971
V O L U M E 27, NO. 6, J U N E 1 9 5 5 ISTERPRETATION OF DATA
T.'ipure 3 is a t>-pical plot of the fluidity and agglutinating behavior of a \\-esrerll Pennsylvania high volatile A coal. I n obtaining the data for this particular curve, a mixture of 1 part of coal and nine parts of inert (loo- temperature char) was used. Size consistency of both coal and char was 35 X 200 mesh. All points of spccific interest are evident in Figure 3. These include: the iniri:il fusion tempei,:iture, -4; the point of maximum fluidity, B; ant1 the maximum r tance point, C. I n addition, the fluidity range ia readilj- a;iparent. Another example illustrating the applicability of the unit for research stlitlie.* is phoivn in Figure -1. .Is is well known, the addition of alkaline d t s t o a caking coal materially alters its behnvioi~during c:tihonization. This is well illustrated in Figure 4, n-hich slion-s the cffect of adding incrensing amounts of soclinm carl,oriate t o :t bed of caking coal and diluent char. Only the uice point is plotted, this point being of primarjinterwt in so far as fluid bed operability is concerned.
coking problems, using techniques similar to those described by BreR-er and Xtkinson ( 1 ) . T h e dilution technlque presents no problem because a few standard coal-diluent ratios suffice to cover a broad range of caking strengths. The incl eased flexibility of the apparatus more than offsets the occacional need for 3, rerun on a borderline sample. Usually a general knowledge of the sample source permits one to choo-e the proper dilution ratio immediately. ACK%OWLEDGRIEST
The author gratefully acknowledges the assistance of 8. .\. Jones for his many helpful suggestions in the design and construction of the described apparatus. LITERATURE CITED
(1) Brewer, R. E., and .Itkinson, R . G.. Tsn. Esc,. CHEY., AS.^,.
ED.,8, 443-9 (1936). ( 2 ) Davis, J. D., Ihid., 3, 43--5 (1931). (3) Gieseler, Ilamin? Diethvlamine
Watet Water Water Water Water Water Water
Dicyclohexylamine Piperidine
mater
ANALYTICAL CHEMISTRY
972
potential usefulness of mixture A in conjunction with each of the 54 other niistures, the strips were rearranged in order RI X 100 of ascending Ry value for mixture B and A B c D E F G H I J r< L M N o P Q its potential value in 53 combinations 22 14 4 5 14 1 Arginine 9 3 20 8 11 mas similarly assessed. By repeating 22 12 8 23 10 2 Lysine 9 2 16 13 24 this procedure the complete table was 3 15 13 18 10 15 4 3 Aspartic acid 18 4 14 26 12 18 9 15 4 4 Glutamic acid 5 25 17 20 scanned in about 5 hours and 11 com11 3 14 5 Cystine 0 5 29 23 30 27 6 binations of solvent mixtures were 40 27 21 6 Alanine 8 31 32 32 23 36 24 selected which should, in theory, permit 19 14 32 7 Histidine 5 15 27 36 21 34 23 8 Serine 29 20 31 6 20 26 40 27 29 16 the separat,ion as discrete spots of 16 or 55 44 39 18 50 55 53 43 57 63 9 Valink more of the 22 amino acids. These 59 51 59 29 54 65 67 59 63 .59 10 Phenylalanine rombinations were: A/C, A/G. A/K, 45 35 23 10 36 40 36 26 45 29 1 1 Proline AIL, B/K, E/G, E/K, E/L, E/(), I/O, 7 25 28 28 20 29 14 12 Hydroxyproline 35 24 20 43 37 46 23 48 55 55 47 .54 42 13 Methionine and Q/S. 36 24 56 14 Threonine 7 28 34 58 55 47 31 This method of R/ analysis is no more 44 35 41 20 38 50 52 43 43 30 15 Tyrosine than a rough sorting device by which 29 18 17 6 20 26 29 17 28 15 16 Glycine 17 Leucine 64 58 52 31 61 66 63 57 .58 57 .. many unsuitable solvent systems can be 64 55 47 27 60 63 62 54 03 5 0 18 Isoleucine reject,ed, since under routine conditions 54 43 46 27 42 59 fi3 57 5.5 no 19 Tryptophan Rj values are not accurately repro20 Norleucine 66 59 . . 63 72 fi.5 59 67 h0 17 9 23 . . B 2.4 31 20 16 8 21 Cysteic acid ducible. Kevertheless the proc,edui,e . 33 15 . . 