Microtechnique of Organic Qualitative Analysis Group Tests for Compounds of Carbon, Hydrogen, and Oxygen D. GARDNER FOULKE AND FR.4SK SCHNEIDER School of Chemistry, Rutgers Unibcrsity, New Brunswick, 3.J., and Department of Chemistry, Queens College, Flushing, N. 1.
BY
MEXKS of the classification reactions described in the preceding paper of this series (I??), an unknon-n organic compound can be identified as an aldehyde, carbohydrate, phenol, acid, ester, alcohol, ketone, ether, or hydrocarbon. B y means of the elementary analysis, it is also possible t o identify it as a nitrogen compound or one containing halogen or sulfur. It is often very advantageous to carry out certain other tests which further limit the field of search and which may give valuable clues t o the identity of the unknown substance. Such tests are included in Kamm’s classification reactions (18) and in Mulliken and Huntress’s sectional and numbered tests (16). T h e microtechnique for these tests is described below.
Fehling’s Test A microtechnique for this test has been described by Ernicli (2) and by Garner (10). Emich carries out the test in centrifuge cones, while Garner uses a microscope slide. In the latter method, the liquid is taken u p by a piece of filter paper, using only one edge. In this way the orange precipitate of copper oxide is collected on that edge and becomes easily visible, even when only a very small amount is present. The authors use a capillary tube for this test, with the idea that semiquantitative estimations could be made simultaneously with the qualitative tests. The capillary has a bore of 0.5 mm. About 2 mm. of the reagent and 10 mm. of the 1 per cent sugar solution are drawn in and one end of the capillary is sealed. After centrifuging to this end, the open end is sealed and the liquids are centrifuged back and forth to mix thoroughly. The capillary is then heated on the water bath (in a test tube immersed in the water bath). The orange precipitate soon appears and can be centrifuged out. If a known quantity of sugar is used, the amount of the precipitate can be compared with that obtained from a standard sugar solution.
Osazone Formation Mulliken and Huntress (15), Kamm ( I S ) , and others (19) subdivide the carbohydrates according to the time required for the formation and appearance of the osazones. llccording to Huntress, the conditions under which the reaction is carried out must be rigidly adhered to, and the errors in measurements must not exceed 1 per cent. The reagent is made by dissolving 1 gram of phenylhydrazine hydrochloride and 1.5 grams of sodium acetate in 7 mi. of v-ater, warming to dissolve. A solution of 1 part of sugar to 3 parts of water is made up. A capillary of a t least 1-mm. bore and 120 mm. long is used. Seven millimeters of reagent are icran-n into the middle of the capillary through one end and then 3 mm. of the sugar solution at the other. The reagent end of the capillary is sealed, and the 2 droplets are joined by centrifuging and then mixed by means of a glass thread. The open end of the capillary is sealed and the droplets are centrifuged to the reagent end again. Owing t o the heating of the sugar end during sealing, some cliarring takes place. The solutions must not come in contact n-itli this charred residue. The tube is placed in a beaker of boiling water, the exact time of immersion noted, and the tube observed for the formation of a precipitate. The time required for the a p pearance of a precipitate is noted. The authors’ experiments showed that the same time is required for the osazone formation when carried out as described above as when the macroprocedure is used. The precipitate can, of course, be transferred to a microscope slide for furt’her niicroscopic examination ( 2 1 , 16).
Garard and Shernian (9)prepared the osazones of small quantities of sugars, but used larger samples than the authors and worked with much greater volumes-10 ml. Fischer and Paulus (Y),in addition to preparing the osazones of 1-mg. samples, presented an analytical scheme for the identification of a sugar or a mixture of two. Wagenaar (20) used a solution of a-naphthol and glycerol as a test for fructose or a di- or trisaccharide containing fructose. Sone of the above investigators used the hlulliken and Huntress reagent or studied the time of osazone formation.
.Icetyl Chloride Test A 1-mni. bore capillary tube is used. Eight millimeters of acetyl chloride and 5 mni. of the alcohol are drawn in and both ends of the capillary are sealed. By centrifuging back and forth the liquids are mixed. The capillary is allowed to stand for 2 minutes, during which the liquid column may separate into two droplets. I n this case these should be centrifuged together. After 2 minutes, the empty end of the capillary is scratched with an ampoule file or piece of broken porcelain and carefully broken off. The same is done with the other end. When the first end is broken off, a slight “pop” can be heard, which indicates that a reaction has taken place. Twenty millimeters of water are added to the capillary, one end is sealed again, and the droplets are centrifuged together. I n the case of alcohols which form slightly soluble or insoluble esters, two layers will form. The liquids are blown out on a slide and the characteristic odor of the ester can be detected. Positive results were obt,ained with nine alcohols tested, but glycerol reacted very slor\\-ly. Morton and Peakes ( f $ ) carried out a reaction with acetyl chloride in capillaries as a step in the detection of triaryl carbinols.
