ANALYTICAL CHEMISTRY
1656 The instability constant, K, may be obtained from corresponding values of x and n, for given c, before precipitation, through Equation 48; and since T , or the expression in the bracket of that equation, is hardly greater than 1, the approximate relatiop is merely Equation 36. K may also be derived from the slope of the titration curve, as a t the inflection point near n = 1, through Equation 56, or a t the point of first appearance of the precipitate of silver cyanide, through Equation 52, which becomes
if P’ is known. The slope a t the point of precipitation of silver iodide may also be used, when P3/c3may be substituted for sp in Equation 66. The thermodynamic constants would follo~vfrom the mass constants as above, the ionic strengths being approximately the same both with and without ammonia. LITERATURE CITED
(1) Kolthoff, I. M., and Sandell, E. B., “Textbook of Quantitative Inorganic Analysis,” p. 546, New York, Macmillan Co., 1952, (2) Ricci, J. E., J. Phys. Colloid Chem,, 51, 1375 (1947).
and in which xp, if not actually measured, may be taken as 2P’/c
RECEIVED for review April 30, 1953. .4ooepted August 14, 1953.
Determination of Dextran with Anthrone TROY A. SCOTT, JR., AND EUGENE H. MELVIN Northern Regional Research Laboratory, Peoria, 111. The anthrone method was used for the determination of concentration of dextran solutions. The effects of the factors involved in the method were studied and a procedure designed to improve the precision was evolved. Sources of error in the procedure were analyzed and it was concluded that the most probable source of error is variation in the blank. Nineteen different chemicals at different levels of concentration were tested for their effects on the analysis. The precision of the method was estimated by calculating standard deviations from 97 sets of duplicate blank determinations and 345 sets of duplicate sample determinations. The standard deviation for the samples, in absorbance units, is 0.0029, correspondingto 0.48% at an absorbance level of 0.600.
T
HE reaction obtained by heating carbohydrates with anthrone in sulfuric acid has been used in this laboratory to determine the concentration of dextran solutions. This reaction, first described as a qualitative test by Dreywood (5),has been converted by several investigators to a quantitative method. Most investigators have used the heat evolved by mixing the aqueous carbohydrate solution with the sulfuric acid solution of anthrone to furnish the heat required for the development of the blue-green color. With this technique, Morse ( I O ) used the reaction to determine sucrose concentrations; Morris (9), for the determination of glucose, glycogen, and lactose; Viles and Silverman ( I S ) , for the analysis of starch and cellulose; Samsel and DeLap (ff), for the determination of methylcellulose; and Bloom and Willcox ( g ) , for the determination of dextran. This “heat of mixing” procedure has been investigated in this laboratory and found to be satisfactory if accuracy no better than *5% is required. For greater precision, it is necessary to keep the temperature rise, due t o mixing the reagents, to a minimum and then place the. reaction mixture in a controlled temperature bath for a specified time. Using this method, Siefter et aI. ( f a )determined glycogen by heating a 1 to 2 mixture by volume of glycogen solution and 0.2% anthrone in 95% sulfuric acid for 10 minutes in a boiling water bath. McCready et al. (8) determined starch concentrations by the same procedure, but shortened the heating time to 7.5 minutes. For the determination of sodium carboxymethylcellulose, Black (1) dissolved the solid sample in 60% (by volume) sulfuric acid, added a 0.1% solution of anthrone in 60% (by volume) sulfuric acid, and heated the mixture for 15 minutes in a boiling water bath.