32 18 23 14 22 ti 22 Cysteine permits the examination of a very large amount of data in a short time and could of course be applied to other fields. Each of the 11 selected combinations of solvent mistures was where the spots were unusually faint or diffuse, Rj values have examined for its ability to separate a mixture of 18 amino acids been omitted. (Nos. 1 to 18, Table 11)on 12-inch squares of filter paper using the ascending technique with the papers held in stainlePs steel TWO-DIMENSIONAL CHROMATOGRAPHY Datta frames (3). Seven microliters of a solution containing 20 micromole of each amino acid per milliliter (8) was applied Complete separation of a complex mixture of amino acids in 1 4 . amounts, keeping the diameter of the initial spot to about on a one-dimensional chromatogram was not expected, but it 2 mm., and each solvent, mixture, used in the order indicated above, was allowed to run for 16 hours. The papers were dried was hoped to effect useful separations in two dimensions using a t 75" C. forabout 15 minutes after development with each solvent, two different solvent mistures. Experiment,al evaluation of then dipped ( 1 2 ) in a 0.25% w./v. solution of ninhydrin in aceall the 1485 different combinations in which the 55 solvent mistone containing i% v./v. glacial acetic acid, and finally heated a t tures could be paired p-ould have been excessively laborious, 75" C. for 5 minutes. and the plotting of " R f maps" ( 1 ) Tvould have been very timeconsuming. A rapid method of sorting the Rf data to indicate a feiv of the more favorable combinations was therefore devised. Experiments showed that when a solvent had been allon-ed to run 13 or 14 inches d o m the paper, t x o amino acids whose Rf values (expressed as percentages) differed by 5 or more were usually separated into discrete spots. This observation was made the basis of a system for the selection of promising pairs of sol6 vents for the separation of particular amino acids. The possibility was taken into account that a two-dimensional separation might be achieved in those cases when a single solvent failed -e.g., if the Rfvalues for two particular amino acids differed by 3 in one solvent misture and b>-4 in another mixture, then twoI dimensional use of these two mixtures should lead ideally to a 2 separation equivalent, to 5 units of Rj(hypotenuse of a 3, 4, 5 right-angled triangle). Hence for the sorting of potentially useful 5 solvent pairs the assumpt'ion \?as made that it should be possible to separate two amino acids if the difference in R f values in either Figure 1. Tw-o-dimensional chromatogram one solvent mixture xere 5 or more, or if the sum of the differobtained on 12-inch-square Whatman No. 1 ence in Rj values in the tm-o mistures were T or more. To compaper using solvent pair E/O pare the solvents in this way the following method was used: Table 11. R , Values of 22 Amino Acids in Solvent Mixtures A to Q
Amino acids applied a t spot f 3
The data of Table 11,extended to cover all 55 solvent mistures, were t,ranscribcd onto 0.25-inch-square paper-covered strips of Duralumin so that each strip bore the name of an amino acid followed in a fixed order by its R j value in each of the 55 mixtures. The strips were fitted into a wooden frame bearing at the top the code letters of the various solvent mixture9 arranged in order and spacing with the Rj values on the strips. The strips were first arranged in the frame in order of ascending Rj value for mixture 4,spaces being left between successive strips if the Rf values thereon differed by 5 units or more. Each group of strips thus represented a group of amino acids which probably would not be separated from one another by mixture A. The potential value of every other mixture to effect, in conjunction with A, a two-dimensional separation of the amino acids in each group was then assessed by examining the appropriate R , values for each mist,ure in turn. Rapid scanning of the columns of Rj values rvas facilitated by use of transparent cursors, and in this way many possible combinations of solvent mixtures could a t once be rejected owing to the large number of amino acids for which the numerical criteria of separation would not, he met. Having assessed the
