ANALYTICAL CHEMISTRY
1570 accurate by the following treatment: The powdered material is placed on the table of the Kofler instrument, melted, allowed to cool, and remelted. The curve of the second melting is taken. Thus the lower rupture of the photocurrent indicates the temperature of the stiffening point. The difference between melting and stiffening point is characteristic for fats-i.e., 7.5" C. for lard and 12.5" C. for beef tallow. The melting points of fats and waxes have been measured in this way. The curve of chloral hydrate is of interest, for here the first maximum means a change of structure. Then passing through a minimum the curve ascends to the maximum a t around 57" C. Measuring in a capillary tube, the melting points of 50.0' to 50.2 C. for naphthylamine purified by recrystallization and of 80" C. for naphthalene purified by sublimation were obtained. Temperature rise occurring during the measuring period is about 3' C. in the case of waxes and 7.7" C. in the case of naphthylamine. This phenomenon indicates the different melting mechanisnls of the two types of material. During the measurement
the regulator resistance of the Kofler instrument was calibrated in a way to keep the speed of temperature increase a t about 1 C. per minute in the vicinity of the melting point. Application of the photocell makes the determination of melting point quicker, more simple, reproducible, and exart. A similar procedure may be applied to determination of evaporation time, particle size, and number of blood cells. ACKNOWLEDGMENT
The valuable help of Thomas Sandor in making the measurements is acknowledged. LITERATURE CITED
(1) Chamot and Mason, "Handbook of Chemical Microscopy," Vol. I, p. 198, New York, John Wiley & Sons, 1940. ( 2 ) Scott, W. W., "Standard Methods of Chemical Analysis," 5th ed.. Vol. 11, p. 2453, New York. D. Van Nostrand Co., 1938. RECEIV D ~June 15, 1949.
Distillation of Antimony Trichloride for Improved Reagent ALEXANDER MUELLER AND SERECK H. FOX with the technical assistance of ROBERT I. DAY, Gelatin Products Division, R . P . Scherer Corporation, Detroit, Mich. UTIMONY trichloride has become well established as a A-chromogenic agent for the photometric analysis of vitafins A and D (1,4,6) and several sterols ( 2 , J ) . It is often difficult to prepare a satisfactory reagent in chloroform, especially from a previously opened stock bottle of antimony trichloride. Exposure of the crystals or lumps to air and moisture causes a discoloration. The addition of drying agents such as acetyl chloride, acetic anhydride, or anhydrous potassium carbonate gives some degree of improvement, but does not give complete assurance of a stable and reproducible reagent.
Table I.
Li2m. Values
Trial 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Average Max. range, % 1Ia.x. deviation, %
on U.S.P. Vitamin A Reference Standard
Undistilled SbCb 12.22 12.42 12.51 12.66 12.27 12.84 12.70 12.80 12.53 12.28 12.81 12.35 12.40 12.39 12.35 12.43 12.34 12.15 12.30 13 06 12 50 7.28 4.48
Distilled SbCla 13.46 13.45 13.20 13.48 13.32 13.27 13.39 13.73 13.20 13.05 13.11 13.09 13.61 13.39 13.49 13.07 13.18 13.05 13.17 13.21 13.296 5.12 3.26
When a stock of antimony trichloride contains impurities or becomes discolored from frequent exposure, it can easily be purified by simple distillation under moderate vacuum. It distills smoothly a t a temperature of 140" to 150" C. and a vacuum of 10 to 15 mm. (obtainable with a water aspirator). The distillate is white and remains colorless for several months. The reagent in chloroform prepared from distilled antimony trichloride is more stable, readily reproducible, and more sensitive.
ANTIMONY TRICHLORIDE DISTILLATIOh
The vacuum still (Figure 1) consists of a 2-liter threenecked round-bottomed flask and a 105" distilling connecting tube mounted for reflux. This is attached to a 30-cm. condenser
Figure 1. Apparatus Distillation of Antimony Trichloride for
V O L U M E 22, NO. 12, D E C E M B E R 1 9 5 0 placed in a vertical position. A jacketed vacuum adapter tube (available from the Vitamin Research Instrument Company, Detroit 3, Mich.) and 250-ml. receiver flask are placed in position. All joints are standard-taper ground-glass. A small amount of silicone stopcock grease is applied to all joints. The 2-liter distilling flask is of ample size for a 3- to 4-pound (1.4- t o 1.8-kg.) charge. A source of steam is conducted through the condenser and the jacketed vacuum adapter tube. This is necessary to keep the molten antimony trichloride from solidifying before reaching the receiver. Other accessories consist of a capillary bleeder tube (for uniform refluxing and for breaking the vacuum) to which is attached a drying tube filled with Drierite; a thermometer a t the top of the condenser; and a safety flask a t the vacuum connection. The still is convrniently heated with a Glas-col hwt(1r CO3IPARISON OF REAGENTS
The ditta presented in Table I on the U.S.P. vitamin A rcfrrt.nce standard (10,000 units per gram) were obtainrd over t i 1w.riod of 2 years in thr coursc of daily standardization. All values reported were performed on the unsaponifiable fraction. The saponification and analytical procedurcs wcre those tlescribed by Oaer et al. ( 5 ) . A suninmry of the rrsults obtained, employing distilled and undistilled antimony trichloride, is shown at tho bottom of Table I. The masimum range is the prr cent diffrrence between the highest and lowest value from the average. The maximum deviatioii is the largest per cent difference between any singlc valuc and the average. By simple comparison of thc two average I,:?~, valric,.: tlic improvc~nicnt in sensitivity is
1571 seen to be 6.36%. The rate of reaction with vitamins .4and 1) \vas not perceptihly different between the two reagents. DISCUSSION
The dist,illed antimony trichloride is collected in quantities of :ibout 100 ml. in 250-ml. flasks, and is stored in t>heflask, tight,ly itopperrd in a cool, dark plact.. Flasks should not be filled OVI'I' half-full brcause of expansion and subsequent breaking upon reheating. Storing the antimony trichloride in separate, small quantities avoids too frcquent exposure to air and mnisturct. When needed for preparation of the chloroform reagent the flask i s placed in a 100" C. drying oven (thr ant,imony trichloride is melted), and the required amount is withdram with a warm pipet. Precautions should be taken to rscludc moist air as much :ts posGble. LITERATURE
cmm
Ewirig, D. T., Powdl, If.J., Brown. 11. A . , and Emmett. A . D.. ANAL.CHEM., 20,317 (1948). (2) Lamb. F. W., Mueller, A, and Reach, G. W., IND.EN(:.C H E M . . ASAI,. ED.,18,187 (1946). (3) Mueller. A. J . Am. Chem. SOC..71. 924 11949). (4) Xield, 6.H., Russell. JV, C., h ' Z i m m e r l i , :4.,J . B d . C ~ P V , , 136,73(1940). (5) Oser, B. L., Melnick, Daniel, and Pader, Morton, IND. E m . C H E X . , A N A L . ED.,15,724(1943). (6) Pharmacopeia of the Pnited Stat,es, S I V , 1950.
