ANALYTICAL CHEMISTRY
.48 (85) Mitchell, H. K., and Haskins, F. A., Science, 110, 278 (1949). (86) Mtiller, R. H., and Clegg, D. L., ANAL.CHEM.,21, 1123 (1949). (87) Nachod, F. C., “Ion Exchange: Theory and Application,” New York, Acadeniic Press, 1949. (88) Xash, H. A., Quackenbush, F. W., and Porter, J. W., J . Am. Chem. Soc., 70, 3613 (1948). (89) Nicholson, D. E., A\ratzcrr, 163, 954 (1949). (90) Osborn, G. H., and Jewsburg-, A , Ibid., 164, 443 (1949). (91) Pacsu, E., Mora, T. P., and Kent, P. W., Science, 110, 446 (1949). (92) Partridge, S. M.,S a t w e , 164, 443 (1949). 193) Patton, 8 . R., and Foreman, E. M., Science, 109, 339 (1949). (94) Peterson, M. H., and Johnson, M. J., J . Bid. Chem., 174, i i o (1948). (95) Phillips, D. M.P., .Vaturr, 162, 29 (1848). (Re) Phillips, D. M. P., and Stephen, J. M. L., Ibid., p, 152. (!GI Pierce, J. V., Page, A. C., Jr.. Stokstad, E. L. R., Jukes, T . H., J . Am. Chem. Soc., 71, 2952 (1949). (981 Pinckard, J. H., Wille, B., and Zechmeister, L., Ibid., 70, 1938 (1948). (99) Plattner, A., and Nager. L., H&. Chim. gcta, 31, 2203 (1948). 1100) Pollard, F. H., McOniie, J. F. W., and Elbeih, I. I. M., S a f u r e , 163, 292 (1949). (101) Potter, V. R., and Heidelberger, C., Ihid., 164, 180 (1949). (102) Pratt, J. J., Jr., and House, H. L., Science, 110, 9 (1949). (103) Ramsey, L. L., J . Assoc. 0 8 c . Agr. Chemists, 31, 164, 441 (1948). (1041 Reichard, P., S a ? u r e , 162, 662 (1948). (103 Riley, F’. T . , Hesselbach, M. L., F’iala, S.. Woods. 51. W.. and Burk, D., Science, 109, 361 (1949). (10G) Roberts, E., and Tishkoff, G. H., Ibid., p. 14. (107) Robinson, D . A , and Mills, G . F., Ind. E ~ QChem., . 41, 2221 (1949)* (10s) Rockland, L. B., and Dunn, M.S.,Science, 109, 539 (1949). (109) Rutter, L., ,Vatature, 163, 487 (1949). (118) Sacconi, L., Gazz. chim. ital., 79, 141, 152 (1949). (111) Sacconi, L., Nature, 164, 71 (1949). f l l 2 ) Schubert, J., Russel, E. R., and Farahee, L. B., Scieace, 109, 316 (1949). i I:? Srhwarts, A. M.,“Surface Active .\gents,” New York. Interscience Publishers, 1949.
_--
(114) Schwartzkopf, O., Cahnmann, H. J., Lewis, A. D., Swilldisk). J., and Wtiest, N. M., Helv. Chim. Acta, 32, 443 (1949). (115) Sommer, H., et al., J . Am. Chem. SOC.,70, 1015, 1019 (1948). (116) Spaeth, E. C., and Rosenblatt, D. H., Science, 110, 258 (1949). (117) Stein, W.H., and Moore, S., J . Bbl. Chem., 176, 337 (1948). . 21, 75 (1949). (118) Strain, H. H., ~ N A L CHEM., (119) Strain, H. H., in “Frontiers in Chemistry,” 1-01.VIII. 1 ) ~ ) 29-63, New York, Interscience Publish?rs, 1949. (120) Strain, H. H.. J . Am. C‘hem. Soc., 70, 588 (1948). (121) Ibid., p. 759. (122) Ibid., p. 1672. (123) Street, K., Jr., and Seaborg, G. T., Ibid., p. 4268. (124) Synge, R. L. M., and Tiselius, A., Acta Chem. S c a d . , 3, 231 (1949). (125) Tiseliusl h.,Arch. Kemi Mineral. Geol., 26 B (1) (19481. (126) Tiselius, A., Chem. Eng. S e w s , 27, 1041 (1949). (127) Tishkoff, G. H., Bennet, R., Bennet. V., and Miller, L. I. .. Science, 110, 452 (1949). (128) Tonipkins, E. R.. Harris, D. I%., Khyni, J. X.. J . A m . Chem. Soc., 71, 2504 (1949). (129) Trueblood, K. IT., and Malmberg. E. W,, .lsa~. C‘HEX., 21, 1055 (1949). (1301 Cpdegraff, I. H.. and Cassidy, H. G., J . Am. ChPm. S o c . , 71, 407 (1949). (131) Urbach, K. F., Scieuce, 109, 259 (1949). (132) Wald, G., Ibid., p. 482. (133) Weiss, D. E., S a t u r e , 162, 372 (1948). (134) Wender, S. H., and Gage, T . B., Science, 109, 287 (1949) (135) Wendler, N. L., Slates, H. L., and Tishler, M., J . Am. (‘hem. Soc., 71, 3267 (1949). (136) White, M. F., and brow^, .J. B., Ibid., 70, 4269 (1948). (137) Wieland, T . , arid Fiwliel., E., ~~ratIirwissenschaften, 35, 29 (1948). (138) Winsten, W. A , . and Kige~~, E,, J . Am. Chem. Soc.. 70, 3333 (1948). (139) Woiwood, A. J., and Linggood, E’. F’., Xuture, 163, 218 (1949). (140) Wolfson, W.Q . , Colin, (’., and Devaney, W.A., Science, 109, 541 (1949). (141) Wynn, V., Suture, 164, 445 (1949). (142) Zechmeister. T,., arid Pinckard, J. I T . . Esperientio. 4. 474 (1948). R E C E I V EXoreniher D 18. 1949
PAPER CHROMATOGRAPHY DORIS L. C L E G G Washington Square College, New York University, ,Vew York,N . Y. 66 A good technique sometimes renders more service to science than the elaboration of highly theoretical speculation.”-Claude Bernard
T
HI- stored in a small space, ready for instant use; can be mounted a s permanrnt records ( a spray of artist’s “fixative” preserves colors against fading too rapidly); photographed wet, if drying causes change or loss in colors of unknowns (148) or interferrnce due to incarease in hackground intensity (77, 148); or photographed dry (143, 14.7) for preparation of slides or simply to store for future referwce. Purity of substance or the presence of unsuspected const,ituents may he easily determined (104). It is also inexpensive; one 9-em. circle n-ill produce one complete separation using the Rutter technique ( 1 7 3 ) or six if the ascending or descending paper strip is employed; or 450 individual chromatograms per day can be accompliPhed using a series of concentric cylinders as described by Williams and Kirby (207). Paper chromatography is useful as a control of synthesis in industrial processes, because rapid q d i t a t i v e analyses frequently can be macle employing this simple b u t extremely effective method. T h e extensive literature devoted to paper chromatography ( ~ V P I Ito
V O L U M E 22, NO. 1, J A N U A R Y 1 9 5 0 has been summarized in several review articles (23, 32, 36, 37, 43, 60, 200, 135, 149, 157, 183, 195, 207, 208-211, 229), perhaps the best and most recently available of which is that of Rlartin (127). This article is beautifully organized and extremely well written, covering the development,s in both columns and paper to about January 1949. A collection of papers covering recent developments in the columns and paper is heing published in the Discitssions oj the Faradnu S o c i d y (60). -4great deal of ingenuity has been evinced in the various niet,hods of handling the paper and in the form or shape of the starting sample: large sheets of paper (81) standing in 5-gallon crocks: strips of various sizes (117, 1 4 4 ) ; 13.5 X 1.8 X 1.0 cm. strips ivhich aro cut narrower at one end, so the wider part fits snugly in a test tube antl the lower edge dips into the solvent ( f 7 l ) ; largcd 2:3 x 23 inch sheets stapled together to stand upright by theii, own sturdiness in a 20-gallon st,ainless steel tank (2274); and the ri~niarltablyeffective Rutter tcchniyue using circles or squares Ivith a \\-icklilw tail dipping into the solution (17 3 ) . Filter paper in various forms, has been utilized as a column ads:orlr)ent: as pulp (28, 205), as powdered cellulose (.34, 153), as powdered IYhatman ashless filter paper tahlets (-94), as mixtures < i f charma1 nntl pulp (205), as cellulose accltate (19), a n t l as strips for dwcending chromatography, producing a "flowing filter papt'r chrom:ttogram" by allowing the solvent to drip off the lower c ~ l of p t h v paper (152, 154). h recent development is the use of filter paper (400 to 450 sheets) with the sample absorbed by 20 to 2.5 s h w t s which are placed on top of the pile: the n-holr is clamped together hrtween perforated stainless steel plates to form an exc*chllent,column (138, 139), and eluted as such. This is really an adaptation of the cotton fahric disk mcxthod of Lenoir (112). One invrstigator has utilized previously boiled string, cotton