Zinc Chloride-Hydrochloric Acid Test Two to 3 mm. of the alcohol to be tested are drawn into one end of a capillary and six times as much zinc chloride-hydrochloric acid reagent is drawn up into the other end. Both ends are sealed and the liquids are centrifuged twice back and forth. The contents are examined for turbidity. After 1 hour the capillary is examined again. I n case a turbidity appears a t either time, the capillary is centrifuged and examined for the presence of two layers. Sormal alcohols will give no turbidity even after 1 hour’s standing. Secondary alcohols Kill become turbid after a time and two layers can be centrifuged out. Tertiary alcohols give an immediate turbidity which may separate out into two layers even without centrifuging.
Bromine Addition Test for Phenols This test has been described by Behrens (1) and Emich (6). It is carried out on a slide and should be observed against first a black background and then a white background. The bromine can be added either as a droplet of bromine water or by inverting the slide over the mouth of a bottk of bromine water.
Phthalein Fusion Test Vsing the same technique as in filling a melting point tube, a crystal of the phenol (approximately 5 micrograms) and an equal bulk of phthalic anhydride are introduced into a capillary 50 to 60 mm. long and of 1 to 1.5-mm. bore, sealed at one end. A droplet of concentrated sulfuric acid is added by means of a capillary pipet. The acid is centrifuged to the solid. The tube is then heated in a sulfuric acid (melting point) bath a t 160’ C. for 3 minutes, After cooling, 2 drops of water are introduced by means of a capillary pipet, centrifuged to the sealed end, and mixed by means of a glass thread. The sealed end is cut off and the liquid bloivn out on a slide. Sodium hydroxide solution ( 5 per cent) is added to alkaline reaction. The bright and characteristic colors of the phthaleins are obtained. Should sodium sulfate crystallize out when the solution is made alkaline, a drop or two of water must be added t o dissolve it.
SEPTEMBER 15, 1940
ANALYTIC4L EDITION
Sodium Bisulfite Test Garner ( I O ) mixes an ether Eolution of the substance with an aqueous solution of sodium metabisulfite in a “settling tube”. The authors carry out this test on a microscope slide. The reagent is prepared according to the directions of Mulliken and Huntress (16). A small drop of the liquid aldehyde or of the concentrated ether solution of the solid is placed on the slide beside a droplet of the reagent. The droplets are joined and mixed with glass thread. In case no precipitate appears on mixing, the finger tip is touched to the underside of t,he slide directly beneath the merged drops to test for the evolution of heat. If much heat is evolved, the slide is laid on a cold surface such as a cold metal block and when cold is examined for a precipitate.
Tollen’s Silver IMirror Test This test has been described by Emich ( 5 ) and can be carried out on a microscope slide or in a capillary. The technique is identical with that for the Fehling’s test. The reagent is prepared according to Mulliken and Huntress (15). This test exemplifies once more the advantages of the microtechnique. On the ordinary macro scale care must be taken to dispose of the test liquids immediately because of their dangerously explosive properties. The minute quantities used in the microtest greatly minimize the danger.
Bromine Addition Test for Hydrocarbons In testing for unsaturation in hydrocarbons according to Kamm, the addition of bromine is of value. Small drops of a carbon tetrachloride solution of bromine are placed in two adjacent depressions of a white spot plate. A drop of the substance (from a capillary pipet) is added to one depression and a drop of equal size of carbon tet)rachloride to the second. The control is necessary when the addition of bromine is slow; this, however, occurs only very rarely. The test can also be carried out in a capillary. The substance is introduced first, and then tiny droplets of the bromine solution are added frnm a capillary pipet until the color remains. In this way a rough idea of the amount of bromine required can be obtained.
Iodoform Test Emich (4)carries out this test in the centrifuge cone, the volatility of the iodoform making the use of the slide inadvisable. The contents of the cone are examined under the mirroscope for t.he yellow hexagons of the compound.
Aluminum Chloride Test A Pyrex capillary tube, about I-mm. bore, is dipped into anhydrous aluminum chloride, so that some of the salt is pushed into the tube, and the excess salt on the outside of the capillary is wiped off. The end which contains the aluminum chloride is heated cautiously, starting at the very tip, so that the salt sublimes into the capillary. When all has sublimed away from the end, it is heated to the softening point and pinched shut with a forceps. Then the tube is held high over the burner, so as to sublime the aluminum chloride farther into the tube but slo~vly,so that it deposits again in a thin layer. After cooling the sealed end is cut off and the other end is dipped into a solution of the hydrocarbon in chloroform (0.5 gram of hydrocarbon in 2.5 ml. of Chloroform). The drop is allowed to slide down the tube to the aluminum rhloride, and the color is observed immediate!y against a whit,? background and again 20 minutes later.