Those papers I\ hich had been irrigated TI ith solvents containing cyclohesylaniine or dicyclohexylamine developed the range of colors given in Table 111 while the remainder gave the usual colors obtained n ith ninhydrin. HOT\ever, if these latter papers a e r e sprayed TI ith a 5% ethanolic solution of cycloheydamine or dicyclohex? lamine before the final drying and development mith ninhydrin, they too displayed the full range of colors. The colors XT ere readily reproducible, provided the final heating \vas carried out carefully, but if it was unduly prolonged the variations were less marked and with cyclohexylamine the M hole sheet tended to be some\\hat discolored. It was found better to heat the papers for 5 minutes a t 75" C. than for 10 minutes at 60" C. All the 11 selected solvent pairs gave good chromatograms on Whatman 90.1 paper, the distribution of spots being similar
'
V O L U M E 2 7 , N O . 6, J U N E 1 9 5 5 in almost every particular to the " R , maps" drawn up from the data of Table 11. I n practice the three most useful combinations were QiX, I/O, and E/0; with each of which it was possible t o detect all the applied amino acids, except leucine and isoleucine, either through separation into discrete zones or because color variations enabled adjacent spots to be distinguished. T h e pairs I:/O anti 110 effected partial separations of leucine from isoleucine, hut, these ivere insufficient to be of great diagnostic value. Figure 1 is a photograph of a chromatogram obtained with solvmt mixtures E anti 0. When 4 pl. of the amino acid solution was allowed to run for 3.5 hours c:ich way in the same solvent mistures on papers only 6.5 inches qu:tre, all the constituents escept leucine and isoleucine could still be distinguished. Furthermore separation of a11 the amino aci& esvrpt leucine and isoleucine and met,hionine and valine \viis effec+ed when 2 p l , of the amino acid mixture was placet1 0.5 inrh from adjacent edges of a 3-inch square anti c k veloprtl for 1 hour cach x a - .
913
the maximum information as to the composition of a mixture of amino acids, much useful informat'ion can also be gained from a small number of one-dimensional chromatograms if the solvent mixtures are suitably selected. Thus all the constituents of a mixture of 18 amino acids could be identified by use of the four solvent mixtures D, H, 11,and P, and Figure 2 shows tracings of chromatograms obtained TT-ith these mixtures on Whatman S o . 1 paper after development for 16 hours by the descending technique a t 20" i 1' C., 3 pl. of a solution containing 20 micromoles of each of the 18 amino acids per milliliter being applied to each paper. The \ d u e of solvent mixture J for the detection of tq-ptophan is also illustrated. .4lthough glycine and hydrosyproline ('XI be distinguished using solvent misture H, the colors are rather similar and the latter amino acid is better detected using solvent misture F. This is also illustrated in Figure 2. The amino acids are indicated by the code numbers given in Tahle 11, hut spots representing unresolved mixtures have not h r r n numbered. Sumbering of adjacent spots indicates that tlir appropriate amino acids n-ere readily distinguished by their characteristic colors (see Table 111), the colors obtained v i t h mixtures F and H being due to the cyclohesylamine therein and those with mixture AI and P to the dicyclohesylamine present. I n the case of mixture D, which contained neither of these amines, treatment of the paper with dicj-clohesylamine before development n.ith ninhydrin permitted distinction of leucine and phenylalaninp in the terminal spot. Use of dicj-clohesylamine also aided the identification of tryptophan with solvent mixture J. T h e reasons for the striking influence of cyclohcsylamine and dicyclohexylamine on the range of colors given 11y various amino acids with ninhydrin were not investigated, b u t it may be significant that, the colors w r e produced only on filter paper and not in the t w t tube.
