75s
V O L U M E 2 2 , NO. 6, J U N E 1 9 5 0 Table V. Sample Weight, Grams 1.6 4.0 1.6 0.5 1.0 2.0 1.0
Hydrolysis of Vinyl Acetate in 2.5 pH Reagent Hours 0.5
0.5 18 1.5 1.5 1.5 9
Condition
C. Room temp. Room temp. Room temp. 100 100 100 100 0
Weight % Reaction 0.5
quantitatively gave results of comparable precision and accuracy by the semimicroprocedure. For the analysis of acetals, special 50-ml. heavy-walled Pyrex brand bottles are used which employ the same caps as the 350ml. Pyrex brand bottles.
0.6
26.0 93.7 96.0 94.2
95.7,96.2
established by direct analysis of careful9 distilled cyclohexanone. This sample analyzed 99.6 * 0.2% by the Karl Fischer procedure (8) and 99.7 * 0.4% by the pyridine-bromophenol blue method ( 2 ) . (A total of 0.4% cyclohexanol plus water was found by analysis. The results on this sample are not included in Table 11.) Analyses of thirteen individual samples gave an average correction factor to 99.6y0 of 1.032 with a deviation of k0.003. Similar experiments were made in establishing the correction factors to be used in the determination of the other ketones as noted in Table 11. In general, acid recovery in the presence of purified oxime was considered the more reliable means for establishing these factors, becac-e the values obtained were not dependent on purity of the ketone as determined by another method. SEMIMICRO APPLICATIONS OF T H E 2.5 pH REAGENT
The present visual method was adapted readily to a semimicro scale. The sample, containing up to 1 me. of free carbonyl compound, is weighed into a 50-ml. glass-stoppered borosilicate glass bottle containing 20 ml. of the hydroxylamine reagent. After 15 minutes a t room temperature, the solution is titrated with 0.1 N sodium hydroxide in 90% methanol until at an adjusted total volunie of 35 ml. the hue of the sample matches that of a blank containing 20 ml. of hydroxylamine reagent plus 15 ml. of 90% methanol. All the compounds listed in Tables I and I1 which reacted
ACKNOWLEDGMENT
The authors are grateful to the following for their aid in carrying out the experimental program: C. E. Ashby, W. L. Autman, D. G. Barlowe, Alan Cobb, Walter Hawkins, R. E. Kitson, J. M. Lupton, A. N. Oemler, and L. W. Safranski. LITERATURE CITED
(1) Brochet. A., and Cambier, R., Compl. rend., 120,449-54 (1895). (2) Bryant, 13’. M. D., and Smith, D. M., J . Am. Chem. SOC.,57, 57-61 (1935). (3) Byrne, R. E., Jr., As.41.. CHEM.,20,1245-6 (1948). (4) Eitel, A.,J.prakt. Chem., 159,292-302(1942). (5) Huckabay, W. B., Sewton, C. J., and Metler, A. V., ANAL. CHEM.,19,838-42 (1947). (6) Mitchell, J., Jr., and Smith, D. M., Ihid., 22, 746 (1950). (7) Mitchell, J., Jr., and Smith, D. M., “Aquametry,” pp. 323-5, New York. Interscience Publishers, 1948. (8) Mitchell, J., Jr., Smith, D. M., and Bryant, W. M. D., J . Am. ‘ Chem. Soc., 63,573-4 (1941). (9) Pressman, D . , and Lucas, H. J., Ihid., 61, 2271-7 (1939); 64, 1953-7 (1942). (10) Richter, V. yon, “Organic Chemistry,” Vol. I, p. 433, New York, Sordemann Publishing Co., 1934. (11) Romijn, G.. 2.anal. Chem., 36,18-24 (1897). (12) Seaman, W., Woods, J. T., and Massad, E. A , , h i A 1 , . CHEM., 19,250-1 (1947). (13) Siggia, S., “Quantitat,ive Organic Analysis via Functional Groups,” pp. 60 f f , New York, John Wley & Sons, 1949. (14) Spengler, H., and Kaelin, A., Hunderl Jahre Schweir. Apofh.Ver. (Centenaire SOC. suisse pharm.). 1843-1943, 542-64 (1943). (15) Thomann, J., Pharm. Ada Heh., 5,11-24 (1930). (16) Walker, J. F., ”Formaldehyde,” p. 98, New York, Reinhold Publishing Corp., 1944. R E C E I V EOctober D 28, 1949. Presented before the Division of Analytical and Micro Chemistry at the 116th Meeting of. the . ~ M I R I C A H CHEMICAL SOCIETY, Atlantic City, N. J.
