V O L U M E 23, NO. 3, M A R C H 1 9 5 1 large number of terpene samples have been investigated and the great majority of them (56 out of 58) have been found impure by examination of chromatograms in five solvents, much of the tedious physical and chemical examination of the sample can he eliminated. The oils examined gave no sign of decomposing or isomerizing on silicic acid. Silicic acid is an excellent adsorbent for this work, because it resolves compounds of very similar t,ype, as illustrated in Table IV. The identification of terpenes should be facilitated by application of the principle of characteristic RF values to the chromatographed samples. In identifying constituents of natural products where the amounts of fractions concerned may be small, the volume of material necessary to secure identification can be substantially reduced. Amounts of oil as small as 0.5 microgram have been detected. Some of the color tests reported here have useful implications for indicating structure-for instance, from Table I1 it is evident that the fluorescein-bromine test reveals the possible presence of ethylenic-type double bonds. Other indications of structure are obtained from a study of Table 11, and many other tests can be applied which have specificity for certain types of structures. As a chromatographic method aside from the advantages to terpene chemistry, many features can be pointed out
425
\+ith Ivss (1r:tPtic revealing agents. These reagents have been used to inthrate traces of impurities in some terpenes which were thought to be pure by reason of physical constants and chromatography \vith other compound-indicating tests. The spray gun used for all of the work except for the sulfuric acid was an artist’s air brush. As shown by Meinhard and Hall (9),the completed chromatograms could be stripped off on Scotch tape and pasted on suitable cards for filing and reference purposes. This was not possible for strips sprayed with cold sulfuric acid, but the chromatograms which were heated after spraying with acid could be saved in the same manner. ACKNOWLEDGMENT
The authors are indebted to Richard Course of this laboratory for assistance in fractionating the oils. LITERATURE CITED
(1) (2) (3) (4) (5)
Boldingh. .J., Ezperientin, 4, 270 (1948). Boulea, V., Bull. soc. chirn. Belyes, 1, 117 (1907). Carlsohn, H., and Muller, G., Ber., 71, 858 (1938). D a t t a , P. E‘., and Overell, B. G., Biochm. .J., 44,XLIII 11949). Datta. Y. P.. Overell, €3. G., and Stack-Dnnne, MI.. .\ratwe, 164, ti73 11949). Flood, €I., Z.nn.ctl. Chem., 120, 327 (1940). Hopf. P. P., J . Chem. Soc., 1940, 785. Kirchner, J . Q., and Keller, G . J., J . Am. Chem. SOC., 72, 1867 (1950j. Meinhard, J. E., and Hall, h’. E . , -4x.4~.GEM., 21, 186 (1949). Ramsey. L. L., and Patterson. W. L., J . Assoc. Ofic. A ~ T . Chemists,31,139 (1948). Rockland. 1,. E., and Dunn, 51. S.,Science, 109, 539 (1049). Sease, .J. JT., J . Am. Chem. Soc., 70, 3630 (1948). Spath, E., and Kainrath, P., Be?., 70,2272 (1937). Wasicky. R., and Frehden, 0..Microchim. Acla, 1, 55 (1937). n’interstein. 4 . . and Stein, Q., 2. physiol. C’hem., 220, 247 (1933). Zechnieistri, L.. “Progress in Chromatography, 1938-1947,” Loiidon, (’hapman and Hall, 1949; New York. John Wiley & Sons. Zechnieister. L., and C:liolnoky, L.. “Principles and Practice of (’hromatol5raph~,”New York. John Wiley & S o ~ i s 1941. ,
The chromtttostrip combines some of the advantages of paper and column chromatography. It adds the rapidity ( a strip may be run in 0.5 hour) and the ease of spot development of paper chromatography to the wide range of adsorbents of column chrom:ttography . The method can be used for rapidly checking solvents and adsorbents for larger columns, because the results obtained are comparable. Although small conventional columns can be used for the same purpose, the proposed method is more rapid inasmuch as one man can easily run a set of 40 strips in an hour’s time. More drastic reagents can be applied to locate compounds than are permissible with paper strips. “Wet tails” (irregular flow of solvent up side of paper due t o touching of the glass wall) and similar annoyances of the flexible paper are avoided. I t is a microchromatographic method and is much more convenient. than a packed column for micro work.