thread, or mercrrized cotton thread ( 1 1 7 ) ) glass wool, and thin asbest os paper which form internally supported columns of very .;mall cross section to acconiplish descending and ascending separations on a definitely micro scale. A slight modification of t,hc descending paper strip method is to attach a thick pad of cellulosr a t the foot of the strip to serve as a collector of the solvent; the - analysis ( 9 5 ) ; (4) a n aqueous suspensiori of alumiiiuni h\-droxidc to separate oxine compounds of aluminum, cadmium. ;;iiic, magnesium, calcium, zirconium, and beryllium (82); (%?)I)qiiizc,l alumina and formaldoxime hydrochloride for a ferro m a n g n n t w spot test (90); ( 6 ) 20'7;) phosphat>ebuffer of p H 6.2 for the resolution of penicillin broths (99); ( 7 ) h y d r a t d oxitlev c ) f aluniinuin, silicon, or chromium used for inorganic chromatography (;f ); ancl (8) starch and fuller's earth for separation o f vitamin A constituents (48). All these are incorporated int,o the. p i r e s of the filtrr paper fibers to complete the st,ahle phase. \.arious methods of evaluating the results have been used, depending on the conipounds being investigated and the facilitie8 of the laboratories concerned. Sometimes the completed chroniatogram is sprayed with a solution to dtlvelop a color and the color is thcn coniparctl photornrti~ic~iilly with a known standard: Rav-
49 enoid pigments (206); sugars sprayed with ammoniacal silver nitrate (26)or ammoniacal silver nitrate with resorcinol and naphthoresorcinol t o identify reducing and nonreducing sugars ('73); benzidine reagent to distinguish between sugars and urates (91); benzidine, ammoniacal silver nitrate, aniline-hydrogen oxalate, or phloroglucinol to identify reducing substances ( 9 2 ) ; reducing and nonreducing carbohydrates and derivatives analyzed by spraying with ly0 aqueous potassium permanganate containing 2% sodium carbonate (148); or sugars are quantitatively weighed (10) and analyzed by hypoiodate oxidation ( 8 5 ) . Sugars ( 7 0 ) and amino acids (156) have been definitely identified by their optical rotation. Radioactive sulfur (190,196),iodine (61,188, 189),and carbon (11, 6 3 ) are first separated from their various compounds on paper and then their positions are det'ermined and quantitatively estimated h y the use of Geiger-Muller counters, or by laying the paper chromatogram on a photographic plate, when a radioautcgraph results. Visual examination revealed the position of t h r components of many mixtures after chromatography oIving to their brilliant colors (ascorbic acid, 124). Zones were detected tiy observing changes in the refractive index of the solution in contact with the part,icles of the adsorbent, ( 3 4 ) . Two glass windows are clamped lietween a U-shaped metal plate and the paper is placed under the thicker polished nindoiv. When observed a t the proper angltb and at a suitable distance, the boundaries of zones are easily seen. Bioautographic study is another extremely useful tool for the quantitative determination of various separated components as in vitamin BI2factors (46, S l 4 ) , vitamin Bg ( 2 f 7 ) ,arid alternate fa?tors capable of supporting growth of L. citromruni 8081 (217). Here, the paper is usually cut in strips, soaked in the solution being tested, and then pressed on an agar plate seeded with t,he selected organisms (46, 214, 215, 217, 218). The paper can also be cut into tiny squarrs a,nd placfd in a tuhe of hactcrial culture (227). X-ray and electron diffraction patterns ( 1 6 4 ) were used to identify various amino acids after they viere leached out from the complet'ed chromatogram (33). The formation of a copper phosphate complex (165) permits identification of amino nitrogen by evaluating the copper in the filtrate. One to 25 micrograms of glycine and valine were determined by this niethod by decomposing the copper complex with diethyl dithiocarbamate, extracting the yellow color with amyl alcohol, and measuring the color against standards (222). 01.imino acids can be neatly determined by absence, because they form copper complexes which do not move with the solvent. The paper is dusted JT-ith powdered basic copper carbonate as a streak along the future path of the first solvent (phenol). The mixture of acids is placed on this paper as usual and also on another paper without the copper compound, and then the two are chromatographed together as two-dimensional chromatograms, using as solvents phenol and then collidine-lutidine. One chromatogram shows spots for each component, but the paper containing the or-amino acid-copper complexes slioi~sspots for all components except thc 0-amino acids, vhich remain at the hottom of the paper because they do not move witah the solvent. Their positions are then found by difference hy mmpxring with thr. standard chromatogram (4.5). Another variation on t>hcuse of copper complexes of amino acids is to develop and dry the chromatogram as usual and then paint with freshly prepared 10% solution of aqueous ferrous ferrocyanide. The pink color of copper ferrocyanide betrays the position of the acid (lysine, 1). Amino acids are usually separated by two-dimensional chromatography and, after drying, the chromatogram is sprayed with ninhydrin solution, which produces various shades of blue or purple in a characterist,ic sequence of spots on the paper ( 3 9 ) . This pattern is so definit,e that some investigators use the relative position on the paper to identify the components present (119, f 7 0 ) . The RP value is the ratio between the distance traversed 1)y the component and the distance traveled by the solvent.
ANALYTICAL CHEMISTRY
SO Inorganic compounds can be separated bj- paper chronilttography and then eluted, or the organic matter can be destroyed and the metallic content determined by use of the polarograph, spectrograph, or colorimeter ( 3 ) . Gltraviolet light is very successful in determining the position of many compounds which fluoresce: pterins (80),oxine compounds of aluminum, cadmium, zinc, magnesium, calcium, zirconium, and beryllium ( 8 2 ) , amino acids arid peptides ( I % ) , purines and pyrimidines ( % I d ) ,and nucleic acid derivatives (125). T'iewed under ultraviolet light, the pale yellow glow of dinitrophenyl amino acids and peptides serves to identify the positions even if it is not a true fluorescence (160). I'urine and pj-rimidinc, derivatives do not fluoresce but quench fluorescence so that, if the). are run using as solvent n-butyl alcohol which does fluoresw, dark pat,ches appear where components are, against the bright background of the paper. Here the al)sence of this p r o p r t y is skillfully turned to account to analyze unknonri suhstancrs. This same method is applied to various cations; tht, chromatograni is sprayed with selected reagents, such as morin, producing spots, some bright, and some dark through the quenching of fluorescence (24, 162). 8-Hydroxyquinoline (alcoholic) sprayed on a chromat,ogram of aluminum and beryllium produces fluorescenw of each, so that their positions are easily t.stttt)lished (147).
siniple, easy, arid extremely effective. One to 300 microgram. of the sample mixture are placed 2 to 3 cm. from one corner of :i large sheet of filter paper. It is developed with a solvent, usually phenol for amino acids, taken out of the atmosphere and dried, rotated go", and inserted into a solvent of somewhat different properties, such as s-collithnr or a collidine-lutidinr* mi\ture.