Analysis and Characterization of Pure Compounds and Mixtures of Compounds Solubility Procedure WILKINS REEVE
AND
ROWLAND ADAMS’, University of M a r y l a n d , College Park, M d .
T
HE solubility of a pure solid compound in any given solvent at any given temperature is as characteristic of the compound as other physical properties such as the melting point or boiling point. However, the measurement of solubility characteristics to establish the identity of compounds or to serve as a criterion of their purity has not come into general use, except where other methods completely fail. This is probably because of the relatively large amounts of material required, the tediousness of carrying out a series of precise solubility determinations, and the difficulty of expressing the results in as simple a form as is done with melting points and boiling points. A critical review of three analytical methods based on solubility procedures is given by Bennett ( 2 ) and one of these, the 1 Present addreas. Jackson Laboratory, E. I. du Pont de Nemours & Company, Wilmington, Del.
Korthrop and Kunitz procedure, is also reviewed by Herriott ( 7 ) . All the procedures described in the literature have been carried out at essentially one temperature. The idea of having fixed amounts of solute and solvent and using the measurement of the temperature of complete solution as a measure of a given component has not been applied to the analysis of solids, although in the case of liquid mixtures, various analytical procedures for the determination of a given component have been based on the temperature a t which the single-phase system changes to a two-phase system (8). Where conventional melting points and mixed melting points fail to prove the identity of two materials or to serve as a criterion of purity, owing to decomposition, the procedure here described based on the temperature of complete solution may often be substituted. Likewise, where thermal analysis of binary mix-
7%
ANALYTICAL CHEMISTRY
Table I.
Solubility Temperatures of Amino Acids in Water
Amino Acid Glycine oL-Threonine &.-Histidme.HC1.H'10 DL-Alanine L-Alanine Taurine obAllothreonine c-Asparagine. HIO or,-Serine oL-Glutamic acid or,-Valine DL-Methionone L-Isoleucine r,-Phenylalanine L-Glutamic acid or,-Isoleucine or,-Phenylalanine DL-Aspartic acid L-Aspartic acid DL-Norleucine L-Tryptophan DL-Leucine
Observed Sol. Temp.,
(2.0
14.0 'at' 6 : 1 23.5 a t 6:1 26.3 a t 6 : l
..... .....
47 6 a t 6 : l
Depression of Sol. Temp. Due Caicd. Sol. to 1% Temp., O C. from Soluble Literature Refer- ImpuDatamb ence rityC, C. 6.7 * 0 . 3 a t 6 : 1 0.42 ..... 0.70 24.8 *'0:3'at 6 : l 25.1 * 0 . 2 a t 6 : 1 39.8 - 0 . 2 a t B : l
..... .....
...
0.77 0.93 0.35 0.63 0.26 0.42
65.2 *'O:Z& 6:l 68.9 * 0.2 a t 6 : l 84.7 * 0 . 6 a t 6 : 1 0.28 92.8 + 0 . 2 a t 6 : 1 94 5 ' a t b : l 0.60 97.0 - 0 . 2 a t 6 : l .. .. 0.49 18.7 * 2 . l a t 2 5 : 1 2.50 ..... 43.6 t 0 . 5 a t 2 5 : l 0.62 66.3 t 0 . 3 a t 2 5 : l 0.27 0.57 67.2 * 0 . 5 a t 2 5 : 1 81.5'ad25:1 78.3 * 0 . 3 a t 2 5 : 1 0.43 0.33 7 0 . 8 a t 2 5 : l 71.4 * 0 . 2 a t 2 5 : l 0.29 ..... 84.5 * 0 . 4 a t 2 5 : 1 ..... 89.5 i 0 . 4 a t 2 5 : l 0.41 ,.,.. 90.0 - 0 . 4 a t 2 5 : l 0.42 Approx. 98.0 -0.2at25:l 0.37 105.5 a t 25:l a Water to amino acid ratio = 6 : 1 or 25:l as indicated. b t Qalues are two thirds of mean deviation. C Calculated from expression log S = a bt ct' by differentiation and substitution of appropriate constants.
.....