Among the workers who have controlled the heat supplied for the reaction, McCready et al. (8) published a time curve for 100’ C., showing maximum absorption a t 6250 A. a t 7.5 minutes. This was substantiated by experimental work done a t this laboratory. Although several authors have advised the use of fresh reagent, only Black (1) has published details of the effects of reagent age and acid concentration on the reaction, and his investigation was limited to cellulose and carboxymethyl cellulose. The authors investigated the influence of these factors on the analysis of dextran solutions and also studied the effects of temperature, anthrone concentration, and various impurities. EXPERIMENTAL
I t has been reported ( 8 , 9 ) that the same absorption is developed from the reaction of anthrone reagent with equivalent amounts of glucose and starch or glycogen. It was assumed that the reaction with dextran would also be the same as with glucose, and National Bureau of Standards dextrose was used in the development of the method reported here. The validity of the assu’mp. tion was established by work with purified dextran. Selection of Wave Length for Absorption Measurements. Figure 1 shows the absorption curve for a 2 t o 1 mixture (by volume) of 0.3% anthrone in concentrated sulfuric acid with O.O037,.gIucose solution heated a t 100’ C. for two periods of time, The
6oor 19
01 4000
I
I
6000
5000 WAVE
LENGTH.
I
7000
ANGSTROMS
Figure 1. Effect of Heating Time on Anthrone-Glucose Absorption Curve 100’ C.. 75% aoid
V O L U M E 25, N O . 11, N O V E M B E R 1 9 5 3
1657
500/-400
300
200
100
0
Figure 2.
5
IO 15 20 HEATING T!ME, MINUTES
25
30
Effect of Temperature on Absorption at 6250 A. in 71.6% Sulfuric Acid
peak a t 6250 A. was chosen for measurement, because the peaks a t 5020 A. and a t 4270 A. require a longer period of heating to develop. Selection of Time, Temperature, and Acid Concentration for Reaction. Time curves were determined for temperatures from 80' to 100' C. a t acid concentrations (in the reaction mixture) of 71.6, 75.4, and 79.6%. (All acid concentrations mentioned in this paper refer to weight per cent unless otherwise stated.) A study of Figures 2, 3, and 4 shows that with decreasing temperature the time curve flattens, the time required to reach maximum absorption increases and the value of maximum absorption decreases slightly. Decreasing acid concentration also flattens the curve and increases the time required to reach maximum absorption, but the value of the maximum absorption falls off rapidly. (The absorption coefficients shown in Figures 1 to 4 are based on a glucose concentration of 1 gram per 100 ml. of the reaction mixture.) The factors considered in the selection of the temperature and acid concentration to be used for the reaction were the flatness of the time curve, the time required to reach maximum absorption, the ease of obtaining the chosen acid concentration, and the vapor pressure of the reaction mixture; 90" C. and 75% sulfuric acid (Figure 3) were chosen as a good compromise of the desired properties in the factors listed. There is little change in slope of the curve for a span of 4 minutes in the region of maximum absorption, and the time (16 minutes) required to reach maximum absorption is not excessive. The 75% acid concentration requires a simple 2 to 1 volume ratio of concentrated sulfuric acid to aqueous solution. 0 Anthrone Concentration in Reagent. Previous workers have used 0.05 to 0.2% anthrone Figure 3. in 95% sulfuric acid as the reagent for carbohydrate determination. Viles and Silverman (IS), using the "heat of mixing" procedure, reported a maximum sensitivity a t 0.l6yOanthrone with cellulose and starch standards. Data presented in Figure 5 show that for maximum precision under the conditions recommended in this paper the anthrone content of the reagent ehould be greater than 0.18 gram per 100 ml. of concentrated sulfuric acid. Application of Beer's Law. Figure 6 shows that Beer's law applies to the absorption of reaction mixtures obtained from aqueous dextran solutions of concentrations less than 8 mg. per 100 ml. The departure from Beer's law was not due to insufficient anthrone, because doubling the anthrone concentration did not change the absorption coefficients a t higher dextran concentrations.