a
. O.D. A
B COURTESY. SCIENCE
Figure 2. Triangular Cross Section of Glass Trough C O U R T E S Y . SCIENCE
Figure 1. Diagram of Glass Trough L e f t . Before cutting R i g h t . Completed channel
Acidic peptides are separated by ion eschaiige resiris, followed by ionophoresis, which separates peptides according to their charges and molecular weight,s into groups having similar mobilities in an electrical field. Then these groups are examined by two-dimensional chromatography (42). Xineteen tlifferent dipeptides of aspartic and glutamic acids wercA identified in a hydrolyzate by this method ( 4 2 ) . .I sperial elutioii inr~thodn-xs d e s scarilxxl (49). Paper chromatograms are also quantitatively evaluated photoelectrically in the case of amino acids (27, 7 6 ) . and metals (136). These results are plotted against the area and compared with siniilar plots of standard acids to get the concentration of the unknown ( 1 6 ) , or measured with a planimeter, and the areas thus obtained are plotted against the concentration (27, 66, 68). The transmittance of monochromatic light by mixtures of dyes has been automatically recorded during the entire process of development (142-14&). Reflectance values of completed, dried chromatograms of mixtures of dyes were automatically recorded (145). Transmittance values are more accurate than reflectance values, because they follow Beer's law more closely ( L I Z ) . Reflectance records taken in this manner have limited value in accurately establishing the location of bands, but in a photometric sense are inferior to transmittance recordings. Alternate methods of continuous scanning, including cathode ray tube scanning, have been discussed (146). A t present, all amino acids cannot be separated by one solvent, but an excellent beginning is accomplished hy using two-dimensional technique and then cutting out the mixtures and further eluting in a one-dimensional chromatogram. The two-dimensional chromatogram is of widespread importance, because it, is
Amino acids occupy definitc', caharactri%tic positions on a111 t nwdimensional Chromatogram (41),and "maps" have been prcpared shoxying the sequence of their positions. Inasmuch ab, originally, they are colorless and, therefore, invisible, the papvr is sprayed with ninhydrin to make a blue to purple spot tiepending on the amino acid concerned, which becomes visiblr 011 heating with steam. The ninhydrin is prepared as 0.1 t o IC; solutions in xater, butanol, or butanol-acetic acid. However! the procedure is not completely specific for amino acids, bccauw any substance containing free amino anti free carboxyl groups will produce the same result. The solvents used for amino acids ai'(usually (1) phenol or butanol-acetic acid, or (2) collidine or c,ollidine-lutidine. Sugars can be successfully chromat,ographetl using the same two-dimensional tcchnique and even the same solvents; however, benzidine or ammoniacal silver nitrate ic used to produce the color. The choice of a developer or solvent is empirical because, as yet, there appears to be no accuratc method of predicting how a suhstancc%will act with a gircii ho lven t. I t is reported that the rate of movement of solvent is about the same, whether ascending or descending methods are used. The exact ratio of change in width of paper to change in rate ha8 iiot yet been definitely established. The paper has pores, which should not be too coarse, but fine and firni-the coarser the paper the faster the rate of flair-. An increase in temperature also causes an increase in movement of solvent and, therefore, of the ions, pH seems to affect the separation both as to completenes6 of separation and sequence of conlponents ( 5 7 ) . The presence oi other ions usually affects Rp values, but not in the case of penivillin and amino acids (182). APPARATUS
A glass trough to hold the solvent, which was designed by Longenrcker (118), is shown in Figure 1; it is easily and inexpensively made from chemical resistmt glass with the aid of adhesive
V O L U M E 22, NO. 1, J A N U A R Y 1950
51
Another glass trough (4jwas made from chemical resistant glass tubing with only the assistance of a gas-air blowpipe and a 6inch file. Mechanical details are given in the original paper. of the technique, the original An apparatus for scanning onedimensional paper chromatopaper should be consulted. grams was reported (76)in which the amino acid paper chromatoThis simple piece of equipgram, after development with ninhydrin, was passed between a ment was quickly p u t to light source and a phototube (Figconstant use, with the imure 4). Rp values and the quantit.y mediate result t h a t i t W&S of unknown were determined by the modified for greater PBSB of use of transmittance values. construction a s shown in FigFor investigators who are anxious ure 2 (181). The main imto collect the effluent uniformly and provement is t h a t the trough gradually, there is a rotating drum is now composed of sheet method which permita 20 spots of metal with a V-shaped cross effluent to be touched to the filter section with welded end plates paper per revolution of the drum and coated with resistant (58). Because about 10 revolutions enamel to eliminate breakagu elapse before the chromatogram is of t,he glass. properly developed, some 200 spots Another apparatus which will have been added a t regular enables one to chromatograph intervals from the graduated syringe. several paper strips simulBecause i t is both tedious and taneously (BlS) is composed time-consuming to develop one of n. Petri dish. Figure 3. Apparatus for chromatogram st, a time, a setup has Both halves face upward One-Dimensional Paper been reported which permits the and are connected by fusing Partition Chromatography GO glass rod, which is wound simultaneous development of many 1. Stninloss steel wire guard with absorbent cotton to nhrornalnorn.ma I\ -I171~ Tha s,nng.~ ". ...-..~. _ ~ . -.,- ~ ----~ -rrratus is shown in Figure 5. A method ( 9 8 ) for treating paper with silicon esters renders the paper impermeable t o water for 11 hours and also causes considerable resisb anee to alkaline solutions. This might well he of use in the formation of barriers and restricted chaunels on filter paper for chromatographic separations. A new analytical technique for Figure 5. Apparatus for Ascending investigations, of anions and cations Filter-Paper Chroby electrochramatography was re3 shom-the a$parXtua as used rnnt"eramS ported in which adsorption is effected with the aid of e.m.f. (107) applied to a column. This might be a fruitful field of investigation for paper separations, especially of inorganic ions. A special pipet, containing a platinum wire of a bore just fitting inside the capillary, permits the deposition of approximately 1 ml. per hour of the mixture being analyeed. It was reported that, beesuse the solution evaporates from the p q e r strips at 60' C., the wet spots attain only 2to 3-em. diameter a t any time, thus concentrating the unknown (199). Another method of making the chromatography of amino acids quantitative consisted of plotting per, cent transmittance on semilog paper against distance along the filter paper strips and messuring the areas under the curve with a planimeter. These transmit,tance values were obt.ained by mounting paper on a slide and passing i t between a n illuminated slit, 6 X 5 nun., and a phototube (#7). A light filter wit,h maximum transmission at 570 mp Figure 4. Photoelectric Photometer for Evaluating Paper Stzip Chmmatowas placed in front of the slit. grams tape in strategic positions.
For the detailed exposition
52
ANALYTICAL CHEMISTRY
’I
11.8 CMS.
200 NUMBER
600
400
OF F I L T E R P A P E R
800
DISKS COURTESY, SCIENCE
Figure 7
A method for producing a column Figure 6. Diagram of Filter-Paper from 400 to 450 Pile Column filter paper d i s h 1. Connection for rubber tube for filling siphon (139) was desig2. Connection for siphon tube 3. Rubber gasket nated by the 4. Filter paper disks containing sample authors as a “chro5. Stainless steel plates 6. Perforated stainless steel plate m a t o p i l e , ” as 7 . Filter paper disk pile 8. Wing nuts shoiin in Figure 9. Bolts a t four corners of steel plates 6. Results of an experiment shoxving the separation of compounds of a knotvn mixture are shownin Figure 7. The length of the horizontal bars used for experimental points indicates the number of paper disks extracted for each analytical sample. A simple piece of equipment is composed of two long stainless steel rods nhich connect t n o stainless steel coils (125). The paper is held by the coils, as shown in Figure 8. -4simple accurate device for measuring RE’values was reported (168) by nhich a scale, divided into 100 parts, was printed on white elastic. One end of the elastic Mas fastened to the end of an aluminum stiip and the other end to a bulldog clip. I n use, the zero mark is placed a t the origin line or spot and the 100 mark stretched out to coincide with the position reached by the solvent front. I?,- values of the spots are then lead off directly from the scale. An apparatus was reported (143, 146) for automatically recording the transmittance of monochromatic light during the process of developmrnt of paper chromatograms of dye mixtures b) capillary ascent. The dye mixture a a s deposited a t the bottom of a restricted channel and the eluant was added a t a point below. The monochromatic light mas focused a t a point above the unknown mixture. The transmittance was picked up by a photomultiplier tube, amplified, and then recorded automatically and continuously by a Brown recorder. The results were reproducible and accurate to + : w An entirely different approach ro:.a. ‘c~dof automatically and * ng the entire process continuously scanning the paper strip of development by the cathode ray scanning technique and presenting the results continuously on a cathode ray oscillograph (146). An alternative method for evaluating a completed chromatogram has been devised in \% hich the paper strip is wrapped around a drum and affised in position with Scotch tape. The drum is COURTESY. SCIENCE
I
I
COURTESY. SCIENCE
Figure 8 rotated a t uniform d o n speed by a synchronous motor. A lamp, lens, and filter SJ stem project a narrow line of light on the strip and the reflected or transmitted light is picked up by a phototube connected to a Brown Elcctronik recorder (141). An example of the information so obtained is shown in Figure 9. I n this case, a disk chromatogram of two dyes was developed by the Rutter technique ( 1 7 3 ) . A diametral strip was cut from the finished chromatogram and mounted on the scanning drum. Reference lines were drawn in pencil: one through the origin or original sample site, and two others equidistant from it, well beyond the confines of the pattern. I n Figure 9 on either side of the central reference line, a band appears representing the location of the ring of blue dye. Beyond these, there is a pair representing the ring of red dye. Finally, there are the outer reference lines. These results were obtained by reflectance. For a n evaluation of concentration they are inferior to results obtained by transmittance, but they afford quantitative information on zone location, zone width, and symmetry. I n this example, there is a notable lack of symmetry M hich would not be a t all evident on simple inspection. If the maximal excursion of the solvent is marked with a pencil line before the solvent disappears by evaporation, these autonlatlc records provide an accurate measure
V O L U M E 22, NO. 1, J A N U A R Y 1 9 5 0 ot R2.values. The sranning can bc rrpeated over and over with(rut attention from the operator.