+ +
tures by means of a melting point curve is not feasible, the substitution of a similar type of curve, based on the temperature of complete solution as determined by this solubility procedure, should frequently be satisfactory. This paper describes a convenient method of determining the temperature at which a solute will dissolve in a fixed amount of solvent, and demonstrates how this temperature, designated as the "solubility temperature," can be of use in the characterization of compounds, in the determination of their purity, and in the analysis of mixtures. The most important new feature of this analytical technique is the use of the temperature of complete solution in a manner analogous to the way melting point data for pure compounds and mixtures have been previously handled. Ale0 of importance is the development of a standardized procedure for accurately and conveniently determining the solubility of relatively small amounts of a substance in a solvent over a wide temperature range. The method requires 50 to 200 mg. of material; however, all can be recovered after the determination is finished, or some other analysis can be carried out with the accurately weighed sample. Less than an hour is required for a determination. All the work described in this paper has been done with amino acids using water aa the solvent, but there are no apparent reasons why the method should not be equally applicable, with water or other solvents, to other classes of compounds. PROCEDURE
The finely ground solid is accurately weighed int,o a 15 X 75 mm. borosilicate glass test tube; care is taken that no solid adheres The wei h t of sample is such that the after dilution with the solfinal volume will approximate 1 vent. A weight of solvent, usually 3, 6, or 25 times that of the sample, is introduced from a calibrated 2-ml. Koch microburet with the necessary corrections for thermal expansion. The addition should be so conducted that no liquid touches the side of the test tube within 5cm. of the top. The tube is immediately and rapidly sealed, using a small pointed gas-oxygen flame. This is done by first sealing a rod on the lip of the test tube while holding the test tube in a vertical plane at about a 60" angle to the as-oxygen flame. The test tube is heated intensely with a s m a t pointed 5ame about 2 cm. below the li while rotating, and the heated ortion is then drawn out t o a &e capillary. After 5 seconds, $\e capillary is sealed off as close t o the t a t tube 8 s t o the walls of the tube.
3,
possible, so that there will be no capillary tube where small amounts of the solid can become lodged. The temperature of complete solution is determined by attaching one or more tubes with rubber bands to the end of a shaft which can be rotated at 60 to 100 r.p.m. and which is mounted 30" from the horizontal so that the end can di into a 7-liter battery jar filled with water. The tubes, attacged a t right angles to the shaft can be immersed 7.5 cm. (3 inches) below the liquid level and still rotate only 30' from the vertical. This furnishes good agitation, for the contents of the tube fall from one end to the other as it is rotated. A strong light is placedso as to illuminate the tubes. The temperature of the bath IS controlled b an internal electrical heater connected through a voltage reguyator. The bath is stirred vigorously to prevent any appreciable temperature differentials. Temperatures are measured by a calibrated 0' to 100' partial immersion thermometer graduated in 0.1 (Kimble Glass No. 43606). As the tube is rotated, the temperature of the bath is rapidly raised to within 2" or 3" of the solubility temperature. This point is easily recognized because most of the crystals have disappeared. The rate of heating is then reduced to 0.1" per minute. As complete solution is approached, the rotation of the tube is stopped for short intervals, so that the contents m y be more closely examined. These short intervals should not amount to more than 30 seconds every 2 minutes. The tem rature at which the last cr tal disaolva is taken as the soluElity temperature. For t u r a prepared from the same sample, thw tamperature is reproducible to 0.2" C. For phenylalanine, valine, aspartic acid, and other amino acids which are not wet too well by water, and which crystallize rapidly when their hot solutions are cooled, more reproducible rwults are obtained by heating the sample tube ra id1 to com lete solution, then removing the shaft with attache8tuL from t i e bath, and rotating 10 minutes in the air with the shaft in a horizontal position. During this period, crystals will form in the sample tube and the tem erature of the water bath will fall rapidly (if the front part of tKe cover is removed) to a few degrees below the expected solubility temperatures. After 10 minutes, the shaft and tu,be are replaced in the water bath and the solubility temperature is determined in the usual manner. APPLICATION TO AMINO ACIDS
Of the important amino acids, four have such small solubilities in water that excessive amounts of water are needed to dissolve them: cystine, tyrosine, diiodotyrosine, and thyroxine. Four others->arginine hydrochloride, Dklysine hydrochloride, prline, and hydroxyproline-are extremely soluble in water. Most of the remaining amino acids fall into two groups. One group has solubility temperatures ranging from 6.7 O to 97 O if a ratio of 6 parts of water to 1 of amino acid is used. The other group has solubility temperatures covering a somewhat similar range if a ratio of 25 parts of water to 1 of amino acid is used. The solubility temperatures for the amino acids in these two groups have been calculated from data in the literature. New data are presented for Dbthreonine and thistidine monohydrochloride. The solubility temperatures of four carefully purified amino acids-DL-alanine, Dtaspartic acid, Dbphenylalanine, and Dkvaline-have also been determined in order to compare these values with the solubility temperatures calculated from the literature solubility data. With the exception of DG phenylalanine, the solubility temperatures for these six amino acids are believed to be accurate to approximately 0.2" C., because the amino acids were recrystallized until their solubility temperatures remained constant. A comparison of the experimentally determined solubility temperatures with the calculated values indicates that the experimentally determined values are often a few degrees higher than the calculated values. In one case (Dtleucine) the difference is 7 O , but, because the calculated solubility temperatures for all the amino acids are based on data obtained below 60" C., this is due to the large extrapolation of the literature data. For the amino acids having solubility temperatures below 60" C., the temperature differential is believed to be due chie5y to the fact that the experimental values are obtained in a dynamic system, wheress the calculated values are based on data obtained under equilibrium conditions.
