950
ANALYTICAL CHEMISTRY
Table 11.
Standardization of Approximately 0.05 N Ferrous Sulfate
(Forward titration employing dimethylferroin indicator. Reverse titration usine ferroin as indicator) F e + + Normality, F e + + Normality, Acid Forward Reverse No. of Formality Titrations Titration Titration I
1
F HtSO,
2
F HtSO,
6 3 6 6 6
0.04781 0.04733 0.04698 0.04749 0.04626
0.04785 0.04734 0.04702 0,04763 0.04630
Diphenylamine Sulfonate Forward Titration 1 F €IC1
6
0.04958
F HCI
6
C ,04972
LITERATURE CITED
0.04961
Potentiometric 2
point by alternate dropwise excess of ferrous and dichromate solution additions. Thirty such reversals over a period of 30 minutes did not affect the sharpness of the color change. A 1-ml. excess of 0.1 N dichromate did not affect the sharpness of the indicator color change in a titrated solution during 1 hour’s time. The use of 0.05 ml. of 0.025 M dimethylferroin in a volume of 150 ml. of solution gives a sharp indicator color change. Comparison titrations of ferrous iron by dichromate using the new indicator with the reverse titration of dichromate by ferrous iron using ferroin as indicator are recorded in Table 11. These latter tests were made in hydrochloric acid solution.
0.04969
acid concentration. Dimethylferroin gives, as color change, a transition from orange to green if hydrochloric acid is present and no phosphoric acid is employed to complex ferric ions. The color change in the presence of sulfuric acid is from red to yellowish green. The indicator may be reversed indefinitely at the equivalence
(1) Case, F. H., Abstracts of 112th Meeting, AM.CHEM.SOC., p. 35L, 1547. (2) Hume and Kolthoff, J . Am. Chem. SOC.,65, 1855 (1943). (3) Kolthoff, Lee, and Leussing, ANAL.CHEM.,20, 585 (1948). (4) Smith, G. F., “Cerate Oxidimetry,” Columbus, Ohio, G. Frederick Smith Chemical Co., 1942. (5) Smith, G. F., and Richter, F. P., “Phenanthroline and Substituted Phenanthroline Indicators,” Columbus, Ohio, G. Frederick Smith Chemical Co., 1944. (6) Syrokomsky and Stiepen, J . Am. Chem. Soc., 58, 928 (1936). (7) Walden, Hammett, and Chapman, Ibid., 53, 3908 (1931). RECEIVED July 22, 1948.
‘Determination of Starch and Cellulose with Anthrone FREDERICK J. VILES, JR., AND LESLIE SILVERMAN Haraard School of Public Health, Boston, Mass. A procedure is presented for colorimetric analysis of starch and cellulose at a wave length of 625 mp, using a 0.1% solution of anthrone in concentrated sulfuric acid. The method is accurate for ranges of 10 to 200 micrograms and sensitive to 2 micrograms of these substances. Because of the instability of the reagent, a known standard must be used with each set of analyses to determine the correct Beer’s law constant. Color intensity studies of the effect of heat upon the reaction between anthrone reagent and starch and cellulose are presented. Spectral transmittance curves of carbohydrate-anthrone colors prepared under different conditions are also included.
D
REYWOOD ( 1 ) initially demonstrated the use of anthrone
in a specific qualitative test for carbohydrates and suggested its possible quantitative use. Morse (3) used anthrone for determining lovi concentrations of sucrose, and Morris ( 2 ) , i n a report that appeared while this article !?as in preparation, investigated its applications t o carbohydrates and some conditions of the reaction. He also studied the relationship of color intensity with various carbohydrates. The authors have applied this reagent to analysis of starch and cellulose (cotton lint) in air samples collected in plants manufacturing cotton textiles. The discussion that follows includes only analyses of these two substances and pertinent facts concerning the reagent and reaction conditions. Anthrone can be made as described by other investigators (1-3) or obtained commercially (Paragon Testing Laboratories, Orange, S. J., Panrone Chemical Company, Farmington, Conn., and National Biochemical Co.. 3106 West Lake St.. Chicago 12, Ill.). For the present work, a commercial product obtained in 1946 from the Paragon Testing Laboratories was used without purification. The test is made by rapidly adding a solution of anthrone (0.05 to 0.20%) in concentrated sulfuric acid t o an aqueous solution or
suspension of the carbohydrate and mixing immediately. Under controlled conditions the amount of green color produced is proportional to the carbohydrate content. The heat produced by mixing acid and water appears to be a necessary part of the reaction. In analyses for cellulose, sulfuric acid (60% by volume) is used to digest the material prior to analysis and therefore is present (in aliquots of 0.5 ml.) mith water and the anthrone reagent. (For dissolving cellulose, 60% sulfuric acid was found to give optimum results regarding rapidity of solution. Cotton is the only form of cellulose refcwed to in this work.) Morse (3) used unfiltered and AZorris ( 2 ) filtered light, for photoelectric determination of carbohydrates. Seither investigator presented any spectral transmittance data for the anthronecarbohydrate color to permit proper selection of wave length for maximum sensitivity. Morris ( 2 ) arrived at an adequate choice (620 mp) from measurements with three light filters. SELECTION OF WAVE LENGTH FOR COLOR MEASUREMENT
The purpose of this study was the proper selection of wave length for colorimetric analysis of starch and cellulose with anthrone. Spectral transmittance curves (Figure 1) of the colors
951
V O L U M E 21, N O . 8, A U G U S T 1 9 4 9
1 -I
1
lot A - W A V E LENGTH
MU-
Figure 1. Spectral Transmittance Curves for AnthroneCellulose and Anthrone-Starch Colors A. B.