THEORIES
.A few theories have been advanced t o explain the mechanism involved, the kinetics of paper chroniatography, and methods of determining quantitatively t h e amounts of the various components. Howevt,r, the time has not yet arrived when a ne\v substance can be plac,c~lon a paper, a developer added, and the rwults accurately pi,ctiicted. The first, formula of any importance to papthr chromatograph>- originated with Flood (69), who rc1ati.ii t h c zonc' raclii t o concentrations
r = f(log c
+ '-1 )
\vhere r is zoiie radius, c is concenttxtiori, atrd J is a constant for the paprr. Hopf (RO) tested this formula using filter paper treated with an bent, and found t h a t Flood's formula was applicable in most cases. H e also derived an equation t o denote the relation t)c>t\veeri two constituents, A and H , of a chromatogram. On tlevelopni(Jnt,the ring for A is bounded b y radii rI and rl, and t h r ring for H is bounded b y radii r3 and r i . T h e initial solution is of known concentrations, a arid b, rrspectivrly. Then a = (+f i i ) K a ,and h = (T': - i i ) K b , and values for lia and Kb can t w calculated. Also
\\here ti,, 6 = ICa/& Glueckauf (79) determined complete isotherms within * I ( is the area of the cross section of the niobile phase, A , is the area of cross section of t h e nonmohile phase, and T is the n u m h of theoretical plates the first solute has passed. Rutter (174) reported that the net resultant flow of solvcwt through the capillary channels of the paper used in chromatoyraphy is in a direction away froni the point of feed and, therefore, sets u p a streaming potential tvhich he measured with a. Cambridge pH meter. The potential gradients listed varied froni 0.0+ t o 10 mv. per cm. The rate of flow of liquid and the nature of the electrodes used in making the measurements appeared to affect the results. Some inquiries into the kinetics of paper chromatographic development, resulted in equations expressing the relation between RF and thc radii of successive zones (144).
~v1wr.er r is zone radius and r1 i.5 solvent radius. I n spite of the efforts of thrse investigators, the theoretical foundations of paper chromatography are in an extrrmely elementary state. In practice, the few generalizations that are the basis for predicting the optimum conditions for a new problem are well nigh useless. Even the selection of appropriate solvent, mixtures is highly empirical and no guiding generalizations are known. \l.e may hope that, in the near future, cBarefully controlled kinetic studies will clarifj- the role played by each of the variables, so that one may devise separations with greater certainty and with less recours(' to empirical trials. APPLIC4TION S
0
0
Figure 9. Automatic Record of Completed Chromatogram of Dye Mixture b y Keflectance
The applications of paper chromatography are primarily analytical and their greatest advantage lies in the minute amounts of material required
54
ANALYTICAL CHEMISTRY
tor examination. The separation of the components of a niixture by diffusion through paper permits qualitative identifiration on the basis of the preferential rate of diffusion. L-nder carefully controlled and specified conditions, the R F tactor, u hich represents the ratio of the distance traversed by the substance to that traveled by the solvent, establishes idrntification. When a pair of components have alniost identical R F values, they may be resolved by further developnient trith another solvent in a direction perpendicular to the first. ThiP process is called “two-dimensional chromatograph! ,” and is being applied to clai ify numerous analytical tangles. 11ORGAhIC IPPLIC ATIO\S
Schoenbein (176, l??), studying the condition5 01 ozone rormation under the influence of electrical discharges, observed *elective adsorption of the components of a mixture by the different heights to which they rose when a strip of filter paper as dipped into the solution. This was the inception of filter paper chromatography, m hich he called “capillary analysis.” Trey (198) first achieved separation of inorganic ions by this technique in 1898. After this time, many years elapsed before any further work was done on paper chromatography until Gordon, Martin, and Synge inaugurated two-dimensional chromatography for the separation and determination of amino acids. Because this technique n’as eytremely effective and equally simple, it spread immediately throughout the scientific norld, and soon many other substances besides amino acids were !wing separated, analyzed, isolated, and purified by this method. Paper impregnated with aluminum hydroxide was used to Deparate inorganic ions by ion exchange (69, 95). This method mas further extended t o the identification of several cationsaluminum, cadmium, zinc, magnesium, calcium, zirconium, and beryllium-as the 8-hydroxyquinoline derivative, the osine. These oxines fluoresce under ultraviolet light, thus indicating their position for further identification (82). Chromic hydroxide caused even better separations than aluminum hydroxide (82). i-arious applications to metallurgy have been listed (154). The use of radioactive tracers has greatly widened the field of applied paper chromatography, because much less than the 1 microgram required for color reactions is necessary for quantitative determination of such materials. Mixed chromatograms \\ ith nonradioactive material (known), in which colored and bioautographic spots correspond in position and shape, definitely identify the tiny tracer accurately. Substances containing radioactive isotopes were separated on one- (189) and twodimensional (11, 61. 65, 188) chromatograms and quantitatively ebtiiiiated by the use of Geiger-Xuller counters (189) 01 by laying the developed chromatograms on a photographic plate and pro(luring a radioautograph ( 6 1 , 6 4 , 161, 1?1A, 193). Rats were injected \\ith and killed after 24 hours, their thyroids were extracted, and the extracts 15 ere chromatographed. .is a result, it was reported that diiodothyroxine and inorganic iodine have activity, but thyroxine has almost none ( 6 1 ) ; the large amount of 1’31 found in inorganic fractions suggested that an appreciable breakdown of organic iodine occurs during hydrolysis because the inorganic I I 3 l that n as initially present 111 the tissue had been removed by treatment n i t h trichloroacetic acid (189); the rate data indicate that monoiodotyrosine 1s a precursor of diiodotyrosine (188). C’14 was administered to Chlorella as Ci4O2 t o determine the dgnificance of the form of compounds in photosynthesis (63). Plant respiration intermediates m r e studied using C14 as a label. The isolation and identification of C14-labeled phosphoglyceric acid from the alga Scenedesmus which had photosynthesized for 3 seconds in C l i 0 2 were reported ( 1 1 ) . Radioactive tracers and paper chromatography u ere used (290) to show that cystine and methionine are important sulfur compounds in alfalfa leaf proteins. Radiomethionine was administered orally to a rat, followed by the injection of benzene
to study the benzene detoxification products (196). The paper chromatogram was read automatically by synchronizing a constant-speed motor pulling the paper strip slo~vly under the Geiger counter, with a tape recorder n hose pen was motivated by counting impulses from the scaler. Lederer (108) separated antimony from all the common metallic ions by acidifying the solution with hydrochloric acid, chromatographing the filter paper strip, developing with water until the chromatogram traveled 10 em., and then adding hydrogen sulfide. This produced a characteristic orange patch of antimony sulfide close t o the original spot, since antimony barely moves with the solvent. He also reported the separation of chloride, bromide, iodide, and thiocyanate ions, using as solvent butanol saturated with 1.5 N ammonium hydroxide, which keeps the halides in ionic form and prevents adsorption on the paper. E F values m r r quoted (111). .Uuminuni and beryllium mixtures were sepaI ated 1%ith 807, n-butyl alcohol-200/o concentrated hydrochloric acid and determined by sprwing x i t h 8-hydroxyquinoline and examining under ultraviolet light, because their oxines fluoresce (147). Lacourt et al. (103, 105) quantitatively determined iron, titanium, and aluminum as chlorides and also cobalt and nickel as chlorides and nitrates. They reported that they could determine 10 micrograms