methyl esters of the estimated boiling point can then be run for direct comparison with the unknown. I n this method fumaric acid is not distinguished from maleic acid, since under the methanolysis conditions employed both are converted t o dimethyl methoxysuccinate (3). It is expected that itaconic acid would also add methanol across the double bond, but this was not verified. Dibasic acids and polyols could also be quantitatively identified according t o the procedures of Esposito and Swann
ACKNOWLEDGMENT
(2) and Esposito ( I ) . These then could be followed by the quantitative method described in this paper. Quantitative results for the dibasic acids are in good agreement with the known composition of the resins (Table 111). Glycol content also agrees with the known composition using an empirical response factor of 1.4 (obtained by experiment). The total moles of glycol should equal the total moles of dibasic acid in a normal polyester, so errors in the determination are easily detected.
The author expresses appreciation t o the Oronite Division, California Chemical Co., for its interest and funds provided for this study. LITERATURE CITED
(1) Esposito, G. G., AXAL. CHEM. 34, 1173 (1962). (2) Esposito, G. G., Swann, hl. H., Ibid., 34, 1048 (1962). (3) Fyolka, Von P., Linz, J., Runge, F., Makromol. Chem. 26, 61 (1968). RECEIVEDfor review August 20, 1962. Accepted December 3, 1962.
Horizontal Chromatography Accelerating Apparatus Separation of Dyes and Indicators J. F. HERNDON, J. C. TOUCHSTONE, G. R. WHITE, and C. N. DAVIS The Malvern Institute, Malvern, Pa.
b The Rf values for 22 dyes and indicators separated by a new centrifugally accelerated horizontal paper chromatography apparatus are given. Clear, reproducible separations can b e effected in as little as 10 minutes. Amixture of seven dyes was separated by the tandem technique.
P
chromatography can be a valuable tool for the analysis and identification of commercial dyestuffs and indicators. Adsorption on various columns has also been widely used. APER
Dye 1 L 3 O / O . 15b 0 1. Congo red 0 0.05 0.11 2. Toluidin blue 0 0.06 0.04 0.06 3. Eosin Y 0 0.17 0.09 4. Phloxine B 0.43 0.20 0.34 5. Fluorescein 0 0 0 6. Amido black 0.73 0.57 7. Bromocresol blue 0.56 0.08 0 ... 8. Saffarin 0.82 0.43 9. Bromocresol green 0.67 0 0 0 10. Janus green 0.60 O / O . 77 11. Bromocresol purple o/o. 73 0.42 0.22 12. Bromothymol 0.48 0.10 0.07 13. Indigo carmen o.ofi;o, 20 0 0 14. h'eutral red 0.09 0.13 0.05 15. Orange I1 0 0 16. Superchrome black 0 0 0 0 17. Erythrosin B 0.85 0.73 0.68 18. Cresol red 0.18 0.50 0.45 19. Methyl red 0/0.09 0 0 20. Bismarck brown 0.76 0.60 0.53 21. Thymol blue 0.17 0.38 0.12 22. Methyl orange a Conditions for separation given in Table 11. b Two figures denote two bands separated.
238
ANALYTICAL CHEMISTRY
0
4
0 0
0.17 0.42 0.75 0
0.91
0
0.93 0
0.94 0.92
6"
0.16
0
0.12 0.93 0.74 0
EXPERIMENTAL
The operation of the centrifugally accelerated horizontal chromatography apparatus has been described in detail (2). The apparatus was operated with an automatic solvent delivery systemzin the present studies. The dyes were divided into-:three groups according to solubility. In Table I dyes 1 to 12 are soluble in distilled water, 13 to 17 are soluble in 10%
Rf Values for 22 Dyes
Table I. 0
require temperature control and equilibration of solvent systems.
Hough, Jones, and Wadman (S), Lederer ( 5 ) , and Zahn (12) have described the separation of dyes by paper chromatography. Pagani (8) and Jungbeck (6) used ascending chromatograms. Sivarajan and Parikh (9) used circular horizont,al chromatograms developed with buffers for separation of reactive dyes. Maccio (6) and Verma and Dass (11) have described the use of reverse-phase systems t o separate fatsoluble dyes. Moloster (7') describes the separation of many different dyes. These methods in general have in common long development times and
0.91 0.31
Solvents and conditions" 5 7 6 0 0 0 0.10 0 0.07 0.30 ... 0.28 0.54 0.60 0.55 0.79 0.66 0.66 0.13 ... 0.12 0.87 0.85 0.87 0.06 0.16 0.11 0.86 0.88 0.91 0 0.05 0 0.90 0.90 0.88 0.87 0.94 0.92 0.44 *.. 0.41 0 o/o. 09 0.04 0.23 0.35 0.35 0
0.24 0.93 0.68 O / O . 15 0.93 0.47
0
0.26 0.88 0.68 O / O . 13 0.91 0.45
...