Specific Grayit? Mulliken and Huntress (16) subdivide the hydrocarbons according to values of the boiling point, melting point, and density; solubility in a number of solvents; and amount of bromine added (bromide-bromate number). The values of the boiling and melting points can be obt’ained by one of the methods listed in the first paper of this series (8). For the purposes of this classification, it is not necessary t’o know the exact value of the density b u t only whet’lier i t lies in one range or another. Therefore, if the sample is compared to a few standards whose densities lie a t the boundary of these ranges, the range in which the density of the unknown is t o be found can readily be determined. This comparison is best and most rapidly carried out by the schlieren method ( 3 ) . The unknown is used as R fluid sample and the standards are used
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as static samples. The standards should be substances in which the unknown is soluble, and which d o not have too low a boiling point and are not too expensive. Since we are concerned here with hydrocarbons, substances like these in character are used as standards t o ensure mutual solubility. The following table lists B number of substances which can be employed: Specific Gravity a t 20° C.
Substanre Octane Diethyl ether n-Decane Methyl isobutyl ketone n-Decul alcohol Acrolein Amyl iaopropionate
0.706 0.708 0.730
0.801 0.830 0.841
0.870
Boiling Point
* c.
125 35.5 173 118 23 1 52.5
160
The values of the specific gravity ranges are those called for in the scheme of Rlulliken and Huntress (16).
Solubility The solubility of the hydrocarbon sample in the solvents used in the classification of Mulliken and Huntress can be determined by one of the methods described in the second paper of this series (17).
Oxidation of Side Chains The permanganate oxidation of the side chain can be used for confirmatory identification. Since a n excess of the permanganate reagent will oxidize and destroy the benzoic or other acid formed, it is important not to use much more than the theoretical quantity of the reagent.
A tube shaped like Figure 1 (left) is used. The bulb is about 10 mm. in diameter, the wide portion of the tube 6 mm. in bore and 5 to 6 cm. in length. For toluene 0.3 ml. of a saturated potassium permanganate solution and 10 cm. of the hydrocarbon are introduced into the tube, which is then sealed a t A . For other hydrocarbons a p r o p o r t i o n a t e amount of the permanganate is used. The tube is heated in a steam bath for 2 to 8 hours with frequent shaking. After the color of the permanganate has disappeared, the tube is cooled and cut a t A , the sealed tip is broken off, and the other portion of the tube is cut off a t B. The funnel-shaDed uortion of the tube is inserted int,o the o h e r part as shown in Figure 1 (right) and centrifuged. The funnel is washed once into the tube. The liquid is transferred to a centrifuge FIGURE 1 cone by inverting the tube into the cone and centrifuging in this position. A flip with the hand usually suffices. After centrifugng, the supernatant liquid is transferred by siphoning into a second centrifuge cone, where it is concentrated on the water bath, using an air stream, t o about 0.3 ml. Should a precipitate appear a t this point, it’ is filtered off. The clear liquid is acidified with hydrochloric acid. The precipitate is separated by centrifuging, washed with water, and finally recrystal!ized from alcohol. A melting point determination is then carried out in the usual manner.
Saponification Equivalent I n determining the saponification equivalent of esters, the diethylene glycol reagent used in the qualitative test described in the previous paper of this series (18) was employed. The procedure is as follows: About 0.15 to 0.2 gram of the glycol-potassium hydroxide reagent is weighed out on a microbalance in a small glassstoppered weighing bottle 10 mm. in diameter and 20 mm. in
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INDUSTRIAL AND ENGINEERING CHEMISTRY
height. Then about 6 t o 10 m of the ester are introduced and the tottle is weighed again. The bottle is stoppered and heated in a brass or copper block (Figure 2) to 60” to 70” C. The stopper is then opened momentarily, closed tightly again, and heated to 13O0,a t which tem erature it is held for 2 minutes. It is agowed to cool, the stopper being occasionally removed momentarily to prevent later dificulty in its removal. The stopper is washed off into the bottle. The contents of the latter are then washed out into a 10-ml. Erlenmeyer flask. Care should be taken to keep the volume of the liquid small, not exceeding 2 ml. It is then titrated with 0.02 N acid, using phenolphthalein as indicator. A blank is run on the reagent t o determine the amount of potassium hydroxide per gram of the reagent. Attempts to measure the reagent volumetrically gave poor results because of the difficultyof getting the same volume of such a viscous liquid from a capillary pipet each time the experiment was tried. Care must be taken throughout the determination t o r t loss of easily volatile esters. hen introducing the sample into FIGURE 2 the weighing bottle, a capillary pipet should be used and the Sam le delivered from the pipet directly overe!t diethylene glycol reagent, BO that solution in the reagent takes place as quickly as possible.