of iron, titanium, aluminum, vanadium, nickel, and cobalt in methyl, ethyl, propyl, isopropyl, and isoamyl alcohol as impurities (104). Burstall et al. (28) proposed methods for separating several cations with ketones. Gold, platinum, and palladium were separated from each other and from iridium or rhenium using methyl n-propyl ketone containing 30% concentrated hydrochloric acid. Chloride solutions of Group I I a (the copper and tin group) n ere separated with ethyl isopropyl ketone containing 1 0 7 concentrated hydrochloric acid. Group I I b compounds were resolved with dry ethyl acetate previously shaken \Tith 2% hydrochloric acid. Xickel and cobalt in steel and minerals were separated and determined on cellulose pulp columns which had been activated by boiling with dilute nitric acid. Scandium was separated from many other metals, including the rare earths, with the salts as nitrates and using ether containing 12.5’30 nitric acid as the solvent by washing through the column of the cellulose pulp. Calcium, barium, and strontium, as chlorides, were separated by an organic reagent, pyridine, containing 4% potassium thyocyanate as the solvent, and their positions were indicated by the use of sprays (5). Aluminum, gallium, indium, and zinc chlorides were separated with n-butyl alcohol containing hydrochloric acid ( 3 ) . The same authors also separated and identified vanadium, mercury, cobalt, copper, iron, manganese, and nickel. Another method of separating the noble metals in aqueous solutions n as reported (109); gold, platinum, palladium, copper, and silver were identified by their different RF values and colors, and also their colors produced with hydrogen sulfide in an atmosphere of ammonia. In this case, an interesting separation occurred in the solvent of butanol saturated with 2V hydrochloric acid. TKOliquid fronts appeared during the development, the first butanol and the second the aqueous phase, which was used as the reference for the RF values. AIercury, silver, and lead chlorides Tvere resolved with 5 S ammonium hydroxide and identified as sulfides by passing hydrogen sulfide vapor through the paper. Lederer (109) separated divalent copper and cadmium with 25q0 ethyl alcohol as the developer. Lead x a s separated from divalent copper, cadmium, and mercury and from bismuth by developing with 1 S aqueous hydrochloric acid, since lead does not travel with the liquid front (110). Cations (24 of them) were separated using various solvents and then the spots were located by hydrogen sulfide vapor or by fluorescence, produced by spraying with a selected reagent, such as morin, drying, and then examining under ultraviolet light. Some cations fluoresce, giving bright spots, and some quench the
V O L U M E 2 2 , NO. 1, J A N U A R Y 1 9 5 0 Huorescwce, yielding dark spots (162). Liiistead (1168 ) stated that the rate of movement of any metal ( R Fvalue) may be by the presence of other ions. Separations of 45 metals and anions, by successive changes of the extract,ing medium, were performed in his laboratory (not yet published). The metals \\.ere dissolved in a solvent, usually an acid, and the individual rnenibers determined 1)y sperific reagents or other conventional means. SUGAR SEPARATIONS B Y PAPER CHROMATOGRAPHY
Simple sugars were separated and detected by ammoniacal silver nitrate ( 7 3 ) ; and in two-component mixtures by using butanol-acetic acid-water as the solvent and aiialyzing by hypoiodate oxidation (8.5). Flood et al. standartlixrd a procedure ( 7 0 ) and were unsuccessful in resolving &sugar mistuws by an optically active alcohol. Benzidine reagent was used to separate reducing sugars from urates (91). Sugars u ere also separated with collidine as the solvent (153); bj- spraJ-irig nith a special reagent of aniline, phthalic acid, and water-saturated butanol, which produced red colors from aldopentoses and greens and broivns from aldohexoscs and uronic acids ( I & $ ) ;using E:hrlich's aldehyde reagent for amino sugars ( 2 ) ; in aspenwood, spruce, and various coniferous pulp fibers by partial hydrolj resulting in mannose and glucose (219); and in sevrral types of pine trees, where they were found to he arabinose and sometimes fructose (220). The structures i ) f nit&ylated sugars \vet'e investigated (Y), :tiid i t was shoivii that methyl irisin contains an appreciable fraction of trimethyl fructoses. These sugars were separated ( 7 1 ) on coluniiis of powdered cellulose (Yd), and quantitatively identified by Rc values (10). They were separated like simple sugars and sprayed with ammoniacal silver nitrate to produce I I I Y ) ~spots, ~ which provided a method of rapid examination of hydrolyzed products obtained from small quantities of methyl Idysaccharides (26). Reducing and nonreducing carbohydrate iterivatives were separated and identified by spraying n i t h 1:; aqueous potassium permanganate containing 27: sodium carIionate to produce yellow zones on a purple background ( I @ ) , :it id those in urino were identified by spraying with benzidine, ammoniacal silver nitrate, aniline-hydrogen oxalate, or phloroglucinol (92). Sugars present in urine ivere identified as arabiiiose, xylose, and fructose (55). 1Iethods of st,udying the sugars present in apple juice, egg white, ailti the fetal blood of sheep (150) and for the examination ( i f blood group -1specific substances from hog gastric mucin and the specific polysaccharides of B. dgseriterim (Shiga) (151) were described. The carbohydrate metabolism of microorganisms was studied using sucrose which was converted into dextran by LPitcorLostoc ?nesenteroides (Leister Institute Strain N o . :3351) (95). The explanation offered was that during the course ( i f the metabolism, large quantities of reducing sugars are formed and the glucose part of the sucrose is polymerized to dextran with simultaneous liberation of the fructose moiety. Sucrose was detected with rewrcinol and estimated after preliminary hydrolysis with oxalic acid. Changes in the sugar content during the growth of the pea were studied by paper chromatography (56) and histochemical reagents were used to tiemonstrate pectin, starch, cellulose, and lignin developmen I, iPPLICATIONS TO MEDICINE AND BIOLOGICAL CIIEJIISTRY
One of the greatest fields for the application of paper chroniatography is in medicine and biological chemistry. The amino acid constit'uents of diphtheria toxin and toxoid hydrolyzates were determined (225') by two-dimensional chromatography, and it was found that tyrosine is present in toxin hut not in the toxoid. I t s usefulness in a routine method of preparing thc toxin from the papain digest (116) was proved. An examination of the chemical nat,ure of purified liver extrart (194) showed the presence of peptide material, free amino acids, and riboflavin ; the percentage of total activity was also studied (178).
5s
Sucleic acids have been studied (Id.?, 202, LO4) and deteriniiit.,l quantitatively both spectrophotometrically (125) and by u l t i w violet absorption (202). Purintls were detected by precipitatiiiil of mercury and conversion to a sulfide. Vitamin A was separated on alumina-treated filter paper and identified with the Cam-l'ricta reagent (47). Sulist,anct:s related to ascorbic wi(1 (vitamin C ) were resolved on paper in several different solvetit-. and then identified with 2,(i-dichlorophenolindophenol,which produces a dark blue background around colorless spots ( I & J . The various vitamin B's have attracted considerable attention (46, 83, 199, 214, 216, 216). T h e microbiological assay n 3 l i paper chromatography has heen used for the study of vitaniiil 136 (21.5) with Saccharomyces carZsbrryensis 1228 on agar ani1 i ) f vitamin HI:! with Lactobacillus leichirinr~nii313 (214) and Luctohtrcillirx ltrctis (Dorner) ( 4 6 ) , wit,h special attention to variou.* relatecl growth factors (179, 214, 216) and their effects on 1 ) : ~ terial nir~taholism. The riboflavin decomposit,ion products \vew shown I)), a paper chromatogram of irradiated samples of i , i l i [ ~ Havin and examined under ultraviolet light, under which thy! shoived a ydlow fluorescence ( 8 3 ) . Penicillin brot,h was chromatographed on paper impregnal i d with 205; phosphate buffer a t pII 6.2 and quantitatively c1rAtei.mined by t,he paper disk plate assay method. Eight penic-illill? were separated from Penicilliuttt chrysoyeriurtz Q176 ( 9 9 ) . Stu(lic+ of two and three carbon compourids containing sulfur and si11I hydra1 groups showed no definite evidenre that the ratio of p t s i i i cillin formed is changed by their presence. Paper chromatography of proteins and enzymes ( ; 7 ! I . < A . 