Anomalous Capillary Melting Points for Sugars Obtained with Borosilicate Glass Capillaries SIR: Capillary melting points for organic compounds have been commonly obtained with the use of softglass capillaries, usually hand-drawn. However, commercially available capillaries are now in extensive use, being inexpensive, conveniently packaged, and highly uniform in shape. These capillaries are made of borosilicate glasses, which a t first thought should not occasion any difference in the melting temperatures of compounds. R e have found, however. that this glass does influence the melting points of some of the sugars. When the melting points of three samples of pure glucose, two of the alpha and one of the beta forms obtained commercially from three different sources, were being checked using thc commercial capillaries (one end sealed) they were found to be 5' to 10' C. higher than the literature values. But the anomalous capillary melting points continued to be obtained *hen other capillaries from another package n ere used. Infrared spectra of the samples verified the anomer structures and failed to show the presence of any impurities. The melting point of a test standard with a certified melting point of 150' C. (micro) placed in one of the commercial capillaries showed that the thermometer was not at fault. Portions of the sugars placed on a Parr-Dennis melting point bar, a Fisher-Johns melting point apparatus, and a Kofler micro hot stage melting point apparatus all melted at temperatures that were in agreement with the published values. The glass of the commercial melting point capillary then became suspect because in the above equipment the latter two apparatus make use of microscope cover glasses and slides which are not made of borosilicate glass and the melting point bar is, of course, all metal. With a return to established practice being desirable to confirm the suspicion, capillaries were hand-drawn from short, open-end sections of soft-glass tubing. These sections, 12-mm. 0.d. mith a I-mm. wall thickness, were first boiled in two changes of fresh distilled water and dried. Melting points obtained with the capillaries thus prepared checked the published values for the sugars. Borosilicate capillaries were also washed and dried (done with openend tubes before sealing and use) ; the melting point still agreed with those from the already-sealed tubes. When ~
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ANALYTICAL CHEMISTRY
sample-containing capillaries, one of soft glass and the other of borosilicate, were attached to the same thermometer in the oil bath, the difference in behavior of the sugar: nab very striking-the sample in the soft-glass capillary mas completely melted while the other was still solid. Similar effects were observed when either pure D-fructose or pure cellobiose was substituted for glucose. I n the latter case, melting was followed immediately by decomposition. This difference in observed melting points cannot be explained by tube dimensions. These were determined by using a micrometer eyepiece microscope. The outside diameter of both sets of capillaries averaged 1.5 to 2.0 mm.; wall thickness for the soft-glass tubes mas 0.05 to 0.09 mm., while that for the commercial ones was about 0.2 mm. The thicker wall of the commercial capillary did not influence the melting points of the glucoses because holding the bath temperature a t 148' and 150' C. did not produce sample liquefaction with these capillaries. Furthermo
elevated melting points were obtained with the soft-glass capillaries when powdered borosilicate glass (obtained by grinding a capillary in a mortar and pestle) was added to the sugar before loading the capillary. These visual observations were confirmed in a differential thermal analysis apparatus. I n Figure 1 are shown the curves for an alpha-n-glucose sample-the shift of temperatures caused by the glass is readily apparent here. There is mention in the literature that the glass of the capillary can have an effect on the melting point obtained. Morton (4) has referred to the reported effect of unwashed soft glass on lowering the melting points of glucose, fructose. mannitol, phenacetin, and other compounds, an effect which is apparently overlooked in other tests on laboratory methods and technique. Here the melting point is lowered because of alkali and products of devitrification remaining on the surface of the unwashed soft glass. However, correct melting points are obtained when the same glass is clean Dieckmann ( I ) , holyever, reported three different melting points for keto-acetyldibenzoylmethane when 100' 200. 3 00' capillaries of three different glasses, 44 Jena. ordinary, and Thuringer, were used. With Jena glass the melting point was some 20" C. higher than with 0 ordinary glass. Jones ( 2 ) found different melting points for pure rotenone when capillaries of Pyrex No. 774, ordi-4 nary soft glass, and Corning electrode glass KO. 015 were used. The melting t8 points were 163-4', 159.5-60.5', and 155-6' C., respectively. This 200° 300° finding was further extended by Jones +-I t 4 and Wood (3) with alpha-toxicarol, 0 beta-toxicarol, and deguelin. Higher > 0 melting points were obtained in borou o 2 silicate capillaries. Jones and Wood z also used six commercially available glasses, plus transparent silica, with -4 alpha-toxicarol. The melting points fell into two groups, 230-1' C. for borosilicate glasses and transparent silica, -8 and 200-7.5' C. for alkaline soft glasses In all of the abovc, alkali again waq the factor involved, but this time -12 coming from the glass itself in sufficient quantity to apparently cause enolization 1160' ~-I 6 of the compound. TEMPERATURE, ' C I n the present instance, both forms of n-glucose, the D-fructose, and the celFigure 1, Differential thermal analylobiose are affected similarly by borosis curves for ( A ) alpha-D-glucose and silicate glass. This could be explained (6) alpha-D-glucose and powdered by assuming that the melting points as borosilicate glass v)
obtained with the soft-glass capillaries might be low because of the effect of alkali-except t h a t t k e melting points of the glucoses as obtained with the softglass capillaries agree with those melting points obtained on the Dennis melting point bar where there are no glass surfaces. Furthermore, the mixture of powdered borosilicate glass and the sugar still gave higher melting points in t h e soft-glass capillary and the differential thermal anilysis apparatus. Perhaps boron from the borosilicate has interacted with the sugars (boron does complex with the hydroxyl group) or has catalyzed some molccular rearrangement. The cause oi the phenomena should be further inve,itigated.
