The Microtechnic of Organic Qualitative Analysis D. GARDNER FOULKE AND FRANK SCHNEIDER School of Chemistry, Rutgers University, New Brunswick, N. J., and Department of Chemistry, Trinity College, Hartford, Conn.
T
HE field of analysis which has received perhaps the least. attention from microchemists, and which at the same time offers the greatest opportunity for a demonstration of
- - shape can sometimes be detected by the difference in behavior
to polarized light. If the substance is nonhomogeneous, it must be purified before the physical constants can be determined. In the case of solids, recrystallization or sublimation is resorted to, and in the case of liquids fractional distillation usually suffices
the economy of time, energy, and materials which micromethods can effect, is qualitative organic analysis. N o s t of the reactions and operations in organic analysis require much more time than the corresponding inorganic (6, 26). procedures. This is due not only to the greater speed of ionic IGNITION TEST. It has been found in a series of experiments reactions as compared to the reactions of organic substances that the ignition test gives as much information when carried (often not even in the same phase) but also to the greater out on a microscale as when larger quantities of the substance are used. The test is carried out by placing a crystal (about 0.01 complexity of the materials dealt with in organic work. mg.) on a platinum microspatula (made by flattening one end of .Any procedures, therefore, which cut down the time required a No. 20 platinum wire for a distance of about 5 mm. and fixing for the operations used in qualitative organic analysis should the wire in a glass rod or needle holder) and holding it in the be welcomed. It is the intention of the authors in this series colorless flame of a microburner (Figure 2). It is desirable to use one of the adjustable types of microburners, since they give a of papers to indicate how micromethods can be utilized to much finer and smaller flame. this end. The sample should not be Figure 1 will indicate the R held directly in the flame, s a v i n g w h i c h can be but the wire should be healed about I cm. from tlieflattened effected with m i c r o p r o end and then moved so that c e d u r e s , The apparatus the sample is g r a d u a l l y shown is the simplest disbrought into the flame. This tillation setup, for both the will prevent loss of the sample by spattering. macro and the micro operation. Actually the separaThis procedure will give tion will be more complete t h e f o l l o w i n g informawith the microtube of Emich tion: (shown in the lower center 1. An approximate idea of the drawing) because an of the m e l t i n g point or a c t u a l fractionation i s sublimation point, if the carried out. FIGURE1. MACRO AND MICRO DISTILL.4TION SETUP substance melts or subI t is not the authors’ inlimes, can be obtained. tention to develop a new 2. When the substance begins to burn, the nature of the scheme of analysis but to adapt the microtechnic to one of the flame should be observed. If i t is smoky, an aromatic comexisting systems. I n their opinion a system based upon a pound is indicated. The luminosity of the flame is also somegeneral classification of organic compounds according to a times of aid in classifying a compound. If the substance exdefinite physical property such as the scheme of Kamm ( d l ) , plodes (sputters) this fact should be noted, together with the Staudinger (SI),or Shriner and Fuson (,So) is to be preferred to odor of any vapors. one which classifies compounds only according to their con3. If any residue is left after ignition, this may be identified. stituent elements. The procedure, therefore, is based on the Since this is in most cases an alkali or alkaline earth carbonate, system of Kamm except where micromethods make i t possible the residue should be tested for alkalinity by dissolving in a drop to omit a step or two. of water on a microscope slide and touching the drop with The micromethods developed thus far in organic qualitative a fiber of litmus silk (11). Identification of the metal can analysis have been confined almost exclusively to specific be carried out either by a flame test or, in case it is not an tests for some compounds and, although they include some of alkali or alkaline earth metal, by a systematic qualitative the oldest tests known to microchemistry, such as Wormley’s analysis (d). The presence of a metal indicates, of course, test for poisons (SS), most of them are for the less common a salt or organo-metallic compound. compounds such as alkaloids. The steps in the scheme of analysis adopted are as follows: preliminary physical examination and purification, determination of physical constants, qualitative tests for the elements, determination of solubility behavior, homologous tests, consultation of literature, and preparation of derivatives. I_=