Table 111. Effect of Cyclohexylamine and Dicyclohexylamine on the Ninhydrin Reaction on Filter Paper
H
P Figure 2.
J
D
M
F
One-dimensional chromatograms
hiiiino .-\?id
Color with Cyclohexylaniine
Color with Dicyclohexylamine
AspaFtic acid Cystine Alanine Histidine Serine Phenylalanine Proline Hydroxyiiroline Threonine Tyrosine Glycine Other amino acid. (Fee Table 11)
Royal blue Orange Purple Gray-green Purple Blue-Frraj Yellon Carmine Gray-purple Slate gray Red-brown Purple
Turquoise Carmine Blue-purple Gray Grav-Durnie Gra,y-brob k-ellow Yellow Gray Gray-brown JTine Purplered
V-ith these smaller p a p m , aspartic acid overlaps lysine, but the turcjuoise blue of the former amino acid enables a ready distinrtion to bc made. On the 3-inch-square papers the yellow color produced by hydroxyproline tends to be obscured by the \vine-red glycine zone if only low concentrations of the former amino acid are present. Threonine gives only a faint grayish spot, but, this deepens on keeping. Serine and histidine which form one elongated spot can nevertheless be distinguished, the gray-purple color produced by serine being quite distinct from the gray given by histidine. Similarly the gray-brown color given by tl-rosine and phenylalanine enables these amino acids to be detected in the overlapping group comprising leucine, isoleucine, methionine, valine, phenylalanine, and tyrosine. Although Whatman S o . 1 paper was used routinely, Whatman Tos. 4 , 5 , 7 , 2 0 , 4 0 , 4 1 , 4 2 , 5 0 ,52,511, and 8 AIhLI, and S. and S. 507 were also examined. With the smaller chromatograms Whatman KO. 20 had the advantage of giving rather more compact spots, although the solvents ran more slowly on it. S o n e of the other papers was better than Whatman 50.1.
The effect of treating amino acid spots on filter paper with cyclohesj-lamine or dicyclohexylamine prior to heating with ninhydrin n-as extended to a small range of amides and peptides. Compounds were chosen which Tvere closely related to those amino acids which gave under these conditions the most striking colors. Whereas aspartic acid gave a royal blue spot after treatment with cyclohexylamine and a turquoise spot after treatment with diq-clohesylamine, asparagine gave an orange spot with both these reagents. Glutamine, on the other hand, gave the same purple color as the free amino acid. Glycylglycine and glycyltyrosine both gave yellow zones with c?-clohesylamine and grayish-yellow spots with dicyclohexylamine while glutathione gave a red-purple color.
ORE-DIMENSIOYAL CHROMATOGRAPHY
ACKKOWLEDG.MEYT
illthough two-dimensional chromatography is the technique of choice when it is dePired to derive from a single experiment
The authors thank C. G. Green for constructing the frame for the analysis and the Datta frames, D . F. Laason for the photc-
DETECTION OF AMIDES AND S I M P L E PEPTIDES
974
A N A L Y TI C A L CHEMISTRY:
9
graphs, and the directors of Beecham Research Lahoratories, Ltd., for permission to publish this paper. LITERATURE CITED
(1) Consden, R., Gordon, A. E.,and Martin. A. J. P., Bioebem. J . (London),38, 224 (1944). (2) Curson, G., and Giltrow, J.,N a t w e , 173, 314 (1954). (3) Datta, p., ~ e n t E. , C., and ~ ~H,, science, ~ ~ 112, 621 i
s.
11 qmi. ~ ~...,
(4)
Hardy, T. L., end Holland, D. 0.. Chemistry & Zndustru, 1952, 855.
