isoleucine-leucine region, 49-51-53. Spackmen et al. (19) have found glucosamine, 59, immediately following leucine, 53, at 30' t o 50' C. Hamilton and Anderson (5) and Moore ( 7 ) have pointed out that the position of the glucosamine peak is relatively unaffected by the p H of the eluting buffer. Thus, the position of the tyrosine, 58, and phenylalanine, 60, peaks may be altered with respect to the amino sugar, 59, by an appropriate change in the time of the buffer shift. 50-cm. Column. On t h e 50-cm. basic column ornithine, 104, a n d 2,4-diaminobutyric acid, 105, form a perfectly symmetrical peak b u t can be distinguished by their somewhat different 4 io- to 570-mp absorption ratios; ornithine, 104, has a higher ratio. Lysine, 109, and kynurenine, 110, have similar elution volumes but the latter tends to tail. Methionine methyl sulfonium salts, 108, also overlap the lysine-kynurenine peak, 109-1 10; however, lysine has a notably higher absorption ratio than the other two compounds. The peaks of 5-hydro~ytryptophan~ 111, and 1-methylhistidine, 112, are superimposed and might well prove difficult to distinguish by ratio. This also applies to the single ammonia-cysteinylglycine peak, 107-107A (of which the latter is probably cpstinylglycine as a result of ready oyidation). Canavanine, 115, appears as a shoulder on the tryptophan-anserine, 116-117, peak which in this study failed to separate from each other. Canavanine,
115, has the highest ratio of the three and this might be of assistance in identification. Homocysteine thiolactone, 121, was found as a shoulder on the trailing side of arginine, 120, and might be overlooked, particularly if the latter were present in considerably larger amounts than the former. Ratio differences are too small to be a dependable means of identifying one in the presence of the other. Of all the compounds examined, 3,5-diiodotyrosine, 122, exhibited the greatest retention on this column and was for the most part eluted just beyond the arginine-homocysteine thiolactone, 120-121, region. 2,3-Diaminopropionic acid (not shown in the figures) was found t o be eluted on the 50-cm. column a t a position just beyond and partially overlapping the ammonia peak. This is in agreement with the unpublished data of Van Etten of the Northern Utilization Research and Development Division, Peoria, Ill. ACKNOWLEDGMENT
The authors thank John F. Thompson and C. J. Morris, U. S. Plant, Soil and Nutrition Laboratory, Ithaca, N.Y., for samples of a- and 8-acetylornithine, meta-carbosyphenylalanine,metacarboxy-a-phenylglycine and the 7-glutamyl dipeptides. Thanks are also due L. Fowden, University College, London. for a sample of 2-azetidinecarboydic acid, and to L. Weil and T. J. Fitzpatrick of this laboratory for the S-carbosyethylcysteine, and isoasparagine. respcctiwly.
LITERATURE CITED
(1) Dreze, A., Moore, S.,Bigwood, E. J., Anal. Chim. Acta 11, 554 (1954). (2) Frimpter, G. W., Bass, A., J . Chromatog. 7, 427 (1962). (3) Gerritsen, T., Lipton, S. H., Strong, F. M., Waisman, H. A,, Biochem. Biovhus. Research Commun. 4. 379 (1961j. (4) Gundlach, H. G., Moore, S., Stein, W. H., J . Biol. Chem. 234, 1761 (1 9.59'1 \ - - - - I .