C.
D. E. F. 6.
90 micrograms of cellulose 90 micrograms of cellulose (reagent 1 day old) 25 micrograms of cellulose 50 micrograms of cellulose (different conditions, see text) 50 micrograms of starch 50 micrograms of starch (reagent 1 day old) Sample E after standing several hours
-
TIME IN BOIL- TIME OF AIR ING WATER COOLING 25'0 IOO°C. c-
501
%
2 401 . W I I :
5 Y
301 201 IO1
1
8
1
1 4
1
1 0
1
1
4
1
1
8
1
1
I2
1
1
16
1
1
20
1
1
24
1
1
28
TIME IN MINUTFQ
Figure 2. Time of Heating or Cooling before Immersion in Cold Water Bath Klett-Summerson colorimeter readings, X = 625 mp Klett reading = (constant) log 1/T = (constant) (optical density)
resulting from the reaction between anthrone and starch or cellulose were determined by a Coleman Model 11 Universal spectrophotometer. These colors were prepared under different conditions to determine their effect on the spectral absorption bands, particularly the wave length of maximum absorption. The conditions involved were: variations in age of the anthrone reagent, water content, anthrone concentration, age of the color, and because two different carbohydrates were used, variations in the carbohydrate. Reference blanks used for transmittance measurements consisted of the same reagents used for the carbohydrate analysis. I n Figure 1, curves A and C are for 90 and 25 micrograms of cellulose, respectively, prepared from 0.5 ml. of 6O0/, sulfuric acid, 1.5 ml. of water, and 4.0 ml. of a 0.2'30 solution of anthrone in concentrated sulfuric acid. A wave length of minimum transmittance of 625 mp is indicated. Curve B represents the same cellulose content and reactants as curve A (90 micrograms of cellulose) except that the anthrone solution used was l day old. The spectral transmittance curve changes with age of the an-
throne solution but the wave length of maximum absorption remains a t 625 mp. The color represented by curve D is based on reaction between 50 micrograms of cellulose, 0.5 ml. of sulfuric acid, 2.0 ml. of water, and 4.0 ml. of 0.1% solution of anthrone in concentrated sulfuric acid. This differed from curves A and C, which contained less water (0.5 ml. less) and twice as much anthrone and different amounts of carbohydrate. Although the spectral transmittance curve shapeis altered by these factors (different water and anthrone content), the maximum absorption band remains a t 625 mp. Curves E and P are spectral transmittance curves for colors prepared from 50 micrograms of starch, 2.0 ml. of water, and 4.0 ml. of 0.1% anthrone in concentrated sulfuric acid. Curve F involved an anthrone solution 1 day old. The similarity of these curves with A and B can be readily noted. The wave length of minimum transmittance is also 625 mp, although a different carbohydrate is used. Curve G is based on the same solution used for curve E after standing for several hours. Fading on standing changes the intensity of color but not the wave length of maximum absorption (625 mp). The use of a filter of this wave length for colorimetric analysis of starch and cellulose with anthrone is therefore recommended and it is probable that this wave length is suitable for reactions of other carbohydrates with anthrone. EFFECTS OF H E 4 T UPON RE4CTION