8 0/0 .71 0
0.23 0.50 0.78 0.10 0.89 0
0.94 0
0.94 0.86 0.48
0
0.31 0
9 0/0.46b 0.34 0.21
O / O . 3gb
0.59 0.18 0.86 0.38 0.84 0.15 0.88 0.79 0 49 o/o, 29 0.45
0.48 0.13 0.77 0.29 0.77 0.15 0.83 0.76 0.44 O / O . 28 0.45
0
0 ...
... 0.90 0.21/ O .63
0.18 0.93 0.23/0.81
0.91 0.54
0.78 0.35
0.91 0.42
0.86 0.70
0
0
...
10 0.27
...
0
0
0.26 0.88 0.53 0/0 .38 0.86 0.60
aqueous methanol, while 18 to 22 are soluble in methanol. The commercially available dyes were made u p in 0.05% solutions without purification. Developing Solvents. 3% sodium chloride ( 1 ) . p H 3. 20.32 ml. of 0.1N hydrochloric acid and 50 ml. of 0.1M Dotassium acid phthalate diluted to 100 h l . with water. p H 7. 29.63 ml. of 0.1N sodium hydroxide and 50 ml. of 0.1M monopotassium phosphate diluted to 100 ml. with water. pH 10. 43.90 ml. of 0 . l N sodium hydroxide and 50 ml. of 0.1M boric acid dilut8edto 100 ml. with water. 1 Butanol - water - NHbOH - 70% ethanol, 200:68 : 2 : 40 (lower layer after equilibration) ( 1 ) . 1-Butanol-acetic acid-mater, 20: 5 : 75 (lower layer after equilibration) ( 1 ) . NH40H-1-propanol. 80: 20, used after equilibration. The dyes ( 5 pl. of solution) were
-
banded on the starting lines of 11-mm.wide strips with a pipet. Whatman 1 and 3 MM papers were used throughout this work. Centrifugal force, solvent flow, and the nature of the media are given in the results.
1.
2. 3. 4. 5.
6. Whatman 3MM
7 . Whatman 3MM
8. Whatman 1
9. Whatman 3MM
10. Whatman 1
STRIP A
-CUTTING
LINES
/
'
-5OLVEHT
FRONT
J ' Lx-C"C--+,-z----4
RESULTS AND DISCUSSION
Table I shows the Rj values obtained for 22 dyes separated b y centrifugally accelerated horizontal paper strip chromatography, using 10 combinations of conditions as listed in Table 11. Generally the class into which the dye fell could determine the condition of choice for separation. Water, saturated with organic solvents, gave greater movement of most of the dyes. Some of the dyes separated in two bands, as in the case of Congo Red, bromocresol purple, neutral red, etc. The conditions investigated gaJ-e no movement of Superchrome Black and only little
Table 11. Conditions for Separation of Dyes of Table I (Developed for 5-em. solvent front movement a t 500 r.p.m. Chamber (25'. C.) presaturated with 3 ml. of solvent. Values represent average of 3 Rf determinations.)
Media Whatman 3hIM Whatman 3MiM Whatman 3MM Whatman 3MM Whatman 1
--
Solvents and conditions Solvent delivery, ml./min. Solvent 0.10 3y0 NaCl Phthalate, pH 3 0.80 0.12 Phosphate, pH 7 Borate, pH 10 0.11 0 07 NHaOH, 80 ml. 1-Propanol, 20 ml. 0.09 NHdOH, 80 ml. 1-Propanol, 20 ml. 1-Butanol, 200 ml. 0.07 H20,88 ml. &"*OH, 2 ml. 70% ethanol, 40 ml. (lower phase) ]-Butanol, 200 ml. 0.07 H20, 88 ml. NHrOH. 2 ml. 70% ethanol, 40 ml. (lower phase) 1-Butanol, 4 ml. 0.1 Acetic acid, 1 ml. HpO, 5 ml. (lower phase) 1-Butanol, 4 ml. 0.06 Acetic acid, 1 ml. HzO, 6 ml. (lower phase)
STRIP E - P O R T I O N I a N E W S T R I P
C O N 0 0 RED,
STRIP C
12
1G
-
N E W STRIP
1 'L ME^^^^^^^^$ ORLUOE
PORTION
-
I
S O L V E N T FRONT
lI
L a WCK
- ---
-~ 7--
S O L V E h T FRONT
WICK
2
-FT =
E
B R O k 5 R E S O L GREEN'
E
;
4
I
. c
1
CRESOL R E O
Figure 1 . Three-dimensional separation of dye mixture Chamber ('25' solvent
A.