( 5 ) Harris, G.. J . Inst. Brewing, 58,417 (1952). (6) Hsrrir, G., and Pollock, J. R. 1.Zbid.. . 59, 20 (1053). (7) Jepson, J. B., and Smith, I., .Valure. 172,I100 (1953). (8) Levy, A. L., and Chung, D., . h a ~ Cnsu., . 25, 396 (1053). (0) Redfield. R., Biochem. et Biophys. Ada. 10, 344 (1 953). (lo) ~ ~ ~ k L,l B,,~ and ~ dunderwood, , J, c,, Cnmr.. 26,.
1557 (1054). A.,and Oreskes, I., Science, 119, 124 (1054). ~(11) Scrifer. , (12) Smith. I., Natuve, 171,43 (1053). RECEIVED
for review July 10, 1954.
loeepted February 1. l%X.
Twenty-Stage Molecular Distillation Unit F. W. MELPOLDER, T, A. WASHALL, and J. A. ALEXANDER The Atlantic Relining Co., Philadelphia, Pa. A 20-stage countercurrent molecular still has been developed for the distillation of high boiling samples. The use of the still is expected to permit a more complete analysis of heavy petroleum products. The still, with a capaoity of 1500 ml., consists of one large and 19 small still pots connected in series. The unit can he operated as an equilibrium-type still for samples less than 260 ml. and as a hatch distillation unit for larger volumes. Using a test mixture of Oetoil Ihis(2-ethylhexy1)phthalatel and Octoil S Ibis(2-ethylhexyl)sebacate], an efficiency of 0.8 theoretical plate per stage was found. Automatic safety controls allow the still to operate unattended.
distillate and reflux wa8 provided for efficient fraotionittion. This design is similar in principle to the method used by F a a e e t t and McCowen (S), except that pumps between stages were eliminated.
I
N T H E past few years several new itnalytical tools have been
developed for the separation and analysis of high boiling petroleum stocks. As the first step in a separations program it is often advantageous to prepare narrow boiling fractions in order t o enhance the degree of separation in subsequent operations. Because of the thermal instability of hydrooarbons at the higher temperatures, it is necessary t o carry out the distillation at the lowest practicable pressure. Scveral types of high vacuum rectifying stills have becn described recently. Byron, Bowman, and Coull ($) have developed a rotary rectifying column consisting of two concentric tubes. The inner tube is cooled and rotated while the outer tube is heated and stationary. Brewer and Madorsky ( 1 ) obtained a pnrtid separation of the meroury isotopes in a 10-cell countercurrent reflux still. Fawoett and McCowen (S) developed a countercurrent redistillation unit for continuous distillation. A multicolumn, countercurrent falling-film still was developed by Mndorsky ( 6 ) . The individual stills consist of a cancentric.evaporata- surrounded by a water-cooled condensing are3. Each column is provided with a combination magnetic pump and reservoir. A multistage still developed by Mair, Pignocca, and Rassini (6) consists of 8 large pot and a reotifying Eection contsining 50 stages. I n the following work, an attempt was made to design a simple multistage molecular still possessing a relabively high separation effieienoy.
-
1.
.
contents of t h a t pbt before %assing downward as reflux to the next lower pot. T h e result is that the lighter components are progressively distilled to the higher stages, while the heavier camnonents are carried down to the loiver stages. Chamber e is merely B reflux unit, The d$tillate from pot 20 is returned to that pot as reflux through tube f. Tubes f also
DESIGN O F MULTISTAGE STILL
. .
1
.
..
. . ~
I
~~
~~
vapo;was
to reduce seriously the fractionation efficiency of the column. A new type of still was desianed, therefore, to eliminate the flow of vapor- from any particular stage. Twenty glass molecular stills of the type described by Mulliken and Harkins 171 were connected in series as a sinele unit with snitabk tnhes io; transfer of distillate and reflug. Countercurrent BOW of ~~
~~~~~~
Figure 1.
Still assembly