V O L U M E 22, NO. 6, J U N E 1 9 5 0
157
The temperature at which a solid dissolves in a given amount of solvent is a characteristic property of the solid, and has h e n designated the “wlubility temperature” when it is determined under the described conditions. Like melting points, solubility temperatures can be used to characterizecompounds, as a eriteriaof purity, and to analyze mixtures. They are especially useful witb solids which have unsatisfactory melting points due to decomposition. A convenient procedure for rapidly and aoourately detemnininp solubility temperatures is described, and their usefulness in the characterizationand analysis of amino acids is illustrated.
The solubility of kvaline changes relatively little with temperature and is such that it falls between the two groups given in Tsble I. In addition, the literature solubility data indicate that the mode of crystallizstion influences the solubility (6). The more atable form should have e. solubility temperature of a p proximately 37” with B water to >valine ratio of 12 to 1 (data from Figure 1 of 6). It is suggested that future workers determine solubility temperatures tiith water to amino acid ratios of 3, 6, 12, 25, or 50 or multiples thereoi, so thtut dets irom different laboratories will be on B comparable bssis. RS AND REPRODUClBlLlTY
mperature is being slowly raised during the course or B m u ~ u n ytemperature determination, the temperature of complete solution determined in this way must be somewhat higher than the corresponding temperature determined under equilibrium conditions. In the determination of melting points, there is B similar difference between the “melting point” determined by the common es.pillmy tube method, and the true melting point determined under equilibrium conditions. With nlrthreonine and okallothreonine, on which much of the pioneer-
ing work was done, the differenoe appears to be only a fraction of B degree. F i t h other amino seids, this difference may amount to more than B degree, but it will be a constant under any given set of experimental conditions for any particulm smino acid. The extent of this temperature differential depends on three factors, among others: the siae of the crystals dissolving, the ease with which the crystals me wetted by the solvent, and the rate st which the temperature of the bath is raised. The crystal siae can be controlled by grinding the sample to a fine powder, or by dissolving the smnple in the sealed tube and then cooling to obtain some small crystals. Errors due to the fact that crystals me difficultly wet by the solvent might be minimized by using a wetting agent, but it is preferable to use a pure solvent and to accept this small temperature differential. If the temperature of the bath is raised at B more rspid rate than 0.1‘ C. per minute, the solubility temperature will be somewhat higher than under the specified conditions. With the more soluble amino xida, sueh 88 threonine and all+ threonine, the rate of solution of the smino %ids is surprisingly rspid. Thus a finely powdered threonine-sllothreonine sample, having B certain solubility temperature, could be obtained in the form of large crystals by allowing the sealed tube to stand for several days sfter the completion of the run. When the solubility temperature was redetermined, a value only about 1‘ high was obtained, in spite of the fact that the surface mea of the crystals exposed to the solvent had been reduced by many thousandfold. With the less soluble amino acids, espechlly those not wet well by water, crystal siae is much more important and should be as s d as possible. Thus in the case of ~ l r phenylalanine, high resulta will be obtained u n l w the sample is first dissolved and thesolubility temperature determined on the freshly formed erystale. Even when this procedure is followed with this amino acid, the reSU1tS are less accurate than in the ease of the other amino acids, and occa4ionally results 1”high will be obtained. The somewhat empiriosl nature of solubility temperatures in appsrent from the above discussion. The small differencea between solubility temperstur? obtained by the described prccedure and the true tempersduea of complete solution, determined under static, ideal conditions, &re due to several factors which will vary from compound to compound. The precision obtained in the determination of solubility temperatures depends upon the socurscy with which the solvent is added to the sample, upon sealing the tube without loss of