169, 192) shoned that enq-rnes are strongly adsorbed by pap(.i at salt concentrations far heloa those required for precipitatioii, and t,hat their movement is also influenced by the preseiicc, 0 1 other proteins, a form of displacement adsorption (192). ('onsiderable enzyme resolution (138) and protein fractionatioii (191) can he attained by the use of a filter paper chroniatopilv by salting-out, adsorption and adsorption displacenient. 1J1i affects RF valurs because niotion is almost nil to pH 5 and t l i t v l it increases (97)>and benzidine reagent brushed on the paper cau colors useful in identification. Some interesting mic,rohiologic~iI assay investigations were perfornietl wing Lcitcortostoc ? d / ' O / ' f J / ' / m t (4.5, 2 1 7 ) and some bioautogi,aph methods. in tvliich it iva> discovered that thymidine and possibly three or four i ~ t h c ~ i ~ growth factors \\-ere prcsent (217 ) in natural materials; that thyniidine responds to ail alkali-stable growth-promoting factor in concenti~atedlivrr extract aiid not to R,? (2.5): and that it possible functional relation exists Ihtv ~ ' citrovorum n factor anti thymidine and pteroylglutamic arid-if used alone, growth \vas not seriously affected, but if uscsd togt.thcr. growth was g i w t l ~ ~ stiniulatcd . Paper chromatography has btwi applied to the clarification ui man>- perplexing questions in the biological field: quantitative, tleterniiriation of ergothioneine in biological fluids (225): anal! sis of biological a1,ylaniines ( 1 8 7 ) ; detection of one or more hitherto unsuspected amino acids in the cholera Vibrio (1.51; and deterniinat,ion of the amino acid constituents of tissurs ani1 chromosomes (isolated from salivary gland cells) of Urosophilu virilis, using acid hydrolyzed adults and larvae ( 1 7 ) . Distribution of free amino acids in mouse epidermis, in various phases of growth (1?0), was decided by the relative position 011 the chromatogram and by comparison with a reference chromatogram, as a result of which it is believed t,hat carcinoma can be sharply differentiated from the tissue of nonmalignant hyperplastic epidermis produced by the application of methyl cholanthrene. Estimation of some free amino acids in the tissues of the rat (6) demonstrated the presence of aspartic acid, glutamic acid, glycine, and alanine by spectrophotometry of the colorc produced by ninhydrin on t,he completed chromatograni. The amino acid pattern in portal blood and jugular blood in dog? (61) was shown to consist of three types: t,hose which rose and fell after the protein meal, those tending to rise slightly through-
ANALYTICAL CHEMISTRY
56 out the 5-hour period, and those t h a t did not change during the experiment. The free amino acids in Haemolymph of Calliphorcr rrythrocephala Meigen ( 6 5 ) were identified as alanine, tyrosine, proline, valine, isoleucine, histidine, phenyl alanine, glycine, leucine, lysine, serine, and aspartic acid. A study of the chemistry of cacao fermentation (Forestero variety) ( 7 4 ) showed t h a t (1) fresh bean pulp juice contained glucose, fructose, and sucrose; ( 2 ) citric acid was the only free acid in the pulp; (a) glutamic and aspart,ic acids and asparaginc existed also in the pulp; and (4) tannins and other color constiturnts were extracted. Investigation of the amino acids in thc tohacco mosaic virus indicated the ahsence of histidine, hydroxyprolinr, and methionine, b u t the presence of fifteen others (87 j . T h e f r w amino acids and peptides in frog embryos (Kana pipi p n s ) (115) yielded only faint smudges of glutamic acid. Thc amino acids in FIaemolyniph of the dragonfly (168) nymph, .I eschrra c ~ a n ~ showed u, the presence of serine, glycine, lysine, ulaninr, tyrosiue, valine, leucine, and proline. The estimation of free amino acids in t,he tissue of rats showed the presc>nw of glycine, alanine, ani1 glutamic and aspart,ic acids ( 5 ) . The release of free and bound forms of biotin from proteiil (20) indicated that the solution libcrated from liver b y the, cirystal pepsin acted as a single compound, whereas that ol)tainrx:l from the countercurrent zxtraction of arid-hydrolyzed liver wa.: resolvrd into several components. The toxic suI>stanc(x, glutcsn, \\-as concentrated from -igrnizrd flour (\vhicli c~ausesfits in dogs) iind resolved into glycine, serine, alanine, and glutamine (2.91. I’eptides from insulin (17 5 ) on oxidation yielded four fractions of h and one of B, which are not all completely identified, Identification of Bence-Jones protein of niultiple myelomatosis in amino acids by two-dimensional chromatography, using phenol and collidine as the solvents, indicated that no methionine was present, and all other c01n1i1on amino acids were tiemonstrateil in the protein hydrolyzate rscrpt hydrns>.proline(60). iPPLICATIONS TO \\11\0
4(:II) SKP4K41‘IO\S
The us(% of paper for the resolution of mixtures o f aniiiio acids was first proposed in 1941 (81) and its rapidity of srparation. qualitativc~ arid quant,itative results obtained, simplivity an(l reproducibility, all caused its immediate widespread adoption. A spot of t,he mixture is placed about 2 cm. from one corner of :I large sheet of paper and its lower edge is dipped int? the solvent (usually phenol) and developed for 12 t o 48 hours. The paper is then dried and rotated BO” and the edge is immersed in anothe~. solvent of someivhat different partition coefficient (collidine). This separates t,he amino acids efficiently, and, after spraying with ninhydrin in a n atmosphere of steam, the blue to purple colors produced identify the various acids by t,htBir position on the paper. They can also be identified by cutting out the colored sections, eluting t,hem, and titrat,ing against 0.01 -V barium hydroxide solution, using bromophenol blue a s the indicxtor, or by thvir fluorescence in ultraviolet light. hpplications of this method are very numerous and result,s have beeti inraluatile in elucidating structures, identifying compounds. srizuating impurities, a n d discovering nrw compouncb. The ninhydrin used as the spray t,o bring out the color of the amino acids is in 0.1 to l.Oyc solut,ions in water, butanol, 01’ butanol-acetic acid, and is reported t o he sensitive t o 0.1 t o 25 grams of compounds detectable (166). A modified ninhydrin is suggested to be made of 2cc ninhydrin solution in methyl Cellosolve water at, p H 5 (citrate buffer), which contains stannous chloridr as a reducing agent (106, 140). The latest innovation is the inclusion of 0.14; ninhydrin solution in the collidine or butanol-acetic acid used as t h e second developer; then only heat is necessary t o produce the well known colors of the amino acids after the chromatogram is completely developed (146). Some paper seems t,o have a peptide contaminant (22h‘). n-hirh can be removed by wishing the paper for 48 h o u ~ n-it11 water. T h e phenol used RS the first solvent must be very pure :inti special directions are a.vaiIal)lr as to the I x A s t method of purifirfition (t5,$), so as complrtrly t o c~liminateall rxtraneous colors.