(1)
RECEIVED
February 23, 1950
Volumetric Determination of Calcium and Magnesium in leaf Tissue A . E. WILLSON, Citrus Experiment Station, Lake Alfred, Fla. volumetric procedures for the determination of calcium and magnesium based on a sequestrating reaction with disodium dihydrogen ethylenediamine tetraacetate dihydrate (Versene) have been described for water analysis (2-4). The study of interfering ions by Bets and No11 (9)indicated the possibility of using this reagent for the determination of calcium and magnesium in citrus leaf tissue solutions. 7 IhlPLE
Table I.
Calcium and Magnesium in Citrus Leaf Samples Calcium, 70 Volumetric
Oxalate 2.87 2.82 2.85 2.92 3.01 3.32 3.70 3.77 3.04 2.87 3.26 2.58 2.58 2.2R 2.49 2.58 2.37
2.85 2.75 2.71 2.90 2.89 3.20 3.64 3.82 2.96 2.78 3.16 2.55 2.61 2.24 2.50 2.68 2.33
Magnesium. % Oxine Volumetric 0.38 0.35 0.37 0.37 0.37 0.40 0.45 0.48 0.39 0.39 0.38 0.35 0.37 0.34 0.40 0.40 0.37
0.39 0.35 0.38 0.40 0.39 0.38 0.38 0.34 0.38 0.40 0.44 0.33
0.3G
0.35 0.35 0.37 0.38
Following a nitrir-perchloric acid digestion (S),aliquots representing 0200 gram of the dried leaf tissue were diluted to 50 ml. with deionized water and titrated to the methyl red end point with 1 N sodium hydroxide. T h e titration procedures described by Betz and ?Toll (Z),employing Ericochrome black T for calcium and magnesium and ammonium purpurate for calcium, were applied directly to these solutions. Separate standardizations of the titrating solution were made for the t 4 o indicators. Results for single determinations on a number of different samples are given in Table I. Values for calcium and magnesium by the oxalate ( 1 ) and 8-quinolinol (ouine) ( 7 ) procedures are included for rompiri-on.
Differences between the methods were calculated a8 per cent of the oxalate and oxine values. The mean difference for calcium values was -1.44% and the standard error was 0.134. The maximum difference for the calcium values was 0.14, which is equivalent to about 5% of the oxalate value. Student's t test (6) showed that the mean difference was significant. Schwarzenbach et al. ( 5 ) have reported that magnesium ions interfere with the color change of the calcium indicator. Beta and Sol1 (2) have pointed out that orthophosphate will precipitate calcium a t the pH of the test. During the titration of leaf samples it was noticed that as the end point was approached the indicator changed color completely and then faded to the original color again with each addition of titrating solution. Near the end point the change was slower. -4series of standards containing known amounts of magnesium and orthophosphate was titrated with Versene ( 2 ) ,using ammonium purpurate indicator. Magnesium equivalent to 1% in the original sample had no effect on the titration. Orthophosphate equivalent t o 0.1% phosphorus in a leaf sample caused a slight lag in the end point. This effect increased with increasing amounts of phosphate. Beyond a concentration e.{uivaleiit to 0 26y0 phoqphorus in the leaf tissue (10 p.p.m. of phoqphorus in the 50-ml titration volume) the end point for the titration was too indefinite to be of any value. The mean difference for the magnesium values was equivalent to - 1.64% of the amount determined with oxine and the standard error was 0.61. Values obtained by the volumetric procwiure varied from the oxine procedure over a wide range. Student's t test showed that the mean difference was not significant and was therefore due to random errors in the procedures. There Jvas approximately ten tinies as much calcium as magiiesium in the samples. Therefore the values for magnesium $I ?re based on the difference between two relatively large t i t r e tiom. Under such conditions erratic results may b~ wpected. Iliehl et al. (4)made a study of interfering ions and buffers, and found that a buffer containing sulfide may interfere with the end ]mint by precipitation of the blue-black iron sulfide. It is possi-