B
Time, min.
___-_
-x--LI
C.
C.) preroturoted
with 3 ml. of
Solvent 3 a t delivery rate of 0.1 7 ml./min., developed until front moved 4 cm. Speed 500 r.p.m. using Whatmon 3 MM poper Solvent 7 a t delivery rate of 0.09 ml./min., developed until front moved 6 cm. Speed 500 r.p.m. using Whatman 3 MM p a p e r Solvent 2 a t delivery r a i e of 0.12' ml./min., developed until front moved 6 cm. Speed 500 r.p.m. using portion 2 of strip A
11
13 12
20 19
12
13
10
inohjlity for several of the other dyes listed. The advantage of using a series of different conditions in search for optimum conditions for separation, as well as some indication of purity, is shown with bromocresol purple, which under conditions 1 and 3 gave two bands and under other conditions gave a single band, This could have been due t o either impurity of the dye or reaction of the developing solvent with the dye. Table I11 illustrates that centrifugal force had no effect on the Rf of the seven dyes that were investigated at four rates of centrifugation. Although the R, remained constant, centrifugal force caused faster movement of solvent front and sharper separations of the zones. Figure 1 describes the separation of a synthetic mixture of seven dyes, using
Table 111. Effect of Centrifugal Force on RJ of Dyes (Conditions of 2, Table 11. Development for 5-cm. movement of solvent front. Values represent average of five Dyes Bromothymol Bromocresol Thymol R.p.m. Methyl red Fluorescein blue Methyl orange green blue 0 0.18 f 0.014 0.20 i 0 006 0.22 f 0.009 0.35 i 0.021 0.45 f 0.016 0.57 f 0.018 300 0.18 f 0.008 0.22 f 0.011 0.21 i 0.010 0.37 f 0.002 0.44 f 0.014 0.58 f 0.004 ,500 0.18 f 0.007 0.20 f 0.004 0.21 f 0.010 0.37 f 0.005 0.42 f 0,008 0.58 f 0.011 700 0.18 f 0.005 0.19 f 0.009 0.20 f 0.005 0.38 f 0.008 0.42 f 0.015 0.58 f 0.018
determinations.) Bromocresol blue 0.60 f 0.015 0.58 f 0,010 0.58 f 0.008 0.58 f 0.008
VOL. 35, NO. 2, FEBRUARY 1963
239
the tandem technique and a series of different conditions. The original chromatogram separated into four zones. The second zone contained three dyes, while the fourth consisted of two components. These mixed zones were cut out and rechromatographed under different conditions as noted. This resulted in separation of the individual dyes without eluting and reapplying t o new strips. The identity of the dyes was based on comparison of the R, values with those of the reference dyes run individually. I n the tandem procedure, modified from that of Tuckerman, Osteryoung, and Nachod (IO), the wick strip or the second-dimensional strip and dye-containing strip were overlapped 3 mm. and placed in the groove of the apparatus. An 11 X 20 mm. glass plate was placed over the overlapping zone and taped down.
Uniform transfer of solvent and solute across the joint took place. The combination of uniform solvent delivery, centrifugal force, strip groove modification in the chromatography apparatus, and the use of the tandem technique has made horizontal chromatography a useful tool in the investigation of dye separations.
Electrophoresis,” 2nd ed., Academic Press, New York, 1958. (2) Herndon, J. F., Appert, H. E., Touchstone, J. C., Davis. C. N.. ANAL. CHEM.34,1061 (1962 j. ’ (3) Hough, L., Jones, J. K., Wadman, W. H..J. Chem. SOC.1949.2511. (4) Jungbeck, S. V. F., Fachorgan Textilveredlung 15, 417 (1960). ( 5 ) Lederer, JI., Science 110, 115 (1949). (6) Maccio, I., rlnales D i m . Nacl. Qudm. 9, 52 (1956). (. 7.) Moloster. Z.. 4 n n . Chim. 3. 771 (1958). (8)Pagani, F., Tinctoria 58, 107 (1961). (9) Sivarajan, S. R., Parikh, S . G., Current Sci. (India)28, 323 (1959). (IO) Tuckerman, AI. X., Osteryoung, R. A., Nachod, F. C., $rial. Chim. Scta 19.239 (1958). (11) ’Verma, $1. R., Dass, R., J. Sci. Ind. Res. 17B,301 (1958). (12) Zahn, H., Testil-Prazis 6, 127 (1951).
