml. aliquot of t h e hydrolyzate is neutralized with solid sodium bicarbonate t o a pH of 5.5. T h e stoichiometric equivalent of bicarbonate is added s l o i ~ l y (no stirring) t o the hydrolyzate. Final pH adjustment is made with sodium bicarbonate or sulfuric acid while the mixture is stirred. The neutralized hydrolyzate is refrigerated during the sodium sulfate crystallization (a small crystal of sodium sulfate will seed the process). The supernatant, about 30 ml., is concentrated at 40 to 45" C. on a Rinco evaporator (Rinco Equipment Co., Greenville, Ill.) t o a volume of 5 to 15 ml. The sodium sulfate is again crystallized at refrigerator temperature?, and then the concentrate and the appropriate water dilutions of the concentrate are ready for application t o the paper. Chromatographic Separation. Sheets of Whatman No. 1 paper are cut 22 x 24 inches with t h e machine direction parallel t o the 24-inch dimension. The paper is ruled off so t h a t each sheet consists of two 12 X 22 inch chromatograms. The origin is drawn 2.5 inches from the leading edge; marks are indicated on this line a t I-inch intervals where the standards and unknowns will he applied. The trailing edge of ~~
920
ANALYTICAL CHEMISTRY
Figure 1. A. B.
Schematic diagram of automatic bath
Woter bath Ihbline, No. 3010-12) Timer (Intermatic, No. A-201, Intcmotional Register Co., Chicago) C. Drive motor with reduction geor; limit switches lo Demit 180' rototion D. To heoterr E. To circulation pump in both F. To 120-roil alternating current G. Ground wire H. Flork clomp I . Counter weight
Table 1. Effect of Neutralization Procedure on Recovery of Known Sugar Mixtures
Sugar Added, % 1.0 1.0
1.0 TRANSMISSION
DENSITY
Figure 3.
2.5
2.0
1.5 NET
(PHOTOVOLT
SCALE
Mannose 0.9 Xylose 1.1 Mannose 2.0 2.1 2 0 2.1 Xylose 3.0 3.1 Mannose Xylose 3.0 3.2 Mannose 4.0 40 4.0 4.1 Xylose a Average of three chromatograms.
READING)
Table It.
Typical standard curve
0 X A
Mannose Xylose Glucose
the sheet is serrated with pinking scissors. The sheets in groups of 20 are mashed with 591, acetic acid and rinsed with distilled water, on a large plastic Buchner funnel (2). The sheets are suspended on glass rods and dried in a forced air hood. The standard sugar solution consists of a mixture of glucose, mannose, and xylose (1, 0.5, and 0.5 -y per pl., respectively). The standard is spotted four times on each chromatogram (2.5, 5, 10, and 20 pl.) so t h a t glucose occurs a t 2.5, 5, 10, and 20 y, mannose at 1.25, 2.5, 5, and 10 y, and xylose at 1.25, 2.5, 5, and 10 y. These constitute the points for the standard curve. The unknown (concentrate and concentrate plus appropriate water dilutions) is then spotted on the remaining six places of the chromatogram, so that the sugar standards bracket the unknown in sugar concentration level. The volume of the concentrate is adjusted to between 5 and 15 ml. so that mannose and xylose are Tyithin the limits of their corresponding standards. This can usually be determined by a n approximate knowledge of the pulp composition. The concentrate is spotted a t three places on the origin. As glucose usually occurs in the hydrolyzate a t a much higher level than either mannose or xylose, its concentration is reduced to a level within the range of the standards. The dilution may range from 1:10 t o 1: 50, depending upon the carbohydrate content of the starting material. This is usually determined by hypoiodite oxidation of a n aliquot of the hydrolyzate. The dilution is spotted on the remaining three places on the paper. The papers are developed with ethyl acetate, acetic acid, and water, 44:20:10 v./v., by descending chromatography for 24 hours ( 1 ) . Fresh solvent mixture is made u p just before use. During the chromatographic development the solvent front will run off the end of the paper. The serrated trailing edge allows the solvent to drip off the sheet evenly. The sheets are dried in a forced air hood for 3 t o 4 hours.