(17)
The use of concentrated sulfuric acid and concentrated sulfuricnitric acid mixture for elucidating positions of organic materials on a chromatogram eliminates the uncertainty usually associated
KECEIVEU.June 26, 19,50. Presented hefore the Division of Analytical Chemistry a t t h e 118th hfeeting of t h e . i x E R r c a . u CAEYICAL S O C I E T Y , Chi. csgo, Ill.
(6) (7) (8)
(9) (10) (11) (12) (13) (14) (15)
(16)
Automatic Fraction Collector for Chromatographic Separations DON4LD F. DUKSO, ELWYN I). SClI4LL,
AND
ROY I,. WHISTLER
Purdire University, Lxfayette, Znd.
A
METHOD has been developed recently for the chroniatographic separation of sugars on charcoal (1). For application of the method to separations on a larger wale, an automatic apparatus has been devised which feeds the mixture and the devtAlopers in succession to the top of the chromatographic column and collects each effluent in separate receivers. The device h:ts performed well in the fractionation of hydrolyzates of guaran, xylan, and starch, and may have general application for chromatographic separations. .4s shown in Figures 1 and 2, the apparatus consists of containers for the solutions to be fed to the column, receivers for the effluents, solenoid valves operating in pairs so that when energized one container and the corresponding receiver are connected to the column, an electronic control unit, a float chamber, a light source, a photoelectric unit, and a vacuum pump. The details
of the control circuit and a list of parts are given in Figure 3.
All the materials are readily available and easily assembled. unit can be built for approximately $200, exclusive of labor.
The
OPERATION OF CIRCUIT
The apparatus is readied for operation by filling the containera, connecting the receivers to the distributor, and inserting a previously packed and wetted column. The space in the tube above the adsorbent must be completely full of liquid to ensure a proper start. To initiate the operating cycle the main power switch, SW,, is placed in the “on” position, the reset button, SW,, is pressed, and the momentary contact switch, SWI, is closed. The operation of the apparatus is fully automatic from this point. After a delay of 70 seconds, the first solenoid valve in the upper bank is energized, allowing the solution in the first container to fill the float chamber and flow to the top of the column. At the same
ANALYTICAL C H E M I S T R Y
426
Chromatographic separation of materials i n sizable quantities involves the handling of large amounts of adwrbents and solvents. In order to reduce the labor required in such a preparative prooedure, an electronically controlled apparatus has been devised to feed different developers to a column and collect the effluentsin separate receivers. Essentially, the apparatus consista of a photoelectric system, con-
trolled by a float, which operates successive pairs of solenoid valves through the medium of a stepping relay. The developers are passed through the column i n sufficient volume and i n proper sequence to effect the desired separation. The apparatus has been used for the fractionation of hydrolyzates of guaran, xylan, and starch. I t may have general application to chromatographic separations.
Although the described apparatus has four containers and four receivers, the number may he increased by the addition of solenoid valves in pairs. These pairs of valves are connected to successive positions of the stepping relay in the manner shown in the circuit diagram. At the same time, the number of BUCcessive positions in which the power relay, Rr, is energized must be increased by the number of pairs of valves added. The other modifications include the addition of containers, receivera, and connections to the effluent distributor. The appzmtus will cease t o operate in the ca6e of interruption of the current supply or energiaatition of the stepping relay for
Figure 1. Automatic Fraction Collector
time. the first solenoid valve of the lower hank is energized. con-
to the'column, the float drops and cuts off the light beam. - T h i s initiates the switching cycle, which consists of turning off the light source, de-energizing the first pair of solenoid valves, energizing the next pair of valves, and turning on the light ~ o u r e e after the float has risen to the top of the float chamber. The solution in the second container is applied to the column and the effluent is collected in the second Goeiver. When the float again interrupts the light beam the switching cycle is r e Deated. The auoaratus team o o e r h m when all the solution tn the last cont&er has passed through the column. It is not required that all containers be used during each run, but only that the developers be placed in successive containers. If for example, only the first three containers are utilized for a parhcular run. the amaratus will cease oueration 120 seconds after all
Figure 2.