Stability of Glucose and Dextran Solutions. Glucose solutions and, to a lesser extent, dextran solutions deteriorate unless protected from microbial contamination. It was found that solutions of either glucose or dextran would keep indefinitely a t room temperature if 0.2 ml. of Roccal (10% alkyl dimethylbenzylammonium chloride, obtainable from the Kinthrop Chemical Co., Inc.) or 2 mg. of phenyl mercuric acetate were added for each 100 ml. of solution. -40.06% solution of glucose or dextran, preserved in this fashion, mas diluted twentyfold for use as a standard for dextran determinations. The concentrations of the preservatives used in the diluted standard solutions did not interfere with the reaction with anthrone reagent. Comparison of Glucose and Dextran in Reaction with Anthrone. Six aliquots of a glucose solution were run together with six aliquots of a dextran solution of equivalent concentration. Bt intervals, one each of the glucose and dextran solutions were removed simultaneously from the 90" C. bath, and cooled, and the absorbances were read. The resulting time curves, given by the data of Table I A , were identical within experimental error. Another set of 11 glucose samples were run (16 minutes, 90" C., 75% sulfuric acid) simultaneously with a set of 12 samples of dextran solution of equivalent concentration (Table IB). The agreement of the average absorbances confirm the reports of other investigators that a polymeric carbohydrate behaves in the same way as its hydrolyzed solution in the reaction with anthrone.
APPARATUS.Spectrophotometer, Beckman Model DU Test tubes, borosilicate glass, 25 X 200 mm. Loose-fitting borosilicate glass test tube caps or small beakers Bath, ethylene glycol, maintained a t 90" C. Bath, water, cooling Wire basket, to hold several 25 X 200 mm. tubes Pipet 10-ml., orifice enlarged and fiducial mark adjusted to deliver 10 ml. of concentrated sulfuric acid in 20 to 30 seconds
5
IO
15
20
25
30
35
40
HEATING TIME, MINUTES
Effect of Temperature on -4bsorption at 6250 A. in ?5.4qo Sulfuric Acid
REAGENTS.Standard, pure dextran (if available) or NBS dextrose Distilled water Anthrone reagent, 2 grams of anthrone (4),dissolved in 1 liter of concentrated sulfuric acid (ACS reagent grade). Anthrone samples from several commercial sources were tested and found to vary widely as to stability in concentrated sulfuric acid. Procedure. Place the wire basket containing the test tubes in a rapidly circulating cold water bath (10' to 15" C.). Using the pipet with enlarged orifice, deliver 10 ml. of anthrone reagent into each tube, alloffing the same drainage time for each delivery. Cover tubes with loose-fitting caps. Pipet 5 ml. of sample, diluted to a concentration of 2 to 4 mg. per 100 ml., into each tube, carefully forming a layer above the anthrone reagent. Include two blanks, using 5 ml. of distilled
ANALYTICAL CHEMISTRY
1658 Table I.
Comparison of Glucose with Dextran in Reaction with Anthrone Reagent
Heating Time, hlin.
Absorbance a t 6250 A. (I-Cm. Ceils) Glucose Dextran (3.00 mg./100 ml.) (2.70 mg./100 ml.)
Variation of Absorhance .n-ith Time of Heating (90' C., 7 5 % HpSOd)
A.
0,483 0.536 0.543" 0.541 0.534 0 511
10 14 16 18 20 24
0.484 0.536 0,548" 0,542 0.537 0.510
Samples Heated Simultaneously f o r 16 Ninutes (90' C., 75GC HzSod)
B.
n ,573 0 ,i66 0.573
0 581
0 5f9 0.569 0 572 0 570 0.570 0.573 0 575 0,574 0,672 0.571
0 581 0.568
0.575 0.575 0.573 0.575 0,572 0.573
0.572 Mean 0,572P 0.5732" Standard deviation 0.0033 or 0 . 5 8 %
concentration of dextran standard (if a glucose standard is used, its concentration is multiplied by 0.900 to ive its dextran equivalent. j absortance of sample absorbance of blank absorbance of standard SOURCES OF ERRORS
Instability of Anthrone Reagent. The absorption coefficient a t 6250 A. for dextran, reacted with 1-week-old anthrone reagent, is 5 to 10% lower than when freshly prepared anthrone reagent is used. This decrease in absorption is erratic a?d not strictly proportional to the age of the reagent, so that a standard solution of glucose or dextran must be run siniultaneously with each group of samples. Figure 7 shows that Beer's law applies when l-weekold reagent is used. Other investigators, using a 100" C. bath or the heat supplied by mixing, have noted inconsistent results vith reagent less than 4 hours old, and McCready et al. (8) have indicated that the reagent should not be more than 2 days old. Using the method outlined in the present paper, reliable resulte were obtained using reagcnt ranging from 1 hour to 7 or 8 days old.