Sugar Founda, %
T h e double sheets are cut in half and sprayed singly and evenly on both sides with a mixture of silver nitrate (5 grams), water (50 ml,), and acetone (400 ml.). After the acetone has evaporated, the sheets are suspended for 2 hours in a closed chamber in which the atmosphere is saturated with water vapor. The exact function of this step is not clear, but it is believed to aid in the even distribution of silver nitrate on the paper and to adjust the moisture level on the paper. As the silver nitrate-sugar reaction is catalyzed by a basic medium, the sheets are suspended for 15 minutes in a chamber in which the atmosphere is saturated with ammonia vapors. The excess ammonia is then removed from the sheets in a forced air hood, and the papers are stored overnight in a lightproof box. The optimum development time is approximately 15 t o 20 hours; hom-ever, this is a variable, related to the level of silver nitrate applied to the paper. Estimation of Sugars. T h e sugar concentration is estimated directlv on t h e paper, using a Photovolt densitometer, Model 501A. T h e net transmission density of the sugar is the reading of the spot minus the background density reading of the paper. For each spot, the paper is shifted under the densitometer aperture until a maximum deflection is obtained on the meter. The paper background density is read in the vicinity of the spot; this is usually constant for a particular latitude of the paper but may vary slightly rrith the long dimension of the paper. Each chromatogram has its o m set of sugar standards. However, if a single set of standards is used for each chromatogram, it is difficult to get a good standard curve. Because of the twodimensional density gradient of the spots, the standard deviation of a single reading is too high to warrant this practice. It is better to average all of the standard readings for a given batch of chromatograms-Le., a single day's run. t-sually a run consists of three unknown liydrolvzates, each of which has been spotted on three chromatograms. The Color Development.
% Total Sugar Authors' FPL method" method (4)
Pulp
-1 Mannose Xylose B Mannose Xylose C
Analysis of Wood Pulps
Mannose
0.9 1.6
0.8 1.6
2 3
1.8 "3
3.0
2 7
1 7
1.7 1 9 a -4verage of three chromatograms. Standard deviation of a single measurement about 1 0 7 . Xylose
total day's production then, consists of nine chromatogram< (-I1 double sheets), each x i t h its own set of standards. Thus. there are nine values for each of the four levels of the three sugar standards. The net transmission densities for the unknowns a t the same spot volume level are averaged, and the corresponding sugar concentration in micrograms is read from the average standard curve. The sugar concentrations are converted to a common hydrolyzate volume and each sugar 1s expressed as a percentage of the total wgars found on the chromatogram. RESULTS AND DISCUSSION
Standard Curve. Figure 2 shows a typical chromatogram after i t has been developed n-ith silver nitrate. The salt spot nhich arises through t h e neutralization procedure is clearly visible near t h e origin. yet i t is sufficiently removed so t h a t it does not interfere with the estimation of glucose, mannose, and xylose. A typical standard curve is shown in Figure 3. This curve was taken from nine chromatograms (three hydrolyzates, each in triplicate) so that each concentration level of the three sugars represents an average of nine net transmission densities. As a rule, mannose and xylose fall sufficiently close together so that a single standard curve may be drawn for both sugars; hovever, this is not always the case. I n all cases, the relationship between net transmission VOL. 30, NO. 5, MAY 1958
921
density and the logarithm of the sugar concentration is linear. Effect of Sodium Sulfate on Recovery of Sugars. Four known mix-
tures of glucose, mannose, a n d xylose were weighed out and dissolved in 200 ml. of 18% sulfuric acid (simulated conditions following hydrolysis), Aliquots of 50 nil. were withdrawn immediately, neutralized, and carried through the chromatographic procedure. All of the known mixtures were run on the same day; three chromatograms were run for each mixture. Therefore, the standard curve represented the average of 12 density readings for each concentration level of the three sugar standards. The value for each sugar in the known mixtures was obtained from a n average of nine density readings. The results (Table I)
show that neither sodium bicarbonate neutralization nor paper partition separation in the presence of sodium sulfate seriously interferes with the recovery of the sugars. Analysis of Pulp Samples. Three wood pulp samples supplied by t h e Joint ACS-ASTM-TAPPI Committee on Chromatographic Methods were analyzed by t h e present procedure. The pulps were also analyzed by J. F. Saeman. His method ( 4 ) includes: acid hydrolysis with a relatively short primary and secondary stage; neutralization with barium hydroxide; separation with butanol, pyridine, and water; elution of the sugars from the paper; and estimation of the sugars by copper reduction. The results of both methods are shown in Table 11.