(5) Hamilton, P. B., Anderson, R. A,,
ANAL.CHEX.31, 1504 (1959). (6) Moore, S.,The Rockefeller Institute, Yew York, S . Y., private communication. 1959. ( 7 ) Z b k , 1962. (8) Moore, S., Spackman, D. H, Stein, W. H., ANAL.CHEM.30, 1183 (1958). (9) Moore, S., Stein, W. H.. J . Biol. Chem. 176. 367 11948). (10) Zbid., 192, 663 (1951). (11) Zhid., 211, 893 (1954j. (12) Spackman, D. H., Stein, TV. H., Moore, S., ANAL. CHEU. 30, 1190 (1958). (13) Stark, G. R., Stein, W. H., Moore, S., J . Biol. Chem. 235, 3177 (1960). (14) Talley, E. A., Porter, W. L., J . Chromatog. 3, 434 (1960). (15) Van Etten, C. H., Miller, R . W., Wolff, I. A., Jones, Q.: J , Agr. Food Chem. 9, 79 (1961). (16) Wolff, E. C., Black, S., Downey, P. F., J . Am. Chem. SOC.78,5958 (1956). (17) Zacharius, R. M., Talley, E. -4., J. Chromatog. 7, 51 (1962). RECEIVEDf o r . review June 12, 1962. Accepted September 13, 1962. Eastern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture. Reference to a company or product does not imply approval or recommendation by the U. S. Department of Sgriculture to the exclusion of others that may be suitable.
f
Tri-n-octylamine as Liquid Anion Exchanger tor Chromatographic Separation of Rare Earths on Paper or Cellulose Powder CORRADO TESTA
Centro lnformazioni Studi Esperienze, Milan, lfaly
b The chromatographic behavior of rare earths on paper treated with tri-n-octylamine (TNOA) by elution with LiNOa solutions was investigated. Rare earths form anionic nitric cornplexes whose strength increases with decrease of atomic number. By plotting log
;(
- 1)
vs. the atomic number
of the rare earths, a straight line was obtained, which indicates a mean separation factor of 1.45 for two adjacent elements. The influence on R F values of the concentration of
1556
ANALYTICAL CHEMISTRY
LiNO3 eluting solution and of TNOA treating solution, and the " 0 3 added to LiNOs, was also studied. Some examples of the separation of three or four rare earths are reported, and the possibility of separating milligram quantities of rare earths on beds of cellulose powder treated with TNOA is shown.
C
HEMICAL separation
technique, can be considerably improved by cornbining the conventional chromatographic procedure with an additional Q
phenomenon which makes the over-all process much more selective. Many new selective organic substances have recently received much attention as liquid-liquid extractants; among the most used are: tri-n-octylamine (TNOA) (9, 10, 16, 17), tri-n-octylphosphine oxide (TOPO) (1, 11, 19, 21, ,%?), and di(2-ethylhexy1)orthophosphoric acid (HDEHP) ( I , 18). Interesting results have been obtained in our laboratory by treating the chromatographic paper with T S O A (2, 3, do), TOPO (5), and H D E H P (6).
earth elements with TXOA-impregnated paper was possible, in accordance with the anionic behavior of such elements. Solutions of LiN03 \yere used as the eluents for paper chromatography. An indicative experiment was also carried out with a column of cellulose powder treated with TNOA.
1.6,-
1 2c4 1
4
’”;
0.8
EXPERIMENTAL
0.6t
O 2 I 00-
\
-0.41
Figure 1 .