Previous workers (1-3) have found that the color intensity of the anthrone reaction with carbohvdrates is widely influenced by heat. Experiments were undertaken to determine the optimum reaction conditions with regard to h d , . Consistent results with minimum error rather than maximum sensitivity were desired. Standard solutions containing 100 micrograms of starch or 50 micrograms of cellulose were placed in Klett-Summerson colorimeter tubes and the anthrone reagent was added by means of a pipet or buret. The components were mixed immediately after addition and subjected to air cooling or immersion in a boiling water bath for definite time intervals. Following this interval, the tubes were immersed in a cold water bath for 15 minutes or more. The color was then read on a Klett-Summerson colorimeter using a 625 mp ( + 1 . 5 mp) filter (evaporated metal film filter obtained from Baird Associates, Cambridge, Mass.). For starch, 2.0 ml. of' distilled water and 4.0 ml. of 0.1% solution of anthrone in concentrated sulfuric acid vere used; however, because 6070 sulfuric acid n as used in the analytical procedure for dissolving cellulose, this procedure required 0.5 ml. of 60% sulfuric acid, 2.0 ml. of water, ahd 4.0 ml. of 0.1% anthrone reagent. Reference blanks consisted of mixtures of similar amounts of acids, water, and anthrone reagent which were allowed to air-cool completely. Because the Klett-Summerson colorimeter contains a logarithmic (optical density) scale, dial readings are directly proportional to color intensity or concentration of carbohydrate if the solution obeys Beer's law. The results obtained (Figure 2) shorv consistent readings after 10 minutes of air cooling, although the maximum sensitivity is not obtained. The quantity of heat produced by the reaction of starch with the reagent appeared to be detrimental to the color developed. Thus maximum color development occurred upon immediate cooling in cold water; and in the hot water immersion tests rapid deterioration of color took place. The cellulose reaction with anthrone apparently did not create sufficient heat to develop maximum color, for increased color results on air cooling and still additional color increase takes place with immersion in boiling water. Deterioration of color begins after immersion for 3 minutes in boiling water. Because of the consistent results obtained in both cases after air cooling for lominutes, this method of treatment was adopted for the analysis of these two carbohydrates. Different amounts of water used in the test also result in changes in amount of heat accompanying the reaction and, therefore, affect color intensity. I n the present work 2.0 ml. of water were found to be satisfactory. Tests for cellulose using 2.5 ml. of water resulted in turbidity; this indicates that the minimum concentration of sulfuric acid in the final solution should be 65% for the amounts of anthrone specified.
952
ANALYTICAL CHEMISTRY Table I.
Stability of Starch-Anthrone Color
Time of Standing
Average Klett-Summerson Readings for 50 y of Starch, X = 625 mp
A . R E A G E N T S 4 H O U R S OLD 8 - R E A G E N T S I D A Y OLD C - R E A G E N T S 5 DAYS OLD
Min. 5 10 25
120 120 120 120
45 Hours 1 3 4 24 Average deviation based on 3 samples
120 120
118 113
f
f
* f
f
* *
2 2 2 2
t-W
ii
2 2 2
100 0
0.04
0
1
Figure 4. METHOD OF ADDING R E 4 G E N T S
Variations in the method of adding anthrone reagent may cause variations in maximum solution temperature and affect the color intensity. Morse (3) added all the anthrone reagent to form two layers and then mixed the solutions. The authors’ experience with this method for cellulose and starch showed less consistent results than those obtained with rapid addition and mixing. Apparently it is difficult to form two layers with water and concentrated sulfuric acid without evolution of appreciable amounts of heat which create convection mixing currents. Undoubtedly partial evolution of heat caused by such mixing is a significant variable and causes inconsistencies. Any method of addition that yields consistent results is satisfactory. COLOR STABILITY
Although tests have been made on the rate of color formation of anthrone with a carbohydrate (S),information on the deterioration of anthrone-carbohydrate color has not been presented.