Preliminary Examination The unknown substance should be examined for homogeneity, color, odor, and crystalline structure. A microscope or lens will be of great assistance in this examinstion. If a polarizing microscope is available, impurities which may be very much like the rest of the material in color and
U
FIGURE 2. IGNITION TEST 104
FEBRUAHk- 15, 1938
2SU.4LkTIC.4L EDITION
103
Determination of Physical Constants The final identification of a substance, whether the original compound or one of its derivatives. m a y be made by the deterinination of its physical constants or by a study of its crystalline form under the microscope. T h e physical constants usually employed include boiling point, melting point, density, refractive index, optical rotation, aiid niolecular weight. Of these the first three are important, the others being used only for confirination. MELTISG POIXT. The melting point may be determined in the usual way, since only a few milligrams or less of the substance are required. For smaller quantities the Iiofler-Hilbck electric - ___ hot stage (22) can be used. Only a single tiny crystal is required. (Inasmuch as observation unFIGT-RE 3. der the microscope enables one to determine more accurately when the substance is beginning to melt, the melting point as determined by the use of such a stage n-ill be a little under the value given in the literature.) Blocks of aluminum or copper have been recommended for use in place of the ordinary heating bath (5,23, 2 7 ) . BOILINGPOINT. The method of Emich ( 7 ) gives highly accurate results in the determination of the boiling point. Hays, Hart, and Gustavson (20) describe an apparatus for determining the boiling point at altitudes other than sea level. DESSITY. If sufficient amounts of material are available (0.5 t o 3 mg.) and a microbalance of either the Nernst or the Kuhlmann type is used, a micropycnometer will serve for the determination of the density of liquids (26). In the absence of a microbalance, the suspension method of Emich (14) can be employed. (See also 4. Several micromethods for the determination of density of solids and liquids and also a review of the literature on this subject are given by Willard and Blank, 32.) REFRACTIVE INDEX. The refractive index of liquids can be determined in the usual way with an Abbe or dipping refractometer. With very small amounts of liquids or solids, the ininiersion method ( I O ) can be used. A list of suitable liquid and solid standards can he obtained from any book on microscopy or petrography. In some cases the schlieren method of Emich ( 8 ) can be employed t o advantage. SPECIFICROTATION.The specific rotation can be measured either by the method of Donau ( 6 ) or Fischer (18). MOLECULAR WEIGHT. A number of methods are available for the determination of the molecular weight on a micro.ccalc. The Rast, method (16, 24) is very simple and can be used for all solids and many liquids. Only a fraction of a milligram of sample is used. The osmotic method of Barger can also he used (1.5). A method of determining the molecular weight by a vapor density measurement has been developed by one of the authors (28). OTHERPHYSICAL COKSTANTS. Other constants which can be wed for the identification of a crystalline organic substance are the interfacial and silhouette angles (29).
Elementary Anal? sis ;Iltliough microchemical tests for both hydrogen and carbon have been described ( l e ) ,it is assumed that only organic compounds are being analyzed and tests for these elements can he omitted. Emich ( I S ) describes two methods for the detection of nitrogen in organic compounds-the sodium fusion and the ammonia test. While the sodium fusion when carried out on a microscale greatly facilitates the destruction of the excess sodium and eliminates the danger of a n explosion, it is rather difficult t o get the tiny pieces of sodium into the capillary. Furthermore, the possibility of nitrogen escaping a s the free gas (19) or, in the case of highly volatile compounds, without decomposition of the sample ( 1 7 ) exists in the microtest a s well a s the macro. T h e ammonia test, of course, cannot be applied to all nitrogen compounds. The authors have turned to the magnesium-potassium carbonate fusion method as modlfied by Barkenbus and Baker ( 1 ) . and have developed the following microtechnic :
+
.