Plot of log
(& -
1) vs. z
for nine rare earths Paper treated with 0.20M TNOA, eluted with 5 M LiNOa f 0 . 0 2 M “ 0 3
Analogous techniques have been extended to column chromatography, by preparing columns filled with cellulose powder previously treated with the above reagents (2, 4, 7 , 8). By column chromatography milligram amounts of substance were easily separated. Paper treated with an organic solution of TXOA behaves very much like a film of anionic resin, as it retains hydrochloric, nitric, and sulfuric anionic complexes of the various elements in accordance n ith the strength of the complex itself. Analogously, paper treated with H D E H P behaves generally like a film of cationic resin, because of the availability of an exchangeable hydrogen ion. Therefore, rare earth elements, n hich are selectively extracted in the liquid-liquid process by HDEHP, were separated by paper chromatography with the use of a strong acid of suitable molarity as the eluting agent (6). I n accordance with the solvent extraction behavior, RFJs of rare earths decreased with the increase of their atomic number. Marcus, Nelson, and Abrahamer (12-14) have shown that rare earth elements may form anionic complexes in concentrated solution of an alkaline nitrate. I n fact, some rare earths n-ere separated by using column chromatography with Dowex 1 resin eluted with LiN03 solution ( I S , l4,,or by liquidliquid extraction with tri-iso-octylamine from LiNOB solutions (13). I n both cases the distribution coefficient or the extraction coefficient decreases as the atomic number increases. The aim of the present work was to show that chromatography of rare
Reagents and Equipment. The rare earth nietals or oxide were supplied by Light’s (London) and by Fluka (Buchs S.G., Switzerland); T N O X was supplied b y Fluka. T h e other compounds were of analytical grade. The chromatographic paper was Whatman S o . 1, in sheets for single experiments and CRL/1 type for multiple experiments. Whatman cellulose powder (standard grade) mas used for column chromatography. The experimental chromatographic assembly used for CRL/l paper has been described ( 5 ) . The column for the separation of Ba, Yb, Na, and La had a cross-sectional area of 1.3 sq. cm. and mas fitted a t the bottom n i t h a fritted-glass disk t o retain the powder. Treatment of Paper and Cellulose Powder. The treating solution for the chromatographic paper was a solution of 0.20 to 0.25M TYOAbenzene, equilibrated with twice its volume of 2 X HxO3: after phase separation, the TYOA solution was percolated through cotton lint to remove any trace of the inorganic solution. The sheets n ere then iinmersed in the organic solution for about 30 seconds, allowed to drip, and finally dried by blowing warm air. For the cellulose treatment a 0.50M TN0,4 solution was used. Tnenty grams of dried p o ~ d e rwere added to 100 ml. of this solution, and the mixture was stirred for about 12 hours by an electromagnetic stirrer. After that, cellulose was filtered, dried between two filter paper sheets, and then freed from benzene by keeping it for 2 hour4 at 80” C.
Table
I.
RP
The Dowder was then cooled and stored i i a desiccator. Procedure for CRL/1 Multiple Chromatographic Tests. The spots were deposited by using 0.01 to 0.02 ml. of solution (25 to 50 pg. of the element). The rare earths tested were La, Ce, Pr, Sm, Eu, Gd, Dy, Er, and Yb; Sc, Y, and T h were also tested. The CRL/l sheet, folded to form a cylinder, was placed inside the device described elsewhere (5); then ascending elution was performed until the solvent front reached a distance of 9 em. from the application point. The sheet was finally dried with warm air and developed by means of a 1% solution of 8-quinolinol dissolved in water and ethanol (1 to 1); the spots were clearly visible after exposure of the strips t o ammonia vapor. R F Values a s a Function of LiNOs Molarity. The eluting LiN03 solutions were slightly acidified with H K O j (final molarity 0.002-V) to avoid rare earth hydrolysis. The folloRing Lip\’O3 molarities were tested: 1, 2, 3, 4, 5, 6, 10M. Table I shows clearly that the RF values increase (and hence retention decreases) froin the lighter to the heavier rare earths, in accordance with the behavior of the anionic resin Dowex 1 (12, 14) and of the TIOA extraction (131. RIoreover, the RF values of every rare earth decrease appreciably on increasing the molanty of the L i S 0 3 eluting solution. Yttrium and scandium showed RF values similar to those of heal-ier rare earths, but yttiium was slightly more retained than scandium; thorium did not move from the application point, confirming its strong complexation in a nitrate system ( 3 ) . The fundaments1 aspects of liquidliquid extraction and chromatography on treated papers can be correlated through the quantitative relationship (3) suggcsted by Martin and Synge (15)
