containing from 5 to 140 p.p.m. of propadiene in a propene fraction. Paraffins, olefins, and diolefins did not interfere; acetylerk compounds were absorbed by the liquid phase. All the subshncea were identified by the retention tia of the single pure c’omponenta and controlled by mass spectrometry. These results illustrate that traces of propadiene to a t least 5 p.p.m. can be determined by this procedure. The possibility of detecting traces of propadiene below 5 p.p.m. was investigated. An enrichment procedure, using the same colhmn, utilized the recover!. into a cold trap at the instrument exit of the fraction corresponding to the
propadiene retention time. This separation was repeated several times; the propene to be tested was introduced into the column and collected each time in the same trap as the propadiene fraction, The propadiene.-enriched gas recovered in the trap was determined using the procedure as described, and the propadiene content reported on the basis of the amount of initial gas introduced in the column. Less than 1 p.p.m. of propadiene is easily detected by this procedure. Precision of Method. When propadiene peak areas are very small, corresponding to about 5 p.p.m. of propndiene, the 95% confidence limits
calculated from 10 measurements are +15% of the mean. When the propadiene concentration is 25 p.p.m., the corresponding limits are k3% of the mean. LITERATURE CITED
(1) Bradford, B. W., Harvey, D., Chalkle D. E. J. Znat. PefToZ.41, 80 (1955). (2) . . h e w , d. M., McNesby, J. R., Smith, S. R.. ‘ Gordon, A. S.,- ANAL. CHEW 28, 979 (19565 (3) Johnson, H. W., Stross, F. H., Zbid., 30,1587 (1958). RECEIVEDfor review December 15, 1958. Accepted May 1, 1959.
Separation and Quantitative Determination of Methyl Arabinosides Using a Starch Column with an Improved Automatic Control DWIGHT F. MOWERY, Jr. New Bedford lnsfifute of Technology, New Bedford, Mass.
b A starch column has been found greatly superior to a cellulose powder column for the separation and quantitative determination of methyl arabinosides. As with mannose and the methyl mannosides previously investigated, the absorbances of arabinose and the methyl arabinosides with anthrone in 9070 sulfuric acid can, within the limits of accuracy of the chromatographic separation, be converted to weight present by a single proportionaliiy factor. The accuracy i s comparable to that of the previously reported methyl mannoside determination. A liquid pump is more satisfactory than gas pressure for circulating solvent through the column. An improved automatic control for the fraction collector was designed to operate by impulses obtained from the fraction-measuring siphon. This device automatically handles the forerun, a center run where a wide band of pure solvent emerges, and the final switching off of the fraction collector and the liquid pump.
T
four methyl niannosides have been separated sufficiently for quantitntive determination using a cellulose po\vder column (4). The four methyl arabinosides, however, cannot be completely separated under the same conditions (Figure 1, upper elution curve) or by other solvent mixtures. In all cases HE
the a- and @-pyranosides remained unresolved. A starch column (2) was therefore substituted for the cellulose powder column. In order to maintain an appreciable flow rate through the starch a pressure of about 25 p.s.i. was required. Use of air or nitrogen to provide this pressure resulted in solution of the gas in the chromatographic solvent butanol-pyridine-water-10 :3 :3 (volume)-and evolution of the gas in the lower part of the column where the pressure drops off, soon rendering it useless. Gas pressure was therefore replaced by a stainleae steel bellows pump working directly on the liquid. The equipment for automation of the fraction collection (4) was improved by use of a siphon for collecting fractions of equal size instead of the constant-flow timed fraction system used previously. This made it unnecessary to jacket the column for operation a t a constant temperature. Instead of a timer for automatically starting the turntable after the predetermined forerun, a special fraction-counting circuit was devised to empty a given number of fractions into a beaker before starting the turntable. The Coleman Junior Spectrophotometer was replaced by a Beckman Model B spectrophotometer and an especially constructed tube holder so that readings could be taken and minimum absorbance values could be recorded while the tubes were slowly rotating. A serially numbered set of 13-mm. tubes was made up
so that the errors of consecutive tubes averaged to zero. Replacement of 95% by 90% sulfuric acid (volume) gave a more sensitive color test with arabinosr and the methyl arabinosides. Except for these changes, the analytical procedure was identical to that in the separation and determination of methyl mannosides (4). As with mannose and the methyl mannosides the absorbances of arabinose and the methyl arabinosides are closely proportional to the weight present and, within the limits of accuracy of the chromatographic separation, the same proportionality constant may be used in all cases. Table I gives the results of analysis of known mixtures. The lower elution curve of Figure 1 was obtained with a 1 X 30 inch, 223gram starch column and the improved equipment using the same methyl arabinoside mixture that was chromatographed in a 1 X 30 inch, 130-gram cellulose powder (Whatman Standard) column in the upper curve of the same figure. An important advantage of a starch over cellulose is the relative ease of packing a starch column. Vibration and liquid pressure were used to settle the starch slurry and every starch column packed was satisfactory, whereas only one in six cellulose columns could be used. A starch column can be used continually until the gradually decreasing flow rate renders it unsatisfactory. The VOL 31, NO. 11, NOVEMBER 1959
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order of elution of carbohydrates from a starch column parallels the order on filter paper using the same solvent mixture, making the latter useful for preliminary qualitative work and spot identification. The starch column gives better separation than filter p-per, which, like the cellulose powder column, failed to resolve the a- and fl-methyl arabinopyranosides. Methyl mannosides are also separated more completely on a starch column. An explanation of the sharper bands, improved separation, and greater ease of packing a starch column probably lies in the reduced tendency for Channeling when using the uniform spherical starch particles instead of fibrous cellulose powder.
