plates were held in place with weights. This tank is essentially the same as that described by Dieckert and Reiser (S), except that neoprene gaskets were used in place of plasticine for sealing the tank. This innovation permitted a simplification of the tank and increased the useful life of a given batch of developing solution to a t least 5 days. Developing Solution. One hundred grams of phenol (ACS grade), 200 ml. of ethyl ether (ACS grade), and 126 ml. of acetone were placed in the chromatographic tank and stirred until the phenol had dissolved. Finally, 25 ml. of water was added, giving a clear solution. Spot Test Reagents. After the developing solution was removed one of the following spot test reagents was applied t o the chromatogram. SULFURIC ACID. The chromatogram was sprayed with concentrated sulfuric acid and then heated over a hot plate until charring took place. Heat should be applied cautiously a t first for best results, particularly if rhamnose is present. However, it is usually necessary to heat the chromatogram until the sulfuric acid distills in order to get maximum charring. L-Rhamnose gave a characteristic yellowish tan spot while the other sugars gave brownish black spots. ~ A N I S I D I NPHOSPHATE. E This reagent was prepared according to the procedure of Mukherjee and Srivastava ( 5 ) . The sugars appear as yellow spots on a white background, when the chromatogram is heated for several minutes a t 95' to 100" C. Method. Each sugar was spotted on the impregnated glass paper in 10t o 50-7 amounts as an alcoholic solution. The paper, after drying, was suspended in the tank according to the method of Dieckert and Reiser (3) and, without prior equilibration, was developed by the ascending technique. Two hours or
less were required to develop a chromatogram 10 to 12 inches long and about 30 minutes for a 7.5-inch chromatogram. ilfter development, the ether and acetone were allowed to evaporate. Heat was then applied carefully to remove all of the phenol without igniting the vapors. After the chromatogram was treated n-ith the appropriate spot test reagent and heated, if required, the spots were viewed best by transmitted light. RESULTS AND DISCUSSION
R, values, obtained by chromatographing six sugars singly and by chromatographing a mixture of the same six sugars, are listed in Table I. The agreement between the two sets of values indicates that the sugars migrate similarly, whether chromatographed singly or in admixture.
was permitted to remain in the paper and the sulfuric acid test was applied, a pink spot appeared for each sugar upon the application of heat. This phenomenon resembles the Molisch test. The p-anisidine phosphate test performed satisfactorily on the silicic acidimpregnated glass paper but mas found to be less sensitive than the sulfuric acid test. I n addition, all the sugars investigated gave the same color reactions, contrary to their behavior in a conventional paper system. The technique of glass paper chromatography has proved useful as a rapid means for following the progress of fractionation of constituents of extracts from peanuts, tung meal, and other natural products. ACKNOWLEDGMENT
The authors wish to thank John Table I.
Sugar >Rhamnose D-Xylose >Fructose n-Glucose Sucrose Raffinose
R,
L. White and Katherine M. Formusa for suggesting the use of the neoprene
Values
Single
Mixture
0.75 0 63 0.52 0.43 0 19 0 02
0.74 0.65 0.52 0.44 0.22 0.02
At the present stage of development of the method, the R, values are not reproducible enough to be used alone for the identification of the sugars. As a control, reference sugars should be run concurrently with the unknown. Sulfuric acid proved t o be a satisfactory reagent for locating the spots containing these sugars. If a little phenol
gaskets. LITERATURE CITED
Brown, M., Yeadon, D. A,, Goldblatt, L. A., Dieckert, J. W., AXAL. CHEM.29, 30 (1957). Dieckert, J. W., Reiser, R., Federation Proc. 14, 202 (1955). Dieckert, J. W., Reiser, R., J. Am. Oil Chemists' Soc. 33, 123 (1956). Dieckert, J. W., Reiser, R., Science 120, 678 (1954).
Mukherjee, S., Srivastava, H. C., Nature 169, 330 (1952). RECEIVED for review April 27, 1956. Accepted September 10, 1956. Division of Analytical Chemistry, 129th Meeting, ACS, Dallas, Tex., April 1956. Mention of specific tradenames does not imply endorsement of the product or of its manufacturer.
Quantitative Chromatog rap hic Procedure for Determining Dextrose in Sugar Mixtures EMMA
J. MCDONALD
National Bureau of Sfandards, Washington 25,
b Dextrose can be transferred from a paper chromatogram to glass fiber paper and the sugar subsequently determined in the presence of the glass fiber. Results are given for the procedure as applied to the determination of dextrose in honey.