300 K
rook/
0.08
A
-
100 ) l G u
STARCH
B
-
100 Y G .
CELLULOSE‘
-f
I
0.16
0.12
IN
% ANTHRONE
0.20
GONG. kS0,
Percentages of Anthrone in Concentrated Sulfuric Acid over 5 Days
Concentrated HzSOIas reference blank Klett-Summerson colorimeter readings, X = 625 mp
characterized by a darkening of the reagent over a period of time, Further investigation x a s made with various anthrone concentrations in concentrated sulfuric acid using the Klett-Summerson colorimeter to determine reagent color changes a t 625 mp, over a 5-day period (Figure 4). The results clearly show the greater stability of the more concentrated solutions. STANDARDIZATION AND APPLICATION O F BEER’S L 4 W
Because anthrone reagent exhibits instability, and in the authors’ evperience inconsistencies in sensitivity, the use of a single standard curve is not practicable. Accurate results can be obtained by the use of one or more known standards for each group of analyses. Obviously this method depends upon the application of Beer’s law for all such anthrone solutions. In investigations with solutions up to 9 days old (maximum age of any solution tested) Beer’s lam was followed, as shown in Figure 5. This observation is in agreement with Morris’s (2) that even after 1 month straight-line calibrations were obtained a t 620 mp. Freshly prepared solutions of anthrone (less than 2 hours old) should not be used, as they cause inconsistent results. Reagents a t least 4 hours old are recommended, with one or more known standards for each group of analyses. Such solutions will give a sensitivity of approximately 2 micrograms of starch or cellulose with the following procedures. ANALYSIS OF CELLULOSE AND STARCH
0.04
0.08
0.12
0.16
0.20
Ye ANTHRONE IN GONG. HpS04 Figure 3. Percentages of Anthrone in Concentrated Sulfuric Acid with Cellulose and Starch Standards Klett-Summerson colorimeter readings, X = 625 mp
Tests made with anthrone and starch indicate complete stability (Table I) from 5 minutes to 3 hours, after which slight fading occurs.
Starch. The solid sample is boiled in distilled water, cooled, and made up to a measured volume with distilled water so that a 2.0-ml. aliquot contains 10 to 200 micrograms of starch. If the solution is turbid, part of it is filtered through a dry filter pa er. A 2.0-ml. aliquot in a colorimeter tube is treated with 4.0 mf: of 0.1% anthrone in concentrated sulfuric acid (4 hours to 9 days old) rapidly added from a pipet or buret. The solution is mixed immediately and allowed to air-cool. After approximately 10 to 15 minutes, the tube is cooled completely in a cold water bath. B
ANTHRONE COWCEVTRATIOR AND COLOR FORM 4TIOh
Observations made with solutions of increasing anthrone concentration revealed increased sensitivity. Data from tests made on starch and cellulose with different anthrone solutions are presented in Figure 3. Reference zero standards consist of the same reactants in each case minus the carbohydrate. lla.;inium sensitivity occurs with 0.16% anthrone solution. Greater concentration of anthrone reduces sensitivity. As there is only a slight increase in sensitivity using 0.16% instead of 0.10% anthrone, a 0.10% solution was selected. Slight turbidity has been observed in some cases when anthrone solutions of higher concentration (0.2%) nere useJ.
,5 0On O t 4
w
K
4 0 0 1 3 0 0 k
c A,- STARCH-REAGENT 8;CELLULOSE
Ieo
MICROGRAMS
STABILITY OF REAGENT
h disturbing feature of this method is reagent instability, which has been investigated previously 12, 3 ) . This instability is
Figure 5.
OF
I DAY
OLD
R E A G E N T 9 D A Y S OLD
-
200
160
CARBOHYDRATE
Standard Curves for Starch and Cellulose, Indicating Application of Beer’s Law
Klett-Summerson colorimeter readings, X
-
625 mp
V O L U M E 21, NO. 8, A U G U S T 1 9 4 9 reference blank containing 2.0 nil. of distilled water is treated similarly. At the same time one or more 2.0-ml. starch standards (100 micrograms suitable) receive the same treatment. The colorimeter is adjusted to zero with the reference blank and the samples and standards are then read. When a logarithmic scale colorimeter is used, concentrations are proportional t o scale readings. The pro ortionality constant is determined by the standards used. Coforimeters with a transmittance scale require B semilogarit,hniic calibration curve. Cellulose. The solid sample is digested icoldj in 607, sulfuric acid for 15 to 30 minutes. The solution is made up to a measured volume with 60yosulfuric acid, so that a 0.5-ml. aliquot contains 10 to 200 micrograms of cellulose. If the solution contains a residue, it is filtered through a dry asbestos mat previously washed with 60y0sulfuric acid and then with water. A 0.5-ml. aliquot is then added to 2.0 ml. of water, and allowed to cool. Then 4.0 nil. of a 0.1% anthrone solution are added and the starch procedure above is employed. A reference blank is prepared from 0.5 ml. of 60% sulfuric acid, 2.0 nil. of water and 4.0 nil. of 0.1% anthrone reagent,. Standards are prepared froin 0.5-ml. aliquots of known aniounts of cellulose in 60% sulfuric acid. Mixtures of Cellulose and Starch. Mixtures of starch and cellulose (cotton) in the presence of each other are analyzed m follows:
953 The sample after boiling in water is filtered through a fine porosity sintered-glass filter or asbestos Gooch pad. The filtrate is analyzed for starch and the residue retained by the filter is digested in 60% sulfuric acid for 15 to 30 minutes. This solution i3 then filtered again if necessary and the filtrate is analyzed for cellulose as outlined above. 4CKYOI'LEDGMEST
The authors wish to acknon-ledge the technical assistance of Maria Frenkel. The work was done on a grant provided by the Nashua I I a n u facturing Company and the Saco-Lowell Shops. L I T E R i T U R E CITED
ISD. ENG.CHEM.,ANAL.ED.,18, 499 (19161. (2) Morris, D. L., Science, 107, 254-5 (1948). (3) Morse, E.E., IND.EXG.CHEM.,ANAL.ED.,19, 1012-13 (19471. (1) Dreywood, Roman,
RPCEIVED M a y 3, 1948.