-
, I -
I
>
DETECTOR OF SITROGES, SULFL-R,~
S DHAJJXESS 15
ORGANIC
COMPOUNDS
A capillary tube of Pyres glass of about I-mni. bore is bent as shoxn in Figure 3,9. The magnesium powder-potassium carbonate mixture (1 part of magnesium powder ground in an agate mortar vith 2 parts of anhydrous potassium carbonate) is introduced at one end and packed in with a glass thread, so that the full cross section of the tuhe is filled. The layer of carbonate mixture should be about 5 mm. long, H. The sample (0.1 to 1 mg.) is introduced into the other end, u-hich is then dipped into a vial of ether so that a droplet of ether about 2mm. long rises in the tube by capillary action, C. In the case of liquids, the ether must be int'roduced before the sample. The tube is then sealed at the sample end by heating in a microflame and pinching the softened glass with a forceps. The ether evaporates during the sealing, the vapor displacing the air in the tube. The portion of the tube containing the carbonate-magnesium mixture is heated in the microflame t o gloning, after which the tube is held in a Bunsen flame so that both the magnesium mixture and the sample are heated a t the same time. In this way the *ample is distilled over the glowing mixture. While the tube is still hot and soft, it is pulled out into a straight' tube. After cooling, the tube is cut at the points shown in E. The, piece containing the fused mass is placed in a depression of a spot plate which has been previously moistened, and i3 crushed, preferably with a porcelain spoon or the small end of a porcelain pestle. The moisture film on the plate will prevent the pieces from flying off the plate. -4drop of hot water is then placed on the residue and, after being allon-ed to stand for a fen- minutes, it ir dran-n off into a fine capillary pipet, the point of which is too fine to permit the solid to he drawn up FTith the liquid. In case any solid particle is drawn up with the liquid, the pipet is sealed at the other end, the liquid and solid are centrifuged to this end, the capil1ar.i- is cut just above the surface of the liquid, and tlic clear centrifugate is drawn off in another capillary pipet. This clear liquid is used for the tests for nitrogen, sulfur, and the lialogens. KITROGES. .4 drop of ferrous sulfate solution is added to the test drop on a slide. After being stirred and allowed to stand for a minute, the clear liquid is drawn off into a capillary pipet, and blown out on a white spot plate and a drop of concentrated hydrochloric acid is added. The Prussian blue color indicates nitrogen. Professor Barkenbus has kindly made the follon-ing suggestion regarding the test, for nitrogen in the presence of sulfur. He and Mr. Baker have found, and the authors have confirmed this with the microtest, that when both sulfur andnitrogen are present the test for nitrogen by means of the ferrocyanide reaction becomes doubtful. If, however, the filtrate from the fusion mixture is first tested for the presence of sulfur and then, if this test is positive, for nitrogen by nieans of the ferric t>hiocyanate test, a red coloration will appear if nitrogen is also present. The thiocyanate test is carried out by adding a drop of ferric chloride solution to the filtrate from t'he fusion mixture. HALOGES. The test drop is acidified with nitric acid by inverting the slide on which the drop rests over a bottle of concentrated nitric acid. Silver nitrate is then added either in the form of a tiny crystal or as a drop of the solution. Care must be taken not t o touch the portion of the combustion tube containing the fusion mixture with the fingers, since sufficient chloride is left on the glass from the contact of the fingers to give a positive test for this element. This portion of the tube should be handled only with the forceps in any case. In case a positive test for the halogens has been obtained, it is advipable to make further
INDUSTRIAL h\D
106
EKGIYEEHING CHEMISTRY
tests to obtain an indication of the type of halogen compound under consideration. Details of this procedure Mill be given in a subsequent report. SULFUR.The drop of test solution is acidified \vith acetic acid and a drop of lead acetate is added. The precipitate is ohserved against a white background. -4black precipitate of lead sulfide indicates sulfur. The conipounds used in testing this procedure include: Xitrogen: urea, henzaniide, carbazole, dinitrostilbene disodium disulfonate, dimethylaniline, benzonitrile,nitrobenzene, nicotine, phenylhydrazine, pyridine, 1-naphthylamine, p-toluidine, picric acid, aminoazobenzene hydrochloride, and asparagine. Sulfur: thiourea, benzenesulfonyl chloride, dinitrobtilhene disodium disulfonate, and carbon disulfide. Chlorine: Dichlorobenzene.