R
sugar methods are not completely selective; therefore, the determination of individual sugars in a mixture offers problems not present in the analysis of individual sugar samples. It is desirable to separate
32
EDUCING
ANALYTICAL CHEMISTRY
D. C.
mixtures into their components prior t o analysis, and paper chromatography offers a means of accomplishing such a separation. The advantage gained by thus eliminating interfering sugars prior to a particular analysis is somewhat reduced by the size of sample that can be chromatographed on paper. In spite of this limitation, paper chromatography has been used in the analysis of many sugar products. Following paper chromatographic separation, sugars are generally removed from the chromatogram before quantitative determination. Such a proce-
dure is imperative if analysis is to be made with reagents which have an appreciable effect on cellulose. The colorimetric methods employing anthrone or phenol in concentrated sulfuric acid are representative of this group. Although the use of filter papers of heavy weight, such as Whatman Nos. 3 and 17, has increased the size of sample that can be separated chromatographically, the fibers of these papers interfere with titration when the sugars are determined by copper reduction methods. Also, an appreciable correction must be made for the effect of the'
reagents on the paper. The advantage in accuracy that one normally obtains by methods appropriate for the larger samples as compared to those designed for the determination of smaller amounts of reducing sugar is thus lost. A procedure is described here in which the separated sugar is transferred to inert glass fiber paper, on which it is then introduced into the reaction mixture. The use of this technique is illustrated hy its application to the determination of dextrose in a synthetic mixture and in two commercial honeys. Glass fiber is unaffected by acid as well as alkaline reagents; therefore, the procedure of transferring the sample onto it prior to analysis has broad application. When clear solutions are required for color observations, the glass fiber may be readily removed by filtration through glass wool. QUANTITATIVE TRANSFER T O GLASS FIBER PAPER
The glass fiher filter paper was grade
X934-H, H. Reeve Angel and Co. Before uae the paper was washed with distilled water and dried on the smooth surface of a glass plate.
Figure 1. Filter paper segment of chromatogram
rose samples were transferred to iber by an tlscending flow of through paper chromatogram Its. The latter, cut and creased nm in Figure 1, were placed in a lizing dish containing water and I place by meana of a porcelain Figure 2). On top of each paper it was placed a folded piece of
'.
0.8
0.8 0.8
1.0 1.0 1.2 1.2 1.2
Transfer of dextrose to glass fiber paper
glass fiber paper. The latter consisted of a 1-inch square folded and held in the folded position by use of two slits made on one side of the square. The mat side which contained the short fibers was turned inward, because the sugar concentrates in this area. The lower surface of the folded glass paper rested on the top edge of the filter paper. It is necessary for contact to be made between the end fibers of the filter paper and the glass fiher paper in order to get effective transfer. Three hours were allowed for the water to rise through the paper and for the dextrose to be transferred to the glass fiber. The latter was then placed in the reaction tube by means of forceps. Dextrose was determined by Somogyi's phosphate method (Z), modified as follows to take account of the glass fiber. The 5 ml. of reagent and the 10 ml. total volume were increased to 7 and 14 ml., respectively, in order to cover the glass fiher completely. A proportionally larger quantity of sulfuric acid wtls required a t the neutralizing stage. During titration the reaction mixture was stirred with a glass rod whose lower end was bent and curved so that it just cleared the side walls of the reaction test tube. Dextrose samples ranging from 0.5 to 2.0 mg. may be determined by this method.
Determination of Dextrose after Transfer from Filter Paper to Glass Fiber
lextrose, Mg. ed Found 5
Figure 2.
.46 .51
.79 .78 .79
1.03 1.04 1.22 1.20 1.20
Error, Mg. -0.04 to.01 -0.01 -0.02 -0.01 i0.03 +0.04 +0.02
0 0
Dextrose, Mg. Added Found 1.2 1.2
1.21 1.22
1.5 1.5
1.50
1.6 1.6 1.6 2.0 2.0
1.57 1.58
1.50
1.58
2.07
1.99
Error, Mg. +0.01 io.02
0 0
-0.03 -0.02 -0.02 i0.07 -0.01
Known quantities of dextrose were placed on segments of filter paper, the sugar was then transferred to glass fiber, and the dextrose determined as described above. The results are given in Table I. The precision of analysis is equal to that obtained by thismethod when dextrose is determined directly. Removal of sugar from the filter was confirmed by subsequent spraying of the paper with a 0.3y0 solution of paminohippuric acid in ethanol (1). This reagent is sensitive to 1 y of dextrose. APPLICATION T O HONEY AND SYNTHETIC SUGAR MIXTURE
Three sugar solutions were chromatographed on Whatman No. 3 filter paper. Two were prepared by dissolving 20 grams of a honey in water and diluting to 100 ml.; the third was a synthetic solution of dextrose, levulose, and sucrose containing 5 grams of dextrose per 100 ml. A volume of 20 nl. (0.02 ml.) of each of the solutions was applied as separate spots on the chromatograph paper. The outer spaces contained dextrose, levulose, and sucrose, in that order, and the center areas contained the synthetic sugar mixture and the honey samples. The chromatograms were placed in a closed cabinet and developed for 65 hours a t room temperature by downward flow of the butyl alcohol-ethyl alcohol-water solution. During this time the papers were twice removed, dried, and subsequently returned to the chromatograph cabinet for further development. The chromatograms were then removed and dried a t room temperature. The outer strips, containing the known sugars, were cut from the main portion of the chromatogram and sprayed with the p-aminohippuric acid solution. After drying them in air they were heated 5 minutes a t 120" C. The locaVOL. 29, NO. l , JANUARY 1957