Dichromate Reflux Method for Determination of Oxygen Consumed Effectiveness in Oxidation of Organic Compounds W. ALLAN RIOORE, ROBERT C. K R O S E R , AND C. C. RCCHHOFT U . S. Public Health Service, Cincinnati, Ohio Although the proposed method for the determination of oxJ-genconsumed has definite limitations, nevertheless it will be of value in estimating the strength of industrial wastes and sewage. Hydrocarbons and straight-chain acids are oxidized very slightly. The end products obtained in the oxidation of amino acids vary with the type of acid used. Branched-chain acids and alcohols as well as phenolic compounds are readily attacked. Sugars are quantitatively broken down to carbon dioxide and water. When 50% by volume of sulfuric acid is used in the reflux mixture, chlorides are quantitatively oxidized. The oxygen consumed values of industrial wastes containing high chloride concentrations can, therefore. be corrected for their chloride content.
S
INCE the inception of a biochemical oxygen demand test in 1870 by Frankland (9) and in 1884 by Dupr6 ( 7 ) for the determination of the strength of waste products of human or
suming power of sewage and industrial wastes-namely, potassium permanganate, potassium dichromate, ceric sulfate, a n d iodic acid.
industrial origin, numerous attempts have been made to devise a chemical method that would give the same results in a much shorter time. Inasmuch as the metabolic activities of the flora and fauna in different samples of waste do not necessarily follow a constant rate, a chemical method would not necessarily correlate with the biochemical determination of oxygen demand. However, it is frequently desirable to know in the minimum time the approximate oxygen-absorbing poiver of a waste. -4 chemical method for determining oxygen consumed is most satisfactory for this purpose, although it nil1 give a value that is not comparable to B.O.D. on toxic wastes and will give higher results on stabilized biological treatment plant effluents, because 11 is impossible for a chemical method to differentiate betneen organic matter in biologically stable and unstable forms. Because a chemical method for determining oxygen consumed seems desirable as an additional criterion of pollution control, it is also necessary to study such procedures more thoroughly in order to understand, apply, and interpret the data obtained with them. Of the various oxidizing agents available, in general, only four have been used t o any extent for determ~ningthe ouygen-con-
Potassium permanganate is still used in the recommended procedure ( 2 ) . Stamm (22) carried out the oxidation with pernianganate in an alkaline solution and prevented the reaction of >InOl-- + Mn02 by the addition of a barium salt which a l l o m a better end point to be obtained. Benson and Hicks ( 4 ) ,in determining the pollution in. sea mater, found that application of the Zinimerman-Reinhardt procedure in titrating the excess permanganate gave more reproducible results. Haupt (IO) tried to correlate the permanganate oxygen consumed with the B.O.D. on wastes from paper pulp factories. He found, however, that the chemical method gave much higher results. due to the fact that cellulose is not readily attacked by either dissolved oxygen or bacteria. -4s xvith most chemical methods, variation in conditions affect the result obtained. Matubara (16) found that increased values could be obtained by increasing the boiling time, increasing the concentration of potassium permanganate used, saponifying the fats or oils, and neutralizing water-soluble fatty acids. Lovett ( 1 5 ) states that totally different values can be obtained depending on whether 0.125 S or 0.0125 4 potassium permanganate is used. Kashkin and Karasik (15) added an initial excess of potassium permanganate calculnted to be equivalent to 0.3 to 0.5 m g . of oxygen, and determined the final excess by titration with oxalic acid a t boiling temperature. Shutkovskaya (21) compared the discoloration of the potassium permanganate by the sample