Anthrone reagent used for A was several days old, while t h a t of B was freshly prepared, which explains difference in absorbance a t 16 minutes. (1
water instead of sample. k o include two glucose or dextran standards with each set of samples. After the aqueous solutions have been added to all of the tubes, shake each tube vigorously, while keeping immersed in the cold water bath, until contents are thoroughly mixed and a t about room temperature. Place the basket of tubes in the 90' C. bath for 16 minutes, then return them to the cold water bath until contents are cooled to room temperature.
ANTHRONE, G R A M S PER 100 rnl. OF REAGENT
Figure 5 . Effect of Anthrone Concentration on Absorbance of .inthrone-Dextran 16 minutes, 90' C . , 75% acid
0
5
10 15 HEATING TIME. MINUTES
20
25
Figure 4. Effect of Temperature on Absorption at 6250 A. in 79.6yc Sulfuric Acid
Read immediately the absorbances of the samples, standards, and blanks in 1-cm. absorption cells, using water as a reference. If the absorbances of the blanks disagree by more than 0.005, use the lower for the correction of sample and standard absorbances, as it is probable that the higher is due to carbohydrate contamination. Calculation
C,
=
concentration of dextran sample. (If the original sample required dilution to bring it into the concentration range necessary for a determination, the C, must be multiplied by the appropriate dilution factor. )
Loepus ( ? ) has recommended a "he'it of mixing" procedure which involves mixing 2 inl. of sample solution with 5 ml. of concentrated sulfuric acid and 0.5 nil. of a 2% solution of anthrone in ethyl acetate. The latter solution is said to be stable, which \\auld eliminate the need for a standard solution with each set of samples. Hoivever, standardization T o d d still be required for each bottle of sulfuric acid. in addition t o the handling of a third solution for each determination. dccuiute work would necessitate better control of heating time and bath temperature if a standard vas not included n-ith eacah set of aamples. Variation of Heating Time and Bath Temperature. The inclusion of a dextran solution of known concentration with each set of samples makes the analysis relatively insensitive to heating time and bath temperature, because each of these factors is constant for all samples in a particular set of determinations. I t is, of course, necessary that the bath have good circulation, so that all tubes are heated evenly. Variation of Acid Concentration of Anthrone Reagent. Because of thr inclusion of a dextran solution of known concentration, the acid concentration of the reagent is not an important source of error. However, it is desirable that the time of heating fall on a point of the time curve (Figure 3 ) myhere the slope of the curve is small-i.e., near maximum absorption. Therefore, it is advisable to determine if the use of ACS reagent grade sulfuric acid of unadjusted concentration \\ill change the time curve to the ektent that the heating time would correspond to a point on the curve where absorption is changing rapidly with heating time. The concentration of most .ICs reagent grade sulfuric acid is betmeen 95 and 97%. T h e n diluted with half its volume of
V O L U M E 25, NO. 11, N O V E M B E R 1 9 5 3
1659
water, the concentration is decreased to 74.7 to 76.3%. Interpolation a t these values in Figure 8 s h o w that the times required to reach maximum absorption are 17.4 and 14.2 minutes, respectively. Thus, the 16 minutes' heating used in t.he procedure would, in the m-orst case, be about 2 minutes past the time of maximum absorption. Examination of the curves (Figures 2, 3, and 4) shows that a t this distance from the peak, the slope of the curve is still small. Thus, it is permissible to use ACS grade sulfuric acid as a solvent for anthrone J
