Anthrone in Estimating low Concentrations of Sucrose E. E. MORSE, Spreckels Sugar Co., Woodland, Calif. 1 procedure is presented for the estimation of sucrose concentrations in the range of 10 to 250 parts per million, using a 0.05% solution of anthrone in concentrated sulfuric acid. This method offers the advantages - of increased sensitivity and accuracy over the commonly used Molisch a-naphthol test.
0
YE of the most coriinion tests in sugar processing factories
is the estimation of the sucrose content of various condensed vapors and solutions in which the sucrose content lies in the range of 10 to 250 p.p.m., n-here it cannot be accurately measured by polarimetric means. In such cases, the sucrose content is universally estimated by the LIolisch a-naphthol test. Since this is a ring test, it cannot be adapted readily to transiriittancy measurements and the sucrose content of a solution is usually reported after visual estimation to be a trace, light, or heavy. The use of anthrone dissolved in concentrated sulfuric acid was suggested by Dreywood ( 1 ) as the reagent in a qualitative test for carbohydrates. The extreme sensitivity of this test, plus the fact that it is not a ring test, made it appear to be a promising improvement. The anthrone reagent has been uded by the author to develop a procedure for the quantitative determination of sucrnse in low ronrentrations. PROCEDURE
The solution to be tested (2.0 ml.) is measured into a clean, dry
solved in concentrated sulfuric acid (reagent grade) are added so as to form a bottom layer. The tube is then shaken to effect complete mixing. The transmittancy of the solution for white light is next measured in a hotoelectric coIorimeter. Distilled water is used as the standark: with its transmittancy taken to be 1.000. By reference to a previously prepared calibration curve or‘ table, the concentration of sucrose in the original sample is ascertained. Table I and Figure 1 present calibration data obtained with a Lumetron photoelectric colorimeter, Model 402E, manufactured by the Photovolt Corporation, Ne%-York, N. Y . The sample and the reagent are shaken together as one shakes down the mercury in a clinical thermometer, with care being taken to prevent spilling. If for re‘asons of safety it is not desired that the tube be shaken, a small glass Ptirrer may be used. Inasmuch L L ~the color-forming reaction is highly sensitive to temperature, it is important that the test be performed in a standardized manner. Variations in procedure will change the temperature rise caused by the heat of dilution of the acid and hence change the amount of blue-green coloring matter produced hy a giwn q u ( w s e rwncentration.
Light Transmittancy of Sucrose SolutionAnthrone Mixtures Solutione
++ + + + +
Anthrone reagent distilled water Anthrone reagent 10 p.p.m. sucrose solution Anthrone reagent 2.5 p.p.m. sucrose solution Anthrone reagent 50 p.p.m. sucrose solution Anthrone reagent 125 p.p.m. aucrone solution inthrone reagent 250 p.p.m. Bucrose solution a Anthrone reagent prepared 4 hours previously. b Anthrone reagent prepared 27 hours previously.
P
u
150-
UI
g
100-
/’
Transmittancy 1
onna
0.791 0.659 0,493 0.317 0.115 0.024
50
-
I If it is desired to use a visual comparison method, Lovibond standard color plates may be used in a suitably constructed comparator. I t is possible to make close matches between the plates and the coloring matter produced in the anthrone test. This method offers an advantage if the samples are turbid becauseone ran disregard the turbidity to a large extent in matching colors. Primary standards made up from the anthrone reagent and sucrose solutions of k n o w concentration and sealed in Pyrex tubes were not found to be satisfactory because the blue color gradually fades and the solutions become slightly eloudy. A t the higher sugar concentrations, a slight amount of dark-color4 material coats the inside of the tubes.
The impurities commonly present in the condensed water saniples of a beet sugar factory do not interfere seriously in this test. Solutions of diffusion juice, thick juice, molasses, and Steffen filtrate were prepared, each containing 200 p.p.m. of sucrose The blue-green colors formcd in the first three solutions were closely the same, but an olive-green color was obtained with the Steffen filtrate sample. This is not surprising when the Ion sugar-to-solids ratio of this material is considered. A special calibration would be required for the accurate measurement of the sucrose content of rondensed waters containing Steffen filtrate.
The photoelectric colorimeter provides the most accurate and wnvenient method of color measurement. Various types of inqtruments can be used, depending on the degree of precision required. For the average factory laboratory, a single photorell
-
200-
m
EFFECT O F IMPURITIES
.METHOD OF COLOR MEASUREMEhT
Ihatilled - .-..... - water .. __.
9 4
d
15 X 125 mm. Pyrex test tube and 3.0 ml. of 0.05% anthrone dis-
Table I.
colorimeter in rvhich the tiansmittancy is indicated by the de. Hection of a microammeter is probably suitable. This may be calibrated using the particular test tubes (usually 15- to 18-mm.) Lyhich fit the holder. The data presented in Table I were taken from two runs made on successive days with sucrose solutions vhich were freshly prepared just before the tests were conducted It is difficult to evaluate the precision of the method with onl: two runs, but the difference between these runs amounts to about 2%, hased on the sucrose content.