I
1912
ANALYTICAL CHEMISTRY
I
1
I
I
1
cellulose column 5 ml. fractions
In L = ,.ob 1 O a 2 t
._
/a-furanoside
W
," U B -
0
I I t
"1 1 I 0.6-
EXPERIMENTAL
Chromatographic Column. The chromatographic column (k, Figure 2) consists of a 1 X 30 inch glass tube flattened a t the lower end with a n exit tube sealed into t.he center. The top edge, formed into a thick lip and ground flat, is closed by a stainless steel plate with an '/ginch nipple and union threaded into the center. There are neoprene rubber gaskets (1) between the ground glass lip and the stainless steel plate a t the top, and between the flat bottom of the column and a brass plate carrying two brass rods, threaded at the upper ends and passing through holes in the top plate. The column can thus be clamped securely between the two plates by means of wing nuts on the rods. A '/e-inch thick porous Teflon disk fitting snugly inside the column and resting on the flat bottom supports the starch packing. The column was packed by introduction of successive portions of starch thoroughly mixed with the chromatographic solvent. A 1- to 2-ml. per minute flow of solvent through the colrimn was maintained by means of the bcl'ows pump used later in the operation of the column. The column was packed to th2 top while being mechanically vibrated. and was closed by a second snugly fitting porous Teflon disk pushed down flush with the ground glass lip. The charge was introduced into the column by disconnecting the union, m, removing the solvent in the short tube above the porous disk, introducing the charge into th(, bottom of the tube with a fine-stemmcd syringe-type micropipet, carefully replacing the solvent, and finally rcconnccting the union. A starch column of this kind provides 50 to 100 separations if care is taken to exclude all air and to limit the flow rate to 2 ml. per minute. A faster flow rate than this npprars to caiisr channeling in the column and permancxntly destroys its efficiency. Liquid Pump. The liquid pump (i, Figure 2 ) is a Corson-Ccrveny MicroBellows pump (Research Appliance Co., Allison Park, Pa.) with a S/d-inch stninless steel bellows. Less expensive bellows pumps of this make in which the off-centered cam is supported a t only one
n
I
1
18-furanoside
I 1\
starch column IO rnl.fractions
L-arbbinose
fraction number Figure 1 . Arabinosides eluted with butanol-pyridinewater, 10 : 3 : 3
Ph
n
Figure 2. Chromatographic column with liquid pump and fraction counter
end have proved unsatisfactory for the continuous duty required in the operation of a chromatographic column. All metal in contact with chromatographic solvent is 1/8-inch stainless steel pipe assembled wit,hout the use of thread compounds, but having all
threaded joints silver soldered on the outside. Fraction Collector. The fraction collector (f and la, Figure 2) is a Reco instrument set for volume collection using a 10-ml. siphon. The tube ho!der for small tubes was modified by slightly
enlarging the outer row of holes to take 16 X 150 mm. test tubes. A rubber cover, j, is used over the tubes to e l i i nate dust and reduce evaporation ( 4 ) . Fraction Counter. The fraction counter (Figure 3; a and b, Figure 2) was especially designed and constructed to allow a designated number of fractions of forerun set on the counter, a, to be discharged into the beaker, e, before automatically starting the turntable, f, for fraction collection. By means of a second counter, h, a stationary microswitch, c‘, and a knob, c, attached to the edge of the turntable, provision was made to stop the turntable in the middle of a run and collect in the beaker any given number of fractions set on counter b. This reduces the number of tubes that will be required for a run including a wide band .of pure solvent. The turntable is then automatically activated again by counter, 6 , and microswitch, b’, and fractions are collected until a second knob, d, contacts a second stationary microswitch, d, and switches off the fraction counter, fraction collector control, h, and liquid pump, i, and closes the magnetic stopcock, g (4). I n the wiring diagram of the fraction counter shown in Figure 3 the relay in the upper left corner is an ordinary 115-volt alternating current relay included in the circuit to eliminate double counts due to double pulses received a t certain times from the Reco fraction collector control, h. The delay relays are inexpensive 115-volt alternating current >second theromostatic delay relays and the counters are Veeder Root Standard Series BX