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Literature Cited (1) Behrens, 2. anal. Chem., 42,141 (1903). (2) Emich, “Lehrbuch der Mikrochemie”, p. 229, Munich, J.
F.
Bergmann, 1926. (3) Emich-Schneider, “Microchemical Laboratory Manual”, pp. 40 et seq., New York, John Wiley & Sons, 1932. (4) Ibid., p. 119. (5) Ibid., p. 120. (6)Ibid., p. 131. (7) Fischer and Paulus, Arch. Pharm., 273,83 (1935). (8) Foulke and Schneider, IND.ENG.CHEM.,Anal. Ed., 10, 104 (1938). (9) Garard and Sherman, J. Am. Chem. Sac., 40,955 (1918). (10) Garner, I n d . Chemist, 5, 58 (1928). (11) Hawk, “Practical Physiological Chemistry”, p. 51, Philadelphia, P. Blakiston’s Son & Co., 1931. (12) Kamm, “Qualitative Organic Analysis”, New York, John Wiley & Sons, 1922. (13) m a . , p. 157. (14) Morton and Peakes, IND. ENG.CHEM.,Anal. Ed., 5, 185 (1933). (16) Mulliken and Huntress, “Method for Identification of Pure Organic Compounds”, 2nd ed., New York, John Wiley & Sone (in preparation). (16) Plimmer, “Organic and Biochemistry”, pp. 256, 267, 280, New York, Longmans, Green and Co., 1933. (17) Schneider and Foulke, IND.ENG.CHEM.,Anal. Ed., 10, 445 (1938). (18) Ibid., 11, 111 (1939). (19) Shriner and Fuson, “Systematic Identification of Organio Compounds”, p. 37, New York, John Wiley & Sons, 1935. (20) Wagenaar, Pharm. Weelcblad.70, 1029 (1933).
Microscopic Identification of Certain Sugars
and Polyhydric Alcohols J
J
JOHN A. QUENSE AND WILLIAM M. DEHN University of Washington, Seattle, Wash.
I
N A RECENT contribution, photomicrographs depicted
the crystal habits of eighteen sugars precipitated from saturated aqueous solutions by acetone, alcohol, acetonitrile, or 1,Cdioxane (IS). These crystals are easily obtained and can now be compared with the photomicrographs for purposes of identification. This paper extends the use of the method t o three rare sugars, three polyhydric alcohols, and a series of twelve binary mixtures. Table I gives optical properties of the sugars and alcohols for purposes of confirmation. I n crystallizing the eighteen single sugars, saturated aqueous solutions were diluted with the miscible solvents t o cause
On dilution with the miscible solvents, one or more of the components may fail to crystallize because of the degree of supersaturation (4, 21), interference of other components and foreign substances (6, 25, S I ) , formation of colloids (B, IS), or the separation of a sirup (23). When mixtures crystallize, deformation of t h e normal habit of the individual crystals may occur because of the presence of foreign substances in solution (7, I S , 14, 27, SI), temperature changes (%), varying degrees of supersaturation ( I I ) , and the previous history of the crystallizing substance (9,29).
TABLE I. OPTICALPROPERTIES Compound Crystal System Elongation Extinction Interference Colors Gentiobiose No data Parallel t o approximately 7 O Low 1st order d-L xose Monoclinic (17, $4, 30) Parallel t o slightly inclined ( f 7 , 30) 1st and 2nd order (17, 30) Parallel (17) 1st order gray (17) Trexalose Orthorhombic (10) Dulcitol Monoclinic (8,80) Parallel t o approximately 40‘ 1st and 2nd order Mannitol a Orthorhombic (Z0,$6) a Parallel a t o slightly inclined Low 1st order Sorbitol a Sorbitol ie described aB needles (fd),hunches, or knobs of intergrown needles (3). The discoverer of sorbitol states that the form of the cSystals would he difficult to determine (0. The habit is similar t o that of mannitol crystallized under some conditions.
2
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crystallization (IS). I n the aqueous solutions of the binary mixtures investigated, the components were present approximately in the ratios of their individual solubilities in water. Because of their mutual solubility effects (IS, 14, 16, 22) the solutions may have been saturated with respect to one of the components b u t not necessarily to the other.
T h a t changes of habit have not obscured t h e identity of the crystals from the following sugar and polyhydric alcohol mixtures, is illustrated i n the photomicrographs. Identification of the individual crystals in the mixtures can be made by inspecting the photomicrographs of the previous contribution (2%