0-Phthalaldehyde is reported to be the specific reagent for tlit. determination of glycine in paper chromatography (155). \+-it,ti acidified ninhydrin, no distortion in colors of RF values of glycine, alanine, threonine, and tyrosine appears even when alkalinity is high, b u t with s-collidine the effect is reversed, the acid spot being near valine-leucine, a d the alkali spot, near glycine (38). The formation of copper phosphate complexes is another method of separation and identification of amino acids (165) and ten such acids were investigated, with the decision that quantitative results can be obt’ained for those amino acids whose copper salts are soluble anti can be applied to others which form insoluble copper salts. The distinctive test, for a-amino acids depends on the fact tlia,t the copper complex, CUCO~.CU(OH)~, forms a comples with a-amino acids whose RP = 0, so t,hat tht. other substances leave them behind a t the bottom of the paper. Twent,y amino acids were investigated (4.5).a-.4mino acids ttnd peptides can also I)r determined by their fluorescence under ultraviolet light (1581. .\mino acids can be separated on pulped filter paper arid charcoal columns with good results (905). Iodine can be directl>-sublinieil on a paper chromatograni or sprayed in alcoholic solution to reveal the position of choline and other .V-methylalkariola~~iines ( 2 1 ) . Furan derivatives (furfuryl alcohol and tetrahylrofurfuryl alcohol) containing 10 to 30% \t-ater produce good separations of amino acids on paprr ( 1 3 ) . reported that the inclusion of 0.5% urea, 8 5 pyridine, furoic acid as stahiliztw sharpens the outlines of the spots 2nd reduces their size. There are many special applications in this field of one- and tno-dimensional ctironiatography, such as identification of 0Itmino adipic acid ( 1 4 ) ; invest,igation of the action of nitrogen trichloride 011 proteins anti attempts to isolate the tosic fartor, which resulted in the conclusion that it probably is not a peptide (12); use of Pauly reagent to sepamte three monohydroxybenzoic acids from one another and from phenols ( 6 8 ) ; separation of isotopic dcrivativrs of amino acids (101); and demonstration of the prewncc, of thyrosint. and diiodothyronine and triiodtr thyronine as ninh,vdriii rcacting products from t,he hydrolyzates of thyroprotein (88). Chromatography of dinitrophenyl amino acids and peptides. which glow a pale yellobv in a fluorescence which is visible hut not a true flnoi~~scence, showed that long nonpolar sidf. rhains cannot be resolved (160). Amino acid constit,uents of rirculin ~ v e r eisolated on po\\-dpred cellulose and identified either 1)y papcr strip chromatograph). or by optical rotation (1.56). Other applications include: determination of presence of esters anti salts of Fatty acids and amino acids in rancid cheese which cause staleness (184); and chromatography of amino acids belonging t o homologoit.; Aeries, which fall on a smoot,h curve characteristic of that wriw and in the order of the molecular weight (163). Preliniinary separation by ion exchange resins, followed by ionophore&, stylarates the peptides according to charges and molecular n-rights into groups having similar 1110bilities in an electrical fic.lt1: these groups were investigated by paper chromatography, aiid ninetem different dipeptidrs of aspartic and glutamic acid \ v ~ r tidentified > in the hydrolyzate ( 4 2 ) . Sorleucine was tliscort~rt~d to he absent in the spinal cord, contrary to t,he general opinion ( 4 0 ) ; steroids a s h\-tirazoncss of Girard’s Reagent T IVWC scfiparated and the dried chi~oinatogram was dipped ill iodoplatiiiate solution t o remove t h r Imckground, so the K r values rould be calculated (828); a-glutamyl peptides were synthesizd anti their purity was confirnied by paper chromatography (113); the copper phosphate-amino acid complex method was used to identify 1 to 25 micrograms of a-amino nitrogrn (222 j. -imino acids were separateJ first by adsorption into dicarlmsylic, Ilasic, anti nrutrrtl on anion and then on cation c~xchtriigerwins ani1 then the eluate was separated on paper strips, denionst rating tlic, presence of the amino acids by ninhydrin, ant1 quantitativc~lydetermining them by transmittanrc, on an elwti,onic tl(~risitometer(16). Studies n-tw made of hytirol!.zatw of g i ~ ~ n ~ i i ~(18.5): i d i n of protein fractions (flowing
V O L U M E 22, N O . 1, J A N U A R Y 1 9 5 0 filter papri' chromatographic technique) (162): of the amino acid caoniposition of plant leaf protein fractions, showing t h a t hydroxylbsent from leaf proteins (122),and ident,ified the amino nt, (1Zf j ; and of enzyme hydrolyzed proteins (202). Quant,itative detrrmination of the components of many mixr u t w w a s reported on keto acids (XI), purines and p y r i m i h e s lstY:3, 2OJ), amino acids of salmine ( 8 4 ) , S-substitutrd amino itcitis (78 ), purine and pl-rimiditie derivatives (89j, lysine ( 1 ), iwatine and rrcatininr (132-1;34), amino nitrogen (283), amino arid mixtures (229),and micro eytimation of amino nitrogen (221j. Various espt~ririir~nta w r e follon-eti l)y paper chromatography: aniitio avids, pep1 idra, anti compounds of tryptophan (186), tictioit of alkalies on wool and tiwniinated ~vool( 3 5 ) ,production i l i ' amino acids after a short esposurc? to C"02 during photosynthesis (180), anti a technique for demonstrating, by chromatography, the nitrogenous lipide constituents, sulfur-containing amino aritl.u, reducing sugars (SI),and rrlation of manganwe antl ino1yi)rlrnum to the free amino acid content of cauliflower ( 8 6 ) . There are several good reviFw artirlrs on amino a d s (f28, l.30, ZOO), especially Martin's ( 1 2 7 ) . \lISCELL4Yl.mI'S
~l'l'Ll(:\'rloYS
Ix-13utyric. tirids \v~lrediscovrrcd i t i plant lraves (.52). I'arious Irganic acids \vert: reaolvrd hy using i )utanol-a.cetic acid mixtures ; t i i d drterted 1 ) ) ~spraying the tlrirJ c.hroniatogram with an inilicator ( 119). Potassitmi h y t h s a n i a t e derivat,ives of organic acicls iv(brr separated 1)). suita!)le solvents (isobutyric acid anti 6
phetiol) anti t h r papi'r n-as thrri sprayed with dilute frrric chloriclt,, which prorlucrtl purplr spots of the derivatives on a yelloni,>ic~kgrounci( 6 2 ) . This niethocl is a. very good means of identif!.iitp orgmic acids o f oiir t o eight carbon atoms. Man\- mist u r w of nonvolatile organic acids were resolved and separatrd i i i t o thcLir i*oniponr'nts (120). Ionization and adsorption of the acids kiy thts paper were suppressed b y swamping both the stationary ancl moliilo solvc,nt phases with a volatile acid. Formic ell defined hands of t h e test acids ( t o travel down and characteristic Rli valurs. Acids in carrot raietl and identified by RF value (120). F a t t y were also srparated ( 2 6 7 ) . Dyes were investigated with cotton fabric disks (lfd),and in hinar)- and ternary nristurtls ( I ; ? ) + absorbed on fihrous materials ( 7 2 ) , and on as(yst1iiiiig paper strips with automatic recording of the per cent n.nnsnii.c.ion (149, 14,:). ('onipou~idsof many otlirr unusual substances were investig a t r d ti!. pnpcr chromatograpliy : pterins (go), identified b y their c.haractc~~~istic fluorescence: tiihydroionones (96); anthocyanines ( 8 ) ; liyosrine and hyoscyamine (5,9):flavins (44); flavonoid pigments in plant extracts (806);various lignins ( 7 ) , which were itlentifid b y their fluorescence; and synthesis of amino acids by ~ l i t T ~ ~ r , i t~yr ipr w of hnt.tpria (102). SC' \ l \ l h H Y
1'apc.i. c~hloniatogi~aphy has providrd an estrrmely x :d in some types of xvork, not ably biorheniistry requiring analy.is or separation on the microgram scale, it is almost indispensatdr. \Ian? procrdures are eminently practical for routine work. T h r ronsidrrable degree of empiricism that is st,ill involved is a draw})arkin attacking a new problem. Further quantitative studirs ,)f a physicochemical nature, including kinetic studies. may rnalilt~ iiiie t o (valuate the various factors involved in a inore quantitativc :ind prrdictable fashion. BIB LIOGKAI'H Y
.Ul)siiese, -4. A , and Lein. M., Science, 110, 163 (1949). ( 2 ) ;Iniinoff, D., and Morgan, IT. T. J., .Tatitre, 162, 579 (1948). ( 3 ) . \ d e n , T. T'., Burstall, F. H., Davies. G . I-ndt,Baruh, and Gillard, S u t u r e , 163,999 (1949). Landua, A. J., and-lwapara, J., Science, 109,385 (1949). Lecoq, Bull. soc. roy. sei. Lidge, 13,20 (1944). Anal. C h i m . Acta, 2,261 (1948). Lederer, &I., Lederer, M.,.Vattire, 162,776 (1948). Ibid., 163,598 (1949). Lederer, M.,Science, 110,115 (1949). Lenoir, J., Bull. soc. chim., 1942,475. LeQuesne and Young, Suture, 163,604 (1949). LeRosen, &\. L., ANAL.CHEM.,19,189 (1947). Li, C., and Roberts, E., Science, 110,425 (1949). Linggood. F.V., and Woiwod. -4. J., Brit. .I.Esptl. Path., 29,
_2R8 _ _ I1 Q48). --,\--
(116A) Linstead, R. P., Satztw, 163,598 (1949). (117) Longenecker, Sf‘. H., ASAL.CHEY.,21,1042 (1949). (118) Longenecker, W.H., Science, 107,23 (1948). (119) Lugg, J. W. H., and Overell, B. T., ,Vature, 160,87 (1947). (120) Lugg, J. IT. H., and Overell, B. T.. ditstralian J . Sci., Res. Ser., Al,98 (1948). (121) Lugg, J. TV. H . , and \Teller, K. d.,Biochem. .I.,38,408 (1948). (122) Ibid., 42, 108 (1948). (123) Ma, M. R., and Fontaine, T . D., Science, 110,232 (1949). (124) Mapson, L. M.,and Partridge, S.M . , S a t u r e , 164,479 (1949). (125) Markham, II.. Symposium on Flavor in Dairy Roducts, Division of Agricultural and Food Chemistry, 116t,h Meeting of -4u.CHEM.SOC., Atlantic City, N. J., 1949. (185) Synge, K.L. M., Biochem. J . , 44,542 (1949). (186) Synge, It. L. M.,and Tiselius, .\.. Acta Chem. Scantl., 3, 231 (1949). (187) Tabone, Robert, and Trowtler, Bull. soc. chim. biol., 30, 5-17 (1948). (188) Taurog, A,, Chaikoff, I. L.. and Tong, W., J . BioZ. Chem., 178, 997 (1949). (189) Taurog, A,, Tong, IT-., and Chaikoff, I. L., Satzcre, 164, 181 (1949). (190) Thomas, Hendricks, and Hill, Symposium on Sulfur in Agrirulture, Division of Fertilizer Chemistry, AM. CHEM.Soc., - l t lantic City, K. J., 1949. (191) Tiselius, A, Arkiv Kemi. Mineral. Geol., 26B,1 (1948). (192) Tiselius, A., Chem. E n g . S e u ~ s27, , 1041 (1949). (193) Tishkoff, Bennett, Bennett, and Miller, Science, 110,452 (119419). (194) Tishkoff, Zaffaroni, and Tesluk, J . B i d . Chem., 175,857 (1918:. (195) Tolley, G., Mfg. Chemist, 20,215 (1949). (196) Tomarelli, R., and Florey, K., Science, 107,630 (1948). (197) Tomisch, E. G., S a t u r e , 160, 164 (1947). (198) Trey, H., Z . anal. Chem., 37, 743 (1898). (199) Vrbach, K. F., Science, 109,259 (1949). (200) Vega, E’. A. de la, Galenica Acta ( M a d r i d ) , 2,85 (1949). i20l) Verdier, C. H. de, and Agren. Gunnar, Acta Chem. Scand.. 2, 783 (1948). (141) (142) (143) (144) (145)
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59
V O L U M E 2 2 , NO. 1, J A N U A R Y 1 9 5 0 202) Vischer, E., and Chargaff,E., J . Biol.Chem., 168,781 (1947). 1203) Ibid., 176, 703 (1948). 204) I b i d . , p. 715. 20.5) Wachtell, 3. L., and Cassidy, H. G., J . Am. Chem. Soc., 65, 665 (1943). LW) Wender, S. H., and Gage, T. B., Science, 109,287 (1949). 207) Williams, R. J., and Kirby, H. M., Ibid., 107, 481 (1948). 208) Williams, T. I., “Introduction to Chromatography,” London, Blache and Son, 1946. Y O Y ) Williams, T. I., Mfg. Chemist, 19, 291 (1948). (210) Williams, T. I., Research (London),1,400 (1948). a l l ) Williams, T. I., SchooZ Sci. Rev., 26, 154 (1945). . 21, 1338 212) Winslow, E. If., and Liebhafsky, H. A., A B U ~CHEW, (1949). 213) Winsten, W.A., Science, 107, 605 (1948). 214) Winsten, W.A , , and Eigen, E., J . Biol. Chem., 181, 109 (1949). 215) Winsten, W.A . , and Eigen, E., Proc. Soc. E @ . B i d . Med., 67, 513 (1948). 218) Winsten, K,A , , Eigen, E., and Oser, B. L., Symposium on Vitamin Bln and Related Factors, Division of Agi icultural
and Food Chemistry, A.M. CHEV.Soc., Atlantic City, N. J., 1949. Winsten, W.A, and Eigen, E., Symposium on Growth Substances and Metabolism, Division of Biological Chemistry, AM.CHEY.Soc., Atlantic City, N. J..1949. Winsten, W ,A., and Spark, A. H., Science, 106, 192 (1947). Wise, Green, and Rittenhouse, T A P P I , 32, 335 (1949). Wise and Rittenhouse, Ibid., 32, 397 (1949). Woiworl, A. J., Biochem. J . , 42, xxviii (1948). Woiwod, A. J., S a t w e , 161, 169 (1948). Woiwod, H. J., and Linggood, F. V., Ibid., 163, 218 (1949). Wolfson, TT. Q., Cohn, C., and Devaney, W..1.,Science, 109,541 (1949). Work. E., La,icct, 256, 652 (1949). Wynn, I-,, Satcue, 164, 445 (1949). Tacowitz, H., Norris, L. C., Heuser, G . F.,f’roc. Soc. Exptl. Biol. J i e d . , 71, 372 (1949). Zaffaroni, A , . Burton, R. B., and Keutman, E. H., J . Bioi. Chem., 177, 109 (1949). Zechmei$ter, Ann. *\-. 1’. Acad. Sei., 49, 145 (1948). RECEIVED Decemhrr 17, 1949.
DISTILLATION ARTHUR ROSE The Pennsylcania State College, State College, P a .
T
HE same general trends iri analytical distillation have prevailed in 1949 as in recent years, and many of the statements made a year ago ( 7 1 ) still apply. Greatest activity has been ,~hownin the design and testing of apparatus for the specialized ,.rinditions of vacuum and micro or seniimicrodistillation. Addiri~iiialdetails and applications continue to be presented on the ixecisioii analysis of liquid and gaseous hj-drocarhon mixtures. The combination of distillation with other processes such as adwrptiori and extraction and identification of final fractions by such properties as infrared spectra are proving increasingly fruit-
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il 11
STILL HEADS
The most prolific single topic has been reflux dividers and conlieriser heads. It would appear that existing designs are all some\\.hat unsatisfact,ory, or that derails and techniques are not propi.t,ly worked out. A t the same time, the problems seem simple ixriough from a superficial point of view, so t h a t many workers are rc.mptcti to t r y working out a solut,ion. Most of the new designs have been tested under a very limited range of conditions, and there are few objective comparisons. Tests of some devices in the ituthor’s laboratory indicate that artual reflux ratios are often far from n h a t is intended. K h e n proper weight is assigned to simiilicity, reliability, ease of repair, and all-around utility, i t appears that morc attention might be given to careful techniques with the ~ l d e types r of heads and reflux dividers. Doty ( 1 7 )has described a vacuum-jacketed vapor-dividing retlux head t h a t eliminates numerous sources of error. Hakala (54) reported total condensation and variable take-off tvithout use of a stopcock. An electronic reflux ratio timer has been introduced \JY E‘isher ( 2 2 )and a device for low distillation velocities bj- T n fiesta ant1 Achon (89). Xord (63) has contributed t o the problem of reflux division of streams containing water and an immiscible liquid. Diehl and H a r t ( 1 1 ) devised a very low holdup head for i’acuum columns. Their reflux divider, as re11 as t h a t of Human and Mills (40, 4 1 ) , features a long-stemmed glass valve. GoldsI,arry and Martin (50) have patented a head with pressure-equalizingline to prevent backflox of condensate. PACKING MATERIALS AND COLUMN EVALUATION
S o entirely new packing mat,erials have been described during the year. S e w test d a t a deal chiefly with analytical columns of relatively large diameter. Cannon (9) has reported t h a t 0.16 X
0.16inchpiotruded iiichel packing gives aheiglit equivalent theoretical plate of 1.4 to 1.5 inches in a 2-inch diameter column, and 1.8 to 1.9 inches in a 4-inch column. Fisher and Bowen (21) have compared lIc3Iahon packing (screen in the shape of a saddle) with several others, all in a 4-inch diameter column. Circular screen disks x e i e tested as packing by John and Rehberg ( 4 4 ) in columns of 17-, 2 5 . 4 , and 50.8-mm. diameters. Dixon ( 1 4 )tried out 1/16- to ‘/r-inch metal gauze cylinders in columns of varied diameter and has ieported as high as 84 theoretical plates per foot. X succeeding ai ticle (15) described the distillation of a multicomponent mivture with separation of materials having boiling point differences as small as 2.4” C. French ( 2 4 ) has given an application of gauze ring packing to distillation of coke oven by-products. No further theoretical discussions have appeared on the complexities of evaluating column packing. Miller and Woodle (66) presented a paper a t the September meeting of the AMERICIY CIIEXICAI.SOCIETY on the use of n-heptane-2,2,4-triniethylpentane as a test liquid. Evaluation a t reduced pressure has been reported by tn-o sets of authors with conflicting result?. Berg and Popovac (Y), using helix packing and n-octane-toluene as a test mixture, found no appreciable difference in number of theoretical plates over a iange of pressures from 20 to 760 mni. Feldman, llyles, TTender, and Orchin (19, 6B), using a valiety of packings, worked iTith n-heptane-methylcyclohexane a t atmospheric pressure and dodecane-cyclohexylcyclopentanea t reduced piessures. Their resultq confirmed the prediction of Bvron. Bo\\ man, and Coull ( 7 ) of a broad maximum in fractionating efficiency somewhat belon- atmospheric pressure, 15 ith niarhedly lo\+er efficiencies a t lower pressures. Y o r e work is needed to clarify this situation. SPINNER COLUIINS
Several spinner columns have been developed and some test data presented. This variety of column piomises to be of real value in precise vacuum distillation. Donne11 and Kennedy ( 1 6 ) have supplied additional details on a small conccnttic tube rolumn, with stationary inner tube, which can separate 15-ml. Samples into 0.5-ml. fractions for infrared analysis. Cruthirds, Jones, and Seyfried (10) reported H.E.T.P. values of 2 inches a t atmospheric pressure and 4 inches a t 0.1-mm. mercury pressure for a column 6.5 mm. in diameter with i/r-inch Chrome1 spinning band. Ten- to 200-ml. samples can be distilled in this apparatus. Larger rotating band columns have been described by Jost (45) with di-