Schematic Arrangement of Components of Automatic Fraction Collector
A. B.
C.
D.
E. F.
Cd,,," ~~~~~~~~
6. Control unit H . EWuentdirtributor, aee F i g w 4 f o r detaile 3. V5-0 Bolenoid valve, Skinnu Chuck Co.
K.
the float in this period will not &activate t6e circuit.-
L.
M.
Effluent receivers Vacuum pump Air rent
V O L U M E 23, NO. 3, M A R C H 1 9 5 1
427
To photocell terminals Common
To V5-t valves in re-
spective pairs
Photocell unit Light
Base plug for vacuum pump
Figure 3.
Circuit Diagram of Control Unit
S W i . Toggle switch, rated 12 amperes a t 125 volts SWs. Momentary contact switch, rated 3 amperes a t 125 volts SWs. Momentary contact switch, rated 12 amperes a t 125 voltk R I . PR4A, heavy duty power relay, Potter and Brumfield R2. PRSA, heavy duty power relay, Potter and Brumfield Rs. L-205 power relay, Detect-0-Ray Corp. R4, Rs, RI. MR2A, medium duty power relay, Potter and Brumfield Re. Series R stepping relay, 24 positions, with electrical reset and two gang switch, Guardian Electric Manufacturing Co. R7. MR14, medium duty power rela), Potter and Brumfield Di, D4. 115h010 thermostatic delaj relay, 10-second delay, Amperite Co. Dz. 115N060 thermostatic delay relay, 60-second delay, Amperite Co. Ds. 115h0120 thermostatic delay relay, 120-second delaj , Amperite Co.
more than 120 seconds. I n the latter case, a safety device consisting of a delay relay, D3,and a power relay, R,, will operate to interrupt the current supply, thereby avoiding damage to the stepping relay which is rated for a maximum energization period of 5 minutes. This safety device will operate if the light beam
ment using a 44 X 265 mm. column of adsorbent. The weight of material which can be adsorbed and the volume of each of the developers required for the fractionation are determined. The large scale separation is carried out with a column which must have a ratio of length to area a t least equal to that of the small test column. When this condition is met, the larger column is operated on the basis of the ratio of its volume to that of the test column. The weight of material which can be adsorbed is equal to the weight determined for the test column multiplied by the adsorbent volume ratio. This same ratio determines the volume of each developer required. For use with the apparatus, glass tubes ranging from 64 X 565 mm. to 80 X 920 mm. fulfill the required conditions of length and area. LITERATURE CITED
(1) Whistler, R. L., and Durso, D. F., J . Am. Chem. SOC.,72, 677 (1950). RECEIVED September 16, 1950. Journal Paper No. 484 of t h e Purdue Agri-
Figure 4. Details of Effluent Distributor
is not restored within 120 seconds of the beginning of each switching cycle because of failure of the lamp filament, of the photoelectric system, or of the float to rise. APPLICATION OF APPARATUS
The data necessary for the fractionation of a large amount of sugar mixture are obtained by a preliminary small scale experi-
cultural Experiment Station, Lafayette, Ind.
Stable Internal Standard Flame Photom et er-Correc tion In the article on “Stable Internal Standard Flame Photometer” [Fox, C. L., Jr., ANAL.CHEM., 23, 137 (1951)] there is a misprint in Table 11, Working Standards for Photometer. The heading for the f i s t column should be MI./l.