either component, and upon the accuracy with which the end point is observed. It is possible to add the solvent with %naccuracy of 1 part per thousand when a oalihrated microburet is used and allowance is made for the temperature of the solvent in the buret. This has been checked by calculating the weight of the water in the sealed-off tube from the weights of the two parts of the sealed-off tube, snd comparing this with the volume of water added. Care must he taken that the sample snd the solvent me deposited in the bottom of the test tube and not on the walls, where they would be destroyed or volatilized when the tube is sealed off. When the tube is sealed, the position in which it is held must be such that the flame does not enter it. I n some
ANALYTICAL CHEMISTRY
758 cases it may be advisable to cool the solvent before sealing off the tube in order to minimize losses; this has not been done in the present work. The temperature of complete solution can be observed within 0.2", providing the solution is such that the last crystals are clearly visible and no insoluble materials are present. If mote than a trace of insoluble material is present, it is necessary to try to estimate the temperature at which no more material dissolves. This, of course, is difficult and inaccurate.
The identity of two solid materials can be established by determining their mixed solubility temperatures; if there is no depression, the two are identical, providing they do not pass through the liquid state before dissolving. In the case of liquids, the solubility temperature procedure may fail. It has been found by Ivan Christoffel of these laboratories that at least and 1one pair of liquid isomers-2-phenyl-2-methoxyethanoi phenyl-2methoxyethanol-have nearly identical solubility temperatures and the solubility temperatures of mixtures of these show no depression. APPLICATION TO ANALYSIS OF MIXTURES
A binary mixture can be analyzed by comparing its solubility temperature with that of known mixtures of the two pure compounds. The solubility temperature-composition diagram of the Dbthreonine-Dballothreonine system is typical (Figure 3).
WEIGHT PERCENT DL-ALLOTHREONINE
Figure 2. Variation of Solubility Temperature of DbAllothreonine in Presence of Various Compounds Water-solid ratio of 3 : l . DL-Allothreonine curve m i for Dballothreonine alone3 in this case, Dballothreonine used relative to amount required for 311 ratio is plotted as abscissa. This gives a curve corresponding to a soluble inert impurity
The reproducibility of solubility temperatures is illustrated by the values obtained for Dballothreonine during its purification by an extended series of recrystallizations. A ratio of 10 parts of water to 3 of amino acid was used. Values found were: crude, 80.7"C.; first crystallization, 81.3"; second recrystallization, 82.5"; fourth recrystallization, 82.7"; sixth recrystallization, 83.4"; eighth recrystallization, 83.5'. EFFECT OF ADDED COMPONENTS
The solubility temperature will be depressed by the presence of soluble impurities because each component of a solid phase dissolves more or less independently of the others, and there is less of the main material to dissolve in the fixed amount of solvent. If the soluble impurity is inert in the sense that it has no effect on the solubility of the main material, the lowering of the solubility temperature can be easily calculated from the temperature-solubility data of the compound in question. In practice, soluble impurities will often affect the solubility of the main component either positively or negatively. Solubility temperatures for Dballothreonine in the presence of ammonium chloride, sucrose, and glycine are given in Figure 2. The effect of a soluble inert impurity is given by the Dballothreonine curve, in which the weight of Dballothreonine used per volume of solvent is the same as the weight of the Dcallothreonine component in the two component mixtures-that is, no second component was used and the weight of the water equaled: (300) X (grams of Dballothreonine)/(weight % DL allothreonine plotted as abscissa). Sucrose is without effect, glycine lowers the solubility temperature somewhat, and ammonium chloride lowers it even more. Even with ammonium chloride, however, the error introduced is not excessive when less than 5% is present. In the presence of larger amounts of a material acting as ammonium chloride does in this case, it would be necessary to construct a solubility temperature-composition diagram as described herewith.