ACKNOWLEDGMENT
The authors n-ish to thank J. F. Saeriian of the Forest Products Laboratory for permitting use of his analytical data. LITERATURE CITED
Durso, D. F., il:[ueller, W. A , , -4s.~~.
CHEW28, 13386 (1956). Hanes, C. S., Isiherwood. F. A , , S a Lure 164, 1107 (1949). McFarren, E. F , Brand, K., R u b komki, H. R ., ANAL. CHEM.23, 1146 (1951). Saeman, J. F., RII3ore, TT. E., Mitchell, R. L., Nillett AI. A , , T a p p i 37, 336 (1954). RECEIVED for review October 16, 1957. Accepted February 6, 1958. Division of Cellulose Chemistry, 132nd Meeting, ACS, New York. ?;. Y., September 1957
Photono met ric Determination of Va nad ium and Chromium CLARK E. BRICKER and STEPHEN S. SCHONBERG Department of Chemistry, Princeton University, Princeton, N. J.
b The number of photons or the time of exposure to a mercury vapor lamp i s measured for determining chromium(VI) and/or vanadium(V). The photochemically sensitive solution of iron(ll1) and oxalic acid containing the chromium and/or vanadium i s exposed to measured increments of radiation and the formation of chromium(lll) or vanadium(lV) is followed photometrically. Because the quantum efficiency of these reductions remains essentially constant over a rather wide range of experimental conditions, the radiation from the lamp can b e considered to generate a titrant at a constant rate. The rate a t which the titrant is formed is determined b y calibrating the lamp with known amounts of chromium or vanadium. The effect o f a number of variables on this reaction has been determined.
G
RAO and Aravamudan (6) have shown that iron(II1) is photochemically reduced in the presence of excess citric acid and can then be titrated with a standard solution of sodium vanadate. They determined oxalic acid by “photochemically oxidizing” this acid quantitatively in the presence of excess iron(II1) sulfate and then titrating the iron(I1) formed (6). Later, Rao and coworkers (8, 9) reported that uranium(V1) is reduced photochemically in the presence of OPALA
922
ANALYTICAL CHEMISTRY
excess lactic acid or ethyl alcohol and the uranium(1V) can be titrated with sodium vanadate. No effort was made to measure the amount of light required for the reduction, but the solutions were exposed to ultraviolet radiation long enough to ensure a quantitative reaction. Previous work with actinometers and with most photochemical reactions in solution has shown that the quantum efficiency of the reaction tends to decrease with increasing time of exposure. This nonlinearity is usually attributed to the internal filter action of a product of the photolysis and/or to the decrease in the concentration of the reacting materials in solution (IO). On the other hand, the total energy of x-rays or gamma rays has been measured by chemical dosimeters which show linearity in the amount of reaction over at least a limited range of energy absorbed. Dewar and Hentz (1) have shown t h a t the amount of iron(I1) oxidized, which was followed either potentiometrically or photometrically, is directly related to the time of exposure to x-rays. I n this paper, a new idea in photolytic reactions is described, in which a constant quantum yield is realized throughout the reaction by having a product from the primary photolysis react with the substance to be determined and thereby return to its
initial oxidation state. This is analogous to the idea used in coulometry a t constant current, where an excess of an ion is added to serve as a n intermediate in the transfer of electrons and thereby a uniform current efficiency is maintained even when the concentration of the substance to be determined is lorn. A solution of iron(II1) and oxalic acid undergoes a photochemical reaction and iron(I1) is produced. The amount of iron(I1) formed per unit time with constant irradiation decreases with the time of exposure. If, however, some vanadium(V) is present with a large excess of iron(II1) and oxalic acid, the iron(I1) formed reduces the vanadium(V) to vanadium(1S’) and iron(II1) is regenerated. With these conditions, the amount of vanadium(1V) produced per unit time of exposure remains constant. Consequently, vanadium can be determined by measuring the time of exposure needed to reduce the vanadium(V) quantitatively. This paper describes such a photochemical method for the determination of vanadium and chromium, separately and in mixtures. Because the number of photons needed to complete the reaction is measured, this method has been called a photonometric determination. APPARATUS AND REAGENTS
A Beckman Model B spectrophotom-