c
E
c c
(
(
1
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FIGURE 4. CAPILL.4RY DETERNIXATIOS O F SOLUBILlll
It is reconmended that the potassium carbonate-magnesium mixture be made up fresh frequently, since it becomes moist and cannot be handled as readily as when dry. I n the case of explosive substances such as picric acid, the sample should be heated yery carefully; otherwise it might explode and blow out the contents of the tube. These explosive substances can be detected \?-hen carrying out the preliminary ignition test,
Determination of Solubility Behavior The procedures of Kamm and others mentioned above are based on the division of organic compounds into several large groups according t o their solubility in a variety of solvents. The particular solvents employed differ t o some extent in the various schemes, but the actual technic remains the same. Since there is no sharp dividing line between “soluble” and “insoluble,” a n arbitrary ratio of solute to solvent must be set. Again this varies according to the solvent employed and the physical state of the solute, but a n average value is 1 part of solute to 25 parts of solvent. If this ratio is exceeded the compound is designated as “insoluble”; if it is less than this value it is designated as “soluble.” Borderline cases are placed in two classes and tested for in each. The ratio of solute to solvent may be determined either by adding increments of the solvent t o a fixed quantity of solute, or vice versa, whichever is more convenient. Work on solubility tests has not as yet been extended t o a large variety of compounds, but the general technic has been worked out. Two technics have been developed for the determination of solubility behavior on a micro scale. The first uses the capillary, the second the schlieren cell. Both methods may be used with slight modifications for either solids or liquids.
VOL. 10, AO. 7
CAPILL~R METHOD. Y In order to determine the solubility behavior of a liquid, a capillary tube of about 0.5-mm. bore and 70 mni. long is dipped into the sample until a droplet about 2 mm. long (0.1 cu. mm.) is drawn up (Figure 4 , A ) . The capillary is then dipped into the solvent until a droplet about 10 mm. long is drawn in, B. The end of the capillary is sealed and the solvent and solute are centrifuged to the closed end. The solute and solvent may now be mixed in one of t r o ways. In the first, a glass thread with the end fused to a droplet, D, is inserted into the capillary and the two liquids are stirred by drawing the thread in and out of the tube and at the same time twirling it between the fingers, thereby giving it a rotary as well as a translatory motion. The second method of mixing consists of sealing the open end of the capillary and centrifugingthe liquids back and forth from one end of the capillary to the other. In this way the liquid of greater density is always t,hrown t o the end of the capillary and on being centrifuged to the other end must pass through the lighter liquid. Complete and thorough mixing is thus obtained. By either method of mixing, the degree of solubility is determined by examining the resulting droplet. If it is perfectly homogeneous and clear, the liquids are completely miscible. If the droplet appears turbid or two separate phases appear, E, the sample has not dissolved. In the latter case, the capillary is cut at the empty end, anot’her 10 or 20 mm. of solvent are added by means of a capillary pipet, and the process of mixing is repeated. If the droplet appears turbid or two phases persist after the addition of 50 mm. of the solvent, the sample is designated as insoluble in that, solvent. The quantity of solvent taken at one time can, of course, be increased or decreased as desired. In the case of solids, the sample may be introduced into the capillary as in the filling of a melting point tube. The weight of the sample can be determined by weighing on a glass or quartzfiber balance (Salvioni, 9),torsion spring balance, or t’he like. About 0.1 mg. should be taken. The solvent is added as with the liquids. Should the solid remain at