Separation of Aluminum, Mercuric, or Ferric Ions from Various Cations by Electrochromatogra phy SIR: Differential electrical migration in solutions of complex-forming reagents serves for the complete separation of one or a few ionic species from multicomponent mixtures (1, d). To exploit some of the possibilities for separations by this technique, lve have tested various complex-forming substances as selective reagents for the separations. The exploratory tests were carried out with individual zones of the various cations in one sheet of paper. I n addition to the ions tested before, radioactive cesium, strontium, and yttrium were also employed. The promising reagents were then tested with mixtures of about 20 cations as described before (2). iiluminum, mercuric, and ferric ions may be separated from complex mixtures. I n the respective solutions, each
of these ions is complexed to form anionic species and is separated from all the others which remain as cations. Aluminum ions were separated in an aqueous solution of 0.06M diethylenetriamine, p H 11. The potential was 10 volts per cm. in paper 70 cm. long. Migration time was 1 hour. The trailing boundary of the aluminum zone migrated about 1 em. tornard the anode from the starting point. Mercuric ions were separated in an aqueous solution of 0.02.11 diethylenetriamine and 0.08V hydrochloric acid, p H 1.7. The potential was 7.9 volts per cm. in paper i o em. long. Nigration time was 40 minutes. The trailing boundary of the mercuric ion zone migrated 1 cm. toward the anode. Ferric ions were separated in an aqueous solution of 0.05M malonic acid,
pH 2.2. The potential was 10 volts per em. in paper 70 cm. long. Aligration time was 2 hours. The trailing boundary of the ferric ion zone migrated 1.5 em. tou-ard the anode. LITERATURE CITED
(1) Evans, C. H., Strain, H. H., b x . 1 ~ . CHEW,28, 1560 (1956). ( 2 ) Strain, H. H., Binder, J. F., Evans,
G. H., Frame, H. D., Jr., Hines, J. J., Ibid., 33, 527 (1961). H.kRL.4N D. FRAME, JR. HAROLD H. STRAIN
Argonne National Laboratory Argonne, Ill. JOSEPH QHERMA
Lafayette College Easton, Pa. BASED on work performed under the auspices of the U.S.Atomic Energy Commission.
Spectrophotometric Determination of Sugars Using p-Aminobenzoic Acid SIR: Recently, Ek and Hultman (1) showed that p-aminobenzoic acid can be used as a colorimetric reagent to determine glucose in body fluids. The same reagent was later used b y Roy ( 2 ) for detecting sugars on paper chromatograms. Roy showed that 1 pg. of glucose, galactose, mannose, xylose, or rhamnose could be detected, if oxalic acid were present in the reagent. The present paper describes a new and improved method, based on the above findings, for the quantitative determination of sugars in the 1- to 200-fig. range. The new method shows greater sensitivity than the commonly used o-aminodiphenyl method described by Time11 (4). I n addition, the p-aminobenzoic acid reagent is very stable and it is virtually nontoxic.
pounds in varying proportions, thus leading to nonlinear calibration curves. Phosphoric acid did not cause any of these complications. The calibration curves 11-erelinear (see below). Further-
a W
PROCEDURE
The sugar sample (I to 200 pg.), contained in 0.5 ml. of water in a glassstoppered test tube, is mixed with 2.0 ml. of a 1.5% solution of p-aminobenzoic acid in glacial acetic acid and 2.0 ml. of a 1.3% solution of phosphoric acid (100%. basis) in glacial acetic acid. The mixture is heated in a boiling water bath for 1 hour. Cpon cooling, the absorbance of the sample is measured against a reagent blank a t 360 mp, using a Beckman DK-2 spectrophotometer or similar instrument.
DEVELOPMENT OF METHOD
Preliminary experiments were carried out to establish optimum conditions for the color reaction. The use of acids stronger than oxalic resulted in higher intensity and better stability of the developed color, but of the strong acids tested only phosphoric acid gave completely satisfactory results. Sulfuric acid yielded a precipitate with the reagent, which sometimes interfered. Hydrochloric acid seemed to lead to simultaneous formation of several colored com170
ANALYTICAL CHEMISTRY
more. the readings obtained were not sensitive to minor variations in reagent concentration or reaction time, nor did storing the reagent for up to 3 days or the developed color sample for up to 4 hours have any significant effect: Addition of water to the reagent reduced the sensitivity, but color and reagent stability and linearity of the calibration curves n-ere not affected. On the basis of this preliminary work, the folloiving procedure m s adopted.
A
GLUCOSE
0 MANNOSE X
Figure 1. sugar
GALACTOSE XYLOSE ARAB,NOSE
Absorbance vs. quantity of
RESULTS
Relationship between Absorbance and Sugar Concentration. Duplicate tests were run a t several different concentrations of five different sugars. T h e results (averages) are given in Figure 1.