ACKNOWLEDGMENT
The technical assistance of A. Cowperthwaite, K. Barthalamus, IT. D . Tillett, L. Tarone, and J. C. Keyser is gratefully acknowledged. The authors are indebted to R. Davis for preparation of the figures. LITERATURE CITED
(1) Block, R. J., Durrum, E. L., Zweig, G., “Paper Chromatography and Paper
RECEIVED for revien- September 4, 1962. dccepted November 6, 1962.
Separation of Lower Aliphatic Amines by Gas Chromatography Y. L. SZE1 and M.
L. BORKE
School of Pharmacy, Duquesne University, Pittsburgh 7 9, Pa.
D. M. OTTENSTEINZ Fisher Scientific Co., Pittsburgh 79, Pa.
b A method has been developed for the gas chromatographic separation of a mixture of ammonia, methanol, and the three methylamines, using a mixture of tetrahydroxyethylethylenediamine and tetraethylenepentamine as the partition liquid. By studying the role of these two partition agents in the retention of lower aliphatic amines, the separation of a multicomponent mixture of 10 amines has been achieved. Particular attention has been paid to the elimination of the adsorption activity of the solid support.
S
of the lower aliphatic amines by gas chromatography is difficult because of support effects and the problem of obtaining the proper solvent efficiency. When dealing n i t h basic nitrogen-containing compounds there i s a tailing effect due t o the adsorption of the sample on the support. This effect is particularly acute in the case of the methyl amines. T o separate the methyl amines and ammonia, James, Martin, and Smith (5) employed a mixture of 5-ethylnonen-2-01 and liquid paraffin nith Celite 545 as the support. Burks and EPARATION
Present address, Department of Biochemistry, University of Wisconsin, Madison 6, Wis. Present address, Johns-Manville Products Corp., Manville, N. J. 240
ANALYTICAL CHEMISTRY
coworkers (1) used triethanolamine coated on C-22 firebrick, while Hughes (4) employed a mixture of n-hendecanol and n-octadecane. I n each case the deactivation of the support was not complete. James (6) further studied the separation of 27 lower aliphatic amines using two columns, one containing liquid paraffin and the other Lubrol MO (a polyethylene oxide). James, Martin, and Smith (6) washed the support, Celite 545, with methanolic sodium hydroxide to reduce the support effects. Ring and Riley ( 7 ) , Decora and Dinneen ( 2 ) , and Smith and Radford (8) reported a more effective deactivation n-ith other amine samples by depositing an alkali hydroxide on the support. I n other cases the deactivation problem was avoided by using Teflon (3) rather than the usual diatomite support. I n this study, particular attention is paid t o the deactivation of the support, the separation of the methyl amines and ammonia, and t o the general separation of the methyl-, ethyl-, and n-propylamines. EXPERIMENTAL
Equipment. Fisher-Gulf Partitioner, Model 160, modified b y placing a flash evaporator between t h e sample inlet a n d t h e column assembly. Column Materials. Chromosorb IT7, 60 to 80 mesh, tetrahydroxyethyl-
ethylenediamine ( T H E E D ) (Fisher Scientific Co., Pittsburgh, Pa.); diglycerol, technical grade and polyglycerol W (20,000 to 30,000 centipoise at 77’ F.) (Colgate-Palmolive Co., New York, N. Y.); Carbowas 400 and 1540 (Union Carbide Chemicals Co.) ; tetraethylenepentamine ( T E P ) (Eastman Organic Chemicals, Rochester, N. Y.). Reagents. All of the amines were reagent grade and were used without a n y further purification. T h e three methyl amines were received as 25% aqueous solutions and were used as such. Column Preparation. T h e liquid phase was applied to the support from a solution of a volatile solvent. T h e solvent was removed using a rotating vacuum evaporator. Potassium hydroxide was applied in a similar manner using methanol as t h e volatile solvent. T h e dried packing was placed in a 4-mm. i.d. column and conditioned for 24 hours at t h e proper temperature with a constant flow of helium through the column. RESULTS A N D DISCUSSION
Deactivation Study. Of the supports available for use, Chromosorb W was chosen because of its relative inertness as compared to Chromosorb P and because of its relatively high column efficiency as compared t o t h e Teflon type supports. Of the amines studied, the methyl amines were the