one end of the capillary after centrifuging instead of passing from one end to another with the liquid, the sample must be stirred with the glass thread as described above. The comparative specific gravity of the solute and solvent can readily be noted in the case of liquids by observing which is nearest the sealed end of the capillary after centrifuging. The most dense will, of course, be farthest from the center of rotat’ionof the centrifuge. method is based on the fact that SCHLIEREN ~ I E T H O DThis . when even a very small quantity of another substance dissolves in a liquid, the refractive index of that liquid is changed. This change in refractive index can be detected very readily by the schlieren phenomenon as described by Emich (8). In determining the solubility of liquids by this method, the solvent is placed in a schlieren cell. (A very good and inexpensive cell is now on the market, obtainable from Rlicrochemical Service, Douglaston, N. Y.) This cell has a capacity of about 0.02 cc. The cell is half filled with the solvent and the solute is added, as in other schlieren experiments, from a capillary pipet. With this procedure as with the capillary method, the relative specific gravities of the solute and solvent may be compared, since the stream of solute flowing into the solvent will descend to the bottom of the cell if denser than the solvent or rise to the surface of the solvent if less den The three degrees of ubility are illust,rated by the action of ethyl acetate (soluble), 2-methyl butanol (intermediately soluble), and bromobenzene (insoluble) in water. In the first case (ethyl acetate), the solute flowed out freely and immediately dissolved with the formation of pronounced schlieren. In the second case (2-methyl butanol), drops formed as the solute left the pipet. These rose to the surface and a few schlieren formed. After about three drops had left the pipet, no further schlieren were observed. In the last case (bromobenzene), no schlieren formed at all and the drops of the liquid settled to the bott’om. In the case of solids, the solute is introduced into the schlieren cell by means of a tiny cup at the end of a glass rod (Figure 5). A capillary tube is allowed t o collapse a t one point in the microflame, and is pushed together a t the same time so that a little ball of glass is formed. This is then drawn out while still soft to form a rod about 5 mm. long and turned to form a U. The shorter tube is cut a t the point indicated, C, to form the cup, D, which holds about 0.1 to 0.5 mg. of sample. This cup is introduced into the schlieren cell as shown in E. The size of the cup must be chosen so that the sample taken fills the cup completely. If, despite this precaution, a small bubble of air remains on top of the cup when it is introduced into the cell, it will be necessary to remove the bubble with a glass thread before the schlieren observation can be made. The usual schlieren are observed if
(4)Detre, D e u f . m e , / . I t ~ u c h c h r . ,49, Y86 (19231. (5) Doiiau, ~11orcatsh..29, 333 (19081. (6) Emich-Schneider, "Jlicrorheriiical Laboratory Jfanual," p . 39 e t seci., S e n . I-ork. John Wile>- tY3 Son.-, 1933. ((7)Itlid., p. 32. (8) Ibirl., g . 40. (9) Ibid., 11. 61. (10) Ibid., r J . 80. i l l ) Ibid., g . 81. I 12) Ibid.,p. 112. (13) Ibid., p. 116. -: (13) Ibid., p. 127. (15) Ibid., p. 136. (16) Ibid., p. 135. (17) Feist, Be,,., 35, 1559 (19021. (18) Fischer. Ber., 44, 129 ( 1 9 1 1 ' . (19) Graehe. f h i d . , 17, 1178 (1884;. 120) Hays, H a r t , a n d Gustai-son, ISD. ESG. CHEAI.,-'.nd. 3 E L . 8. 286 (1936). FI~;LTRE 5 . S C H L I E RD E ~E T E E ~ I I ~ . OF ~ TSOLI-RILITT I~S OF S ~ L I W (21) I < m ~ m ."Qualitatire Organic Analysis," Sen. T o r k , John W l e y & Sons, 1922. (22) Kofler and Hilbck, Mikrocizernie, 9, 38 (1931~. the substance is soluble, either ascending or dewending; nothing ( 2 ' 3 ) Mason, Cizemistru & ~ n d f t s t r y44, , 677 (1925). ( 2 4 Pregl-Fyleman, "Quantitative Organic 3Iicroaniilysis," 2nd can be observed if the substance is insohthle. ed., p. 217, London, J. a n d A . Chiirchill, 1930.