Values as Functions of LiN03 Molarity
Chromatographic paper treated with 0.20M TNOA-benzene. separately with CRL/1 type paper Molarity of LiX03 Rare earth 2 3 4 5 elements Lab7
Ce58
PrSg Smez Eu63
Gde4 Dye6 Er6* Y b70 sc21
yas
Thm
0.63
0.76 0.80 0.88 0.89 0.89 0.89 0.89 0.89 0.91 0.90 0.00
0 33 0.4i
0.63 0.86 0.90 0.91 0.91 0 91 0 91 0.89 0.90
0.00
0.10 0.16 0.25 0.i7
0.85 0.89 0.89 0.89 0.89 0.89
0.90
0.00
0.05 0.0s 0.10 0.42
0.i9 0.83 0.S3 0.86 0.91 0.89 0.88 0.00
0.03 0.04
0.08 0.17 0.23 0.37
0.40 0.60
0.77
Elements considered ____- ~ _ _
6
10
0.02
0.00 0.00
0.03 0.04 0.06 0.08 0.13 0.13 0.33 0.61
0.i8
0.73
0.73 0.58
0.00
0.00
VOL. 34, NO. 12, NOVEMBER 1962
0.00
0.00 0.00 0.00 0.00 0.02 0.03 0.03 0.00 0.00
1557
detail for La, Ce, and Nd, by using a paper treated with 0.25M TXOA and not very concentrated solutions of Lii\'03--i.e., 1.50, 1.75, 2.00, 2.50, 3.00, 3.50, and 4.00M.
R
As shown in Figure 2, three parallel straight lines are obtained for log - 1) us. log Lin-03 molarity.
(A
-0.51
t' / log M LiNO)
Figure 2.
Plot of log
(&- 1)
vs.
LiN03 molarity for La, Ce, and N d Paper treated with 0.25M T N O A
where E: is the extraction coefficient for the extractant used and k is a constant. Figure 1 shows the linearity between log - 1) and the atomic number, 2, of the rare earths considered. The mean separation factor
(A
in this case is 1.45 for two adjacent elements. This value is very similar to that found by Marcus and Nelson for the Don-ex 1 resin (14). The influence of 11iN03 molarity on - 1) has been studied in more
(&
RF Values a s a Function of TNOA Molarity. If t h e chromatographic paper is treated with more and more concentrated T S O A solution, t h e RF values of each rare earth decrease gradually. Lanthanum and neodymium have been eluted with4J1 LiN03 by using papers treated with 0.005 t o 0 . l M T S O A solution. Table I1 s h o w the RF d u e s so obtained. Influence of H N 0 3 Added to L i N 0 3 on RF Values. T h e R F ~values increase on adding HS03 t o t h e L i s 0 3 eluting solution. This behavior has been shown for U, Th, and La (3). Table I11 s h o w the influence of " 0 , added to 3J1 LiN08 on the Rr values for La, Ce, and Nd. It is clear that the free acidity of HS03 lorn-ers the formation of the complex. Chromatographic Separations of R a r e E a r t h s on TNOA-Treated Paper. ELUTIOX KITH LINO,. Two, three, or four rare earth elements were separated by selecting suitable conditions on t h e basis of t h e previously described euperiments. The chromatographic strips ( 2 X 25 em.) were treated with 0.25.11 TNO-4 pre-equilibrated n i t h 2M HS03. The rare earth mixture (25 to 50 pg. of each element) mas eluted by ascending chromatography with Li?;os solutions. Table IV summarizes the separations obtained while Figure 3 repre-
Figure 3. Separation of Th, Ce, Nd, and G d on paper treated with 0.25M
TNOA Eluent 3M LiNOa
sents the chromatographic separation of Th, Ce, S d , and Gd. Chromatographic Separation of Ba, Yb, Nd, and La by a Column of Cellulose Powder Treated with OSOM TNOA. Columns of cellulose powder treated with TSOA (4), T O P 0 (Z), and HDEHP (7, 8) have been used to separate milligram quantities of metal ions : Cellulose powder was treated with 0 . 5 N T S O A pre-equilibrated with 224 HK'Os. The mixture of the elements to separate (20 ing. each) was dissolved 0.002M in 3 ml. of a 8M Lin-03 H S 0 3solution. The columns had been
+
Table II. RF Values for La and N d as Functions of Molarity of TNOA Treating Solution
Eluting agent 4111 LiN03
RF TSOA, M 0 005 0 010 0 025 0 050
La 0.69
0.49
0.72
0 20
0 45 0 31 0.21
0.10 0.07
0 100
i
IYd 0 82
Yb
I
Table Ill. Influence of "01 Addition to 3M LiN03 on R F Values of l a , Ce, and Nd
(Paper treated with 0.20M TS0.4) HNO,
final molarity 0.002 0.020 0.200
1,000
La 0.11 0.14
0.40
0.61
RF Ce 0.15 0.19
0.45 0.69
Sd 0.33
0.42 0.67 0.80
Cokmn volumes
Separation of Ba, Yb, Nd, and La by column of cellulose powder traated with 0.50M TNOA
Egws 4.