Finally, we believe that as glass capillaries are used extensively there is some need for further testing borosilicate melting point capillaries for suitability, particularly in ccnnection with those compounds t h a t are capable of mutarotation, bear hydroxyl groups, or have aldehydic or ketonic groups capable of undergoing keto-enol transformations. ACKNOWLEDGMENT
‘The authors are indebted t o C. 11. Conrad for the differential thermal analyses made on the samples. LITERATURE CITED
( I ) Dieckmann, IT., Chem. Rei. 49, 2203, 2213 (1916).
( 2 ) Jones, H. A.; IND.ENG.CHEM.,ASAL. ED. 13, 819 (1941). (3) Jones. H. A.. Wood, J. W.. J . Am. Chem SOC.63, 1760 (1941).
(4) Morton, -4.A., “Laboratory Technique in Organic Chemistry,” pp. 22-23, McGraw-Hill, New York, 1938. LEONSEGAL DAVIDJ. STANONIS Plant Fibers Pioneering Research Laboratory Southern Utilization Research and Development Division U. S. Department of Agriculture Kew Orleans, La. Use of a company and/or product named b r t h e Department does not imply approval or recommendation of the product to the exclusion of others which may also be suitable.
Determinatiton of Total Cholesterol in Blood Serum with Perc hIoric Acid-P hos ph o ric Acid-Fe rric ChIoride Rea g e nt SIR: The current methods for the determination of tot a1 cholesterol in blood serum are based on either the Liebermann-Burchard reaction (4) or the reaction with ferric chloride-sulfuric acid reagent (6). H o m v e r , the former reaction requires manv strict conditions of performance to obtain a reproducible color developrient ; while in the latter reaction, small amounts of impurities in sulfuric acid frequently give higher chole-terol values ( I , 2, 5 ) . T h e method presenied here is very simple to use with a large number of samples, and gives :holesterol values analogous t o those determined b y Schoenheimer and ,Sperry’s method, which is generally accepted as a standard method for the determination. EXPERlMENTAl
Reagents. Color reagent. Dissolve 8.0 grams of l ~ e C l ~ . 6 H 2(rea0 gent grade) in a mixture of 200 ml. of perchloric acid (70%, reagent grade) and 600 ml. of phosphoric acid (85%, reagent grade). This reagent is very stable and can be stored at room temperature for a long time. Ferric chloride, 0.1 %, glacial acetic acid solution. Ferric chloride, 0.2’%, aqueous acetic acid solution. Dissolve 1.18 grams of FeC13.6H20 in a mixture of 590 ml. of glacial acetic acid and 20.0 ml. of water. Standard cholester 31 stock solution, 0.2 mg. per ml. Dissolve 50 mg. of pure cholesterol (3)in 250 ml. of glacial acetic acid. Working standard cholesterol solutions. Dilute 10.0, 20.0, 30.0, 40.0, 50.0, and 60.0 ml. of the standard stock qolution with glacial ztcetic acid to give a total volume of 61.0 ml. in each case. The working standard solutions thus obtained have equivalent concentrations
of total serum cholesterol of 100, 200, 300, 400, 500, and 600 mg. per 100 ml., respectively. Procedure. To 6.00 ml. of O.lY0 FeC13 solution in a centrifuge tube is added 0.100 ml. of serum. T h e mixture is stirred thoroughly with a stirring rod t o complete coagulation of proteins. The rod is removed and t h e tube is ceiitrifuged a t about 3000 r.p.m. for 10 minutes. T o 4.00 ml. of t h e supernatant solution in a test tube is added 2.00 ml. of color reagent. A blank tube with a mixture of 2.00 nil. of glacial acetic acid and 2.00 ml. of 0.2% FeCI3 aqueous acetic acid solution is also prepared. I n the case of calculating unknown cholesterol values from the absorbance of a single working standard solution, another tube with a mixture of 2.00 nil. of a single working standard solution, 2.00 ml. of 0.2% FeCL aqueous acetic acid solution, and 2.00 ml. of color reagent is also prepared. The contents of all tubes are
mixed well by shaking. Then the tube.; are heated in a boiling water bath for 20 minutes in such a way that the surfaces of the contents are kept under the surface of boiling water. The tubes are cooled in running water. When the contents of the tubes are thoroughly chilled, the absorbance of the sample (and of a single working standard solution) is read at 450 mp on a spectrophotometer with a glass cell of 10 mm. sample path length against the reagent blank. Calculation. T h e cholesterol values of t h e samples are calculated from t h e calibration curve which is described below or conveniently from t h e ratio of absorbance of unknown t o t h a t of a single working standard solution employed. Calibration Curve. Three aliquots of 2.00 ml. of each working standard cholesterol solution are pipuetted into test tubes, and 2.00 ml. of 0.2% FeClr aqueous acetic acid solution and 2.00
Table I. Influence of Composition of Color Developing Agents o n Coloration Concentration of FeC13 Ratio of HC1O4 Color of (%, w./v.) to &Po4 (v./v.) blank tube Amax (mp) E460 mlr 0 1:3 colorless 455 0.350 2: 1 yellow 0.600 1:1 faint yellow 0.560 0.5 1:2 colorless 450 0.500 1:3 colorless 454 0,450 4.53 0.400 1:4 rolorless ‘>. -.1 yellow 379 0.800 1: 1 yellow 0.520 1.0 1:2 Taint yellow 452 0.500 1:3 faint vellow 452 0.440 1:4 colorlkss 453 0.440 1:2 yellow 376 0.450 2.0 1:s faint yellow 0.420 1:s faint yellow 383 0.390 3.0 1:4 faint yellow 363 0.340
VOL 35, NO. 11, OCTOBER 1963
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