The solubility temperature of DL-allothreonine is 91.2 a C. ; mixtures of ~~-allothreonine with DL-threonine will have loner solubility temperatures until a composition correspondin to 37% DL-allothreonine and 63% DL-threonine is reached, a t wkch point the solubility temperature has a minimum value. Mixtures containing increasing amounts of Drrthreonine have higher solubility temperatures until finally the solubility temperature of DL-threonine is reached. With mixtures having compositions represented b the left-hand portion of the curve, the last material to dissorve as the temperature is raised is the Dballothreonine. At the minimum point on the curve, the Dbthreonine and the DL-allothreonine are present in the ratio of their solubilities, and both dissolve together. On the right-hand portion of the curve, the Dcthreonine is the last component to dissolve. Over part of the diagram an observed solubility temperature can represent two compositions. These can be differentiated by determining the change in solubilit,y temperature when a mixture of one of the pure components with the unknown sample is run. In the csse of the Dkthreonine-DL-allothreonine mixture, each side of the solubility temperature-composition curve can be closely approximated by plotting the solubility temperature of each pure component determined separately with amounts of the component equal to that amount which would be present if the mixture of isomers was used. This is represented by the dashed line of Figure 3, and demonstrates how each of these particular isomers dissolves almost independently of the other. "Binary" mixtures containing small amounts of other components can be analyzed successfully, providing the other components are sufficiently soluble to dissolve before the end point is reached. This is done by analyzing for one component by determining the solubility temperature in the usual way, then
~
3 so
0
DL-THREONINE-
W
0
a 80
0
ALLOTHREONINE DL-THREONINE
a
*
I-
DL-ALLOTHREONINE
DL-
ro
I
W
60
t
$ 3
50 40
30
0
30 40 50 60 70 80 WElOHT PERCENT DL-THREONINE
90
I(
t o 60 50 40 30 20 WEIGHT PERCENT DL-AUOTHREONINE
IO
o
IO
20
wo so
eo
Figure 3. Solubility Temperature-Composition Diagram for DL-Threonine-DLAllothreonine System Solid line.
DL-Tbreonin~DL-allothreonine mixture with water to amino acid ratio of 331 DL-Allothreonine alone (left) and DL-threonine alone of amino acid u a e d relative to amount required for 3:l ratio plotted as abscissa
Dashed line. ( r i g h t ) with
V O L U M E 2 2 , NO. 6, J U N E 1 9 5 0 analyzing for the second component by determining the solubility temperature of a mixture of the unknown and the second component. This mixture must contain sufficient of the second component so that the solubility temperature is determined on the par: of the curve where the second component is the last msteriai to dissolve. The percentage of the second component in the mixture can be calculated from the following equation:
pz
P,(W
759 Table 11.
Solubility T e m p e r a t u r e Data
(3 t o 1 ratio
oL-Allothreonine, o/c
..,
9.9 20.3 29.4 39.1 50.4 61.6 69,6 76.6 84.2 92.1 100
W
DETERMINATION OF SOLUBILITY
From solubility temperatures determined with different ratios of solvent, approximate solubility data can be calculated. Data obtained by this dynamic method will approximate data obtained under equilibrium conditions, but they will not be identical. In the case of Dcthreonine, the data closely fit the 0.0058581 0.00M)07t2 over the equation log S = 2.1381 temperature range 14" to 61' C. where S is the solubility expressed in grams per 1000 grams of water and t is the temperature in degrees centigrade. For Dcallothreonine, the equation is log S = 1.895 0.006881 over the temperature range 43" to 91" C. These data are believed to be accurate to within at least 1%.
+
+
+
EXPERIMENTAL
Purification of Amino Acids. The amino acids were rccrystallized as recommended by Dunn and Rockland (6). The Dcalanine (Bios Laboratories, Inc.) was recrystallized six times. Its solubility temperature a t 6 to 1 was: two recrystallizations, 25.2" c . ; four, 26.1' c . ; six, 26.3" c . DL-Aspartic acid (Eastman Kodak Company) was recrystallized four times. Its solubility temperature at 25 to 1 was: two recrystallizations, 70.9" C.; four, 70.8" C. L-Histidine hydrochloride monohydrate (Pfanstiehl Chemical Company) was rerrystallized twice. Analysis calculated for CeH1?0aN&I: C, 34.37; H, 5.77. Found: C, 34.39; H , 5.80. Solubility temperature at 6 to 1: unrecrvstallized, 23.2O C.; recrystallized twice, 23.5' C. DL-ieucine (Mann Fine Chemicals, Inc.) aft,er two recrystallizations had a solubility temperature a t 25 to 1 of approximately 105.5" C. as determined by iminersin the sealed sample tube, fwtened with rubber bands to the en& of an ordinary 110' C. thermometer, in an oil bath and shaking by hand. DL-Phenylalanine (Mann Fine Chemicals, Inc.) was recrystallized six times. Its solubility temperature a t 25 to 1 was: two recrystallizations, 80.4OC.; six, 81.5"C. DL-Threonine (Eastman Kodak Company, Merck, and Interchemical Company) was recrystallized ten times by dissolving
DL-Threonine, %
... ... ... ...
+ A ) - l00A
where Pf equals the per cent of the second component in the original sample, P, equals the per cent of the second component observed in mixture of sample and second component, W equals weight of original sample, and A equals weight of added second component. If the percentage of the first and second components is known, the difference between the sum of these and 100 represents the percentage of the other components present, providing these other components are not present in sufficient amounts to alter seriously the solubilities of the first two components. In the analysis of mixtures consisting predominantly of two previously characterized components, this method has decided advantages over the procedure of Northrop and Kunitz, which fails t o distinguish more than one component when two components are present in the same ratio as their solubilities. Thus the Xorthrop and Kunitz ( 7 ) procedure would show a mixture of 37% allothreonine and 6390 threonine to be a homogeneous single material, especially because the relative solubilities of these isomers ark independent of temperature and only aqueous solvents can be used. If it is not possible to obtain the pure components to construct the solubility temperature-composition diagram, then the solubility temperature procedure obviously fails, whereas the procedure of Northrop and Kunitz will often be successful.
of water to total solids)
...
Solubility Temperature, C 91.2 78.2 76.3 86.8 73.1 81.3 76.8 60.2 61.0 48.6 33.8 37.9 43 3 49.7 5.5.4 61.1
Ratio of Water t o Amino .4cid
Pure DL-threonine
6.00 4.20 3.56 3.00
14.0 39.0 50.0 61.1
Pure DL-allothreonine
6.37 4.61 3.66 3 00
43.1 64.5 78.9 91.2
it in the smallest amount of boiling water, then adding three volumes of absolute ethyl alcohol and allowing it to cool to room temperature and stand 24 hours. The neutral e uivalent by formol titration was 119.4; calculated, 119.1. T i e solubility temperature was unchanged by the last four recrystallizations. m-Allothreonine was prepared by the procedure of Adkins and Reeve ( I ) and recrystallized nine times. The neutral equivalent by formol titration was 119.3; calculated, 119.1. The solubility tem erature was unchanged by the last two recrystallizations. DL-qaIine (Mann Fine Chemicals, Inc.) was recrystallized four times. Its solubilit temperature a t 6 to 1 was: two recrystallizations 94.1' C.; Zur, 94.5"C. Solubility temperature data for Figures 2 and 3 are given in Table 11. ACKNOWLEDGMENT
It is a pleasure to acknowledge the financial assistance of the Biological Laboratories, Organic Chemical Department, E. I. du Pont de Nemours & Company which made this research possible. Many of the solubility temperatures were either checked or determined by Charles Haber. The microanalyses were performed by Mary Aldridge. LITERATURE CITED
(1) Adkins, H., and Reeve, W., J . Am. Chem. Soc.. 60, 1328 (1938). (2) Bennett, G. M.,AnaEysl, 73, 191 (1948). (3) Dalton, J. B., and Schmidt, C. L. A., J . Biol. Chem., 103, 549 (1933). (4) Ibid., 109,241 (1935). (5) Dalton, J. B., and Schmidt, C. L. A., J . Gen. Physiol., 19, 767 (1936). (6) Dunn, M. S., and Rockland, L. B., "Preparation and Criteria of Purity of the Amino Acids," in hl. L. Anson and J. T. Edsall's "Advances in Protein Chemistry," Vol. 111, p. 341. New York, Academic Press, 1947. (7) Herriott, R. M.,Federation Proc., 7,479 (1948). (8) Houben, J., "Die Methoden der organischen Chemie," 3rd ed.. Vol. I, p. 879, Leipsig, G. Thieme, 1925. RECEIVED December 13, 1949.
Correction In the article on "Iodometric Determination of Resorcmol" [ANAL. CHEM.,22, 585 (1950)] in the first column, last paragraph, the next to the last sentence should read: "The mixture was stirred until solution was complete and then was diluted to 0.5 liter." In the second column, first equation, 310should have been used instead of 313. H. H. WILLARD