Flow rate.
1558
ANALYTICAL CHEMISTRY
6 = 13mm., h = 140 rnm. 0.50 ml per minute.
Column dimension,
pre-equilibrated by 50 ml. of such a with f3-U L i N 0 3 ~ barium was eluted first, then ytterbium, follo~ved aft'er a discontinuous elution with 6 to 5.2M LiN03 (gradient, -0.2~11 per column volume); neodymiuni and lanthamum were fin d l y eluted with 4 to 1.4M LiNO3 @adient, -0.2M per column volume), Figure 4 shows the elution curve of this separation. The fractions (4 ml.) were analyzed quantitatively by complexometry, while the elements were identified by HDEHP chromatography ( 6 ) , comparing the RF's of the peaks with those of the pure elements.
Table IV.
Chromatographic Separation of Rare Earths on Paper Treated with 0 . 2 5 M TNOA
Rare earths separated Pr-Sin-D y Th-La-Y Pr-Dy-Yb
Th-U-La-Sc Ce-Sm-Ho Th-La-Pr-Eu La-Sd-Er Th-Yb-La Th-Ce-Sd-Gd Ce-Dy-Yb
Lis03 eluent, dl 3 1
6 1
Length, cin.
17 15 16 17
4
17
2 2 G
16
3 5
17 15
16
15
RF values Pr0.13; S m 0 . 5 2 ; DyO.S9 Th 0.01; La 0.62; Y 0.93 Pr 0.04; Dy 0.22; Y b 0.68 Th 0 . 0 0 ; U 0.14; La 0.54; Sc 0.80 Ce 0.08; Sm 0.66; Ho 0.83 Th 0.01; La 0.24; Pr 0.57; Eu 0.88 La 0.18; S d 0 . 5 8 ; Er 0.92 Th 0.00; Yb 0.58; La 0.73 Th 0.01; Ce 0.25; S d 0.63; C k I 0.86 Ce 0.04; Dy 0.44; Y b 0.77
i*.CKNOWLEDGMENT
The author thanks E. Cerrai for valuable suggestions and A. illbini for useful laboratory collaboration. LITERATURE CITED
(1) Blake, C. A,, et al., Proc. 2nd Intern. Conf. Peaceful Uses At. Energy 28, 289 (1959). (2) Cerrai, E., Testa, C., Energia Xucl. ( M z l a n )8, 510 (1961). (3) Cerrai, E., Testa, C., J . Chromatog. 5, 442 (1961). ( 4 ) Zbid., (4) Zbzd., 6, 443 (1961). (5) Ibad., 7, 112 (1962). ( 6 ) Zbid., 8, 232 (1962). (7) Cerrai, E., Testa, C., Triulzi, C., Eneryia h'ucl. ( M i l a n ) 9, 193 (1962).
(8) Ibid., p. 377. (9) Coleman, C. F., Brown, K. B , U.S. .4t. Energy Comm., TID-7555,43-56 (1957). (10) Coleman, C. F., et al., Proc. 2nd
Intern. Conf. Peaceful Cses At. Energy 28,
278 (1959).