1 OOOb
0.783
0,658 0.498
REAGENT STRENGTH AND KEEPING PROPERTY
0.323 0.109
Dreywood ( 1 ) recommended that the strength of the reagent be 0.27&. Preliminary experiments showed that the reagent atrength could be reduced to 0.05% and still give tests of sufficient sensitivity to permit distinguishing between solutions containing
n. 024
1012
D E C E M B E R 1947
1013
10 arid 25 p.p.m. of sucrose. The niore dilute reagent offers ail d v a n t a g e because its bright yellow color is reduced. By using whes of greater diameter, the strength of the reagent may be still further reduced. This offers no partitular gain because an in‘.rrased volume of reagent is required’and, furthermore, the large rvst tubes are inconvenient to handlr i u niaking many routine : I ieasurements. The anthrone used in this work \vas jwpared according to the tiitithod in “Organic Syntheses” ( 2 ) . Solutions of this material in Svjnccntrated sulfuric acid turned a t ) r h i - g r c e n color overnight. Since the presence of an impurity vas suspected, the anthrone $vas rccryrtallized from glacial awtir acid to yield material tyhich -lowly changed to an orang+yrc~rn color on long standing. X wand recrystallization from the sanic solvcrit n-as of benefit, but riiBt a third. Possibly one recrystallization n-ould be sufficient if nn excess of solvent w ~ r eused and the anthrone sloal?- crystallizrd arid well washed with fwsh solvent. In order to test thc keeping pmp(’rty of tht, anthrone reagent, various samples of anthrone in concentrated sulfuric acid were waled in 18-mm. (outside diameter) Pyres tubes and stored at rooin temperature. One sample was stored a t 0 ” to 1 C. The transmittancies of the tubcs relative to distilled water were rnrasui~edat intervals. I n Figure 2 they are plotted relative to ‘he initial transmittancy. These measurements were made in a F’hotovolt Lumetron colorimeter using unfiltered light. The apparent anomaly that material which was recrystallized r h r w times developed color faster than material which was rellized twice is believed to be due t6 the fact that the room ’t>mpcrature was higher during the test period on the former rnaterial. Even solutions prepared from the purified anthrone and re.$gent grade sulfuric acid slowly darkened to an orange color on qtanding. Fresh solutions or older solutions which had been ri4rigerated a t 0 ” gave a negative ferric chloride test for an enol, t)ut solutions which had stood for about 7 to 9 days at room wiiperature gave a positivc enol test. It is believed that the 40w increase in color and the positive enol test are both indicar i i m that anthanol, the tautomer of anthrone, is gradually formed III the solut,ion. Other decomposition products are undoubtedly formed also. For that reason, it is suggested that the 0.057 reagent he freshly prepared every three or four days.
1
22
P P M SUGAR
250
125
so
25 13
2
I
3
5
4
7
6
E
TIME I N M I N U T E S
Figure 3.
Table 11. Sucrose P.p.m. 250 125 50 25 12.5
Rate of Color Development in Solutions of Anthrone Reagent and Sucrose
Rate of Color Development in Anthrone Teat Max. Densit,g
Time to Reach 0 . 9 5 Max. Density
0 . 4 0 Max. Density
Time t o Reach
vi71
Mm.
2.07 1.05
1.8
1.5
1 4 1.0
0.49
2.1 1 8
I .8
1.1 1.1 1.1
0.24 0.13
RATE O F COLOR FORMATION
L)eterniinat,ions were made of the time required to reach the maximum color density after the reagent and solutions containing various concentrations of sucrohe had been mixed. I n these detrrminations, the sucrose solution was contained in one test tube and the reagent in another. ;It zero time the solutions were mixed by pouring thr rcagt’rit into the sucrose solution and then poiiring the mixturr hack into t tit, first tube. Transmittancp
readings were taken a t frequent intervals until the reactions \sei+ substantially complete. I n Figure 3 the optical density (-log transmittancy) is plotted against time after mixing. The tranqmittancies are the values relative to the transmittancy of the. blank. Table I1 shows the time required to reach 90 and 95vc of thc final density. A delay of 2 minutes before measuring the coloi will enqure that 95% of the color has been developed, while about 9 0 5 ii drveloped in 1 minute. COMPARISOY WITH a-NAPHTHOL TEST
!I 2
The main advantage of anthrone is that it gives a more accurate and more sensitive test than a-naphthol. The coloring matter is distributed throughout the n hole body of the solution and i. not concentrated in a shallow ring. Furthermore, the eytent ut agitation of the tubc has no effect on the test as it does nith a-naphthol. Solutions containing 10 and 25 p.p.m. can br readily distinguished from each other. I t is very difficult nith the a-naphthol test to make this distinction.
01
b\
02 -
--Q--c
LITERATURE CITED
I
Figure 2. Effect of Kecrystallization of Anthrone on Color Development in 0.05% Solutions
(1) Dreywood, R., IXD.ESG. C H E M . ,ANAL.ED.,18, 499 (1946). (2) Gilman, H., a n d B l a t t , 4.H., editors, “Organic S ~ n t h e s e s , ” Collective Vo1. I, p. 60, New York, John Wiley & Sons, 1941. RECEIVEDSlay 2 2 , 1947.