* 33
tion of the dextrose, levulose, and sucrose was then observed under ultraviolet light. Using the location of dextrose on the outer strips as a guide, the position of the dextrose on the center portion of the chromatogram was determined. The portions of paper containing dextrose were clipped off and the dextrose on each was transferred to glass fiber paper and analyzed as described above. The results are given in Table 11. The floral origin of the honey samples is not known; however, the dextrose found is of the same order as that reported by J$71~ite and Maher (3). I n products such as honey n here large quantities are available, micromethods of analysis are not generally used because of their lower precision. However, when an individual sugar is quantitatively separated prior to its determination, the accuracy of the results depends solely upon the method of analysis. Paper chromatography piovides such a separation of microquantities. TVhen using the conventional selective methods, one must consider the
Table II. Determination of Dextrose in Sugar Mixture and in Honey
%
Dextrose, Mg. Dextrose in PresDextrose-suerose-levulose mixture Honey I Honey I1
ent
Found
1
1.00 1.04 0.98 0.938 0.930 1.28 1.31
30 (approx,) 23.45 23.25 32.0 32.75
effect of all of the constituents on the results. White, Ricciuti, and Maher (4) made an extensive investigaton of the dextrose and levulose content of 15 honey samples from different floral sources. I n this study they compared the results obtained by five different macroselective methods. By statistical nieans they were able to determine the method that gave the greatest precision. However, because nondextrose constitu-
ents have a different effect ou the individual methods, the authors were unable to tell which one gave results closest to the actual composition of the honey. White and hlaher (3) later partially resolved this problem by separating honeys into their monosaccharide, disaccharide, and polysaccharide constituents by means of carbon-Celite columns. The procedure for transferring a sugar onto glass fiber and subsequently adding the sample and glass fiber to the react8ion mixture is applicable to other sugars of this mixture as well as to any sugar mixture that can be separated on a paper chromatogram. LITERATURE CITED
(I) Sattler, L., Zerban, F. K . , Ax.4~. CHEJI.24, 1862 (1952). (2) Soniogyi, If., J. Biol. Chem. 160, 61 (1945). (3) White, J. IT.,Jr., Maher, J., J . Assoc. Ojic. Agr. Chemists 37, 498 (1954). (4) Khite, J. W., Jr., Riceiuti, C., Maher, J., Ibid., 35,859 (1952). RECEIVEDfor review May 9, 1956. rlccepted October 12, 1956.
Coprecipitation of Radium with Barium Sulfate LOUIS GORDON Syracuse Universify, Syracuse, N . Y KEITH ROWLEY Brookhaven National Laborafory, Upfon, L. I . , N. Y.
b The technique of precipitation from homogeneous solution was used to determine the nature of coprecipitation of radium with barium sulfate. The distribution of radium between the aqueous and solid phases follows the law of Doerner and Hoskins. The value of A, the distribution coefficient in the Doerner-Hoskins equation, was found to b e constant within experimental error over the range in which the fraction, f, of the barium precipitated was 0.03 to 0.96. Its value is 1.21 f 0.009f under the experimental conditions a t 90" C.; its value a t other temperatures obeys the relationship, loglo X = 2 2 0 / ( 2 7 3 t ) - 0.520, for values of t between 50" and
+
90" C.
T
HE coprecipitation of radium with barium sulfate during the slow precipitation of the latter has been previously studied. Doerner and Hoskins ( 2 ) have deduced that the distribution of radium between the solid and liquid phases should obey the relationship:
34
ANALYTICAL CHEMISTRY
where X is the logarithmic distribution coefficient and i and f represent initial and final solution concentrations. Another mode of distribution (10) is given by /Ra++\
D =
\Ba++)orystnl
(E+)
(2)
Ba++
where D is the homogeneous distribution coefficient. The two modes of distribution result from different mechanisms of attainment of equilibrium between solid phase and solution. There are two conditions under which the Doerner-Hoskins distribution equation is obeyed: The surface of the growing crystal is in equilibrium with the hody of the solution-i.e.,
(3)
and the rate of diffusion of ions n-ithin the crystalline lattice is negligible compared to the rate of precipitation. It is also usually assumed that activity coefficients remain constant. Doerner and Hoskins utilized evaporation and cooling of saturated barium sulfate solutions in some experiments to attain equilibrium between the crystal surfaces and solution. The data they obtained were not sufficient to prove the constancy of the logarithmic distribution coefficient over a n-ide range of "fraction of barium precipitated." Narques (6) also subsequently precipitated barium sulfate by an evaporation process; her data are shown in Figure 1,a. The values of the logarithmic distribution coefficients found by Marques for the evaporation process a t 20" C. were slightly higher than those found by Doerner and Hoskins; the latter investigators did not state the temperature at which their esperiments were conducted. Both Marques [cf. (6, Figure l,a)] and Doerner and Hoskins found smaller logarithmic distribution coefficients by precipitation