(11) Rlann, C. K., White, J. C., AXAL.
CHEM.30, 989 (1958). (12) Marcus, Y., Israel At. Energy Comm., Rept. R/20 (1959). (13) RIarcus, Y., Abrahamer, I., Zbid., IA-608 (1961). (14) Marcus, Y., Nelson, F., J . Phys. Chem. 63, 77 (1959). (15) Martin, A. J. P., Synge, R. L. N., Biochem. J . 35, 1358 (1941).
(16) IIoore, F. L., ASAL. CHI;>?. 32, 1075 (1960). (17) Moore, F. L., U. S. At. Energy Comm., NAS-NS 3101 (1960). (18) Peppard, D. J., et al., J . Znorg. h'ucl. Chem. 4,334 (1957). (19) Ross, W. J., White, J. c., k A L . CHEJI.31, 1847 (1957). (20) Testa, C., J . Chromatoy. 5, 236 (1961). (21) White, J. C., rlSTJ1 Spec. Tech. Publ. N238,27-34 (1958). (22) White, J. C., ROSS,W.J., U. S. A4t. Energy Comm., NAS-NS 3102 (1961). RECEIVEDfor review April 23, 1962. Accepted July 23, 1962.
Use of Countercurrent Distribution in the Quantitative Determination of the Alkaloids of Commercial Veratrine GLENN R. SVOBODA' Department of Pharmaceufical Chemistry, School of Pharmacy, University o f Wisconsin, Madison, Wis.
b The partition characteristics of the Sabadilla alkaloids have been applied to a countercurrent separation of cevadine and veratridine from the other alkaloids in commercial veratrine. The mathematics of countercurrent distribution have been utilized to develop a method for the determination of the equivalent percentage composition of the known alkaloids and analyses for two samples of veratrine are reported.
C
distribution is considered primarily as an effective sqiaration technique. However. a knodedge of the partition characteristics of the individual components of an alkaloidal mixture also allons a quantitative analysis of the total alkaloids without chemical modification, provided chemical artifacts do not appear under the conditions of separation. Veratrine is a commercial preparation OCKTERCURREKT
of the total alkaloids froin the seeds of Schoenocaulen oficinale (Sabadilla) available from S. B. Penick and Co. Several methods of analysis h a r e been reported in the literature (1, 5 ) . These procedures do not take into account the comple.;ity of the alkaloidal mixture present. iiuterhoff ( 1 ) assumed that veratrine was composed solely of a mixture of the alkaloidal esters, reratridine and cevadine, the two major components. On this basis an analysis of the steam-volatile veratric and angelic acids available upon ester hydrolysis would lead to erroneous composition values. Xacek et al. (5) have pointed out that such a procedure (1) does not take into account the presence of other ester alkaloids such as cevacine ( 4 ) , which liberates acetic acid upon hydrolysis. illso, duterhoff ( 1 ) does not take into account the nonester alkanolamines such as sabine (S),veragenine (Q), and veracerine (4,or the vanillic acid ester of veracevine, vanilloylveracevine (8).
The paper chroniatographic methods suggested by Macek ( 5 , 6) are useful for qualitative and quantitative investigations of the major components of S a b adilla but are unsatisfactory in the identification of minor components. Rfacek et al. (6) have noted the difficulty of placing large quantities of veratrine on paper and then identifying a component which was less than 1% of the alkaloidal mixture. The partition characteristics of many of the alkaloids found in Sabadilla had been previously studied in this laboratory ( 7 ) . On the basis of this work it seemed feasible t o utilize a series of countercurrent distributions t o separate the major alkaloidal components of the mixture to facilitate a quantitative estimation of the minor constituents by the application of the mathematics of countercurrent separations. 1
Present addrese, Freeman Chemical
Corp., Port Washington, Wis.
VOL. 34, NO. 12, NOVEMBER 1962
1559