_ > I
The schlieren permit a very sen6itiYe and rapid tleteriiiiriation of the solubility of a suhstance. The authors are working a t present with a large nuiiiber of compounds, 1)oth as solutes and solvents. The resnlts of this rrork nil1 be reported in a subsequent paper. Literature Cited (1) Barkenbus a n d Baker, IXD. ESQ. C H m f . , Anal. E d . , 9, 136 (1937). (2) Benedetti-Piohler a n d Spikes, "Introduction t o t h e Microtechnique of Inorganic Qualitatire .Inalysis," Douglaston, N. T., Microchemical Service, 1935. ( 3 ) Dennis and Shelton, J . Ani. Chern. SOC.,52, 3128 (1930).
(26) Ibid., D. 222. ( 2 7 ) Rassoa, 2. a n o i y . Cherrr., 114, 117 (1920). (28) Schneider a n d Saschek. Rochester Meetlne, Seitteriiber, 1937. (29) Shead, ISD.ESG. CHESI.,Anal. E d . , 9, 496 (1937). (30) Shriner and Fuson, "Systematic Identification of Organic Coninonnds." New T o r k . J o h n TYilev & Sons. 1935. (31) Stiudinger, " I n t r o d u c t i o n ' t o Qualitati\-e Organic .In:tlysis," Berlin, Julius Springer, 1923. 132) Willard a n d Blank, J . Chern. Education, 10, 109 (1933). (33) Worrnley, "Microcheiriistry of Poisons," Philadelphia, J. B. Lippincott, 1867. RECEIVED September 2 5 , 1937. Presented before the Microchemical Section at the 94th Meeting of the American Chemical Society, Rochester, N. P.,
September 6 to 10. 193i.
Qualitative Separations on a Micro Scale Analysis of the Alkali Group A . A. BENEDETTI-PICHLER i N D JAMES T. BRYANT Chemical Laboratories of W'aqhington Square College, Xew York University, New York. N. Y.
A
S I X FORhIER investigations of this series, an attempt was made to adhere in the transposition to the milligram scale to the procedure of Noyes and Bray ( 7 ) RS closely a. advisable. A brief survey of the outline of the authors' final scheme which is presented herewith will nevertheless reveal considerable deviations. The principal change consists in the substitution of chloroplatinic acid for perchloric acid as a reagent for the separation of potasqium, rubidium, ani1 ceiium from sodium and lithium. The decision on chloroplatinic acid is permissible because of the small quantities of reagents required on the milligram scale. The use of this reagent is preferable from the technical viewpoint, since the chloroplatinatea obtained call be easily converted into chlorides by siniple ignition. Additional changes in the analysis of the potassium subgroup consist essentially of a different arrangement of the order in which the reagents used by Soyes and Bray are applied. The mixture of the chlorides is first treated with iodobismuthous acid which enables an immediate detection of the cesium if present in quantities larger than 5 pg. The immediate isolation of cesium simplifies the remainder of the procedure for the analysis of the potassium subgroup, which now may limit itself essentially to the separation of
potassium aiid rubidium. The triple chlorides with gold and silver, introduced by Elriich as slide tests, are used as confirmatory tebts for rubidium and cesium. As for the working technic, both the tapered (Emich) and the cylindrical (Spikes. 4 ) microconeq are used. Most of the work is performed in Eniich cones, and the Spikes cones are used only in the compaiison of the volumes of precipitates for the estimation of the quantity of the various metals ( 3 ) . In part of the work use is made of cones of clear fused quartz. Experience has shown that sodium is always found in blank analyses, when cones of soft 01 Pyrex glaqs are used for the ignition of precipitates or residues.
Procedure (7 I Filtrate F l 5 l from the ( N H I ) ~ C OgJr o u p
+ HCI + BaC12
BabO,. reject
Filtrate F160:
+
BaCO;, reject
(NHdzC03 I Filtrate F161: Evaporate, ignite the residue
Residue Ri61: + HzPtCle + alcohol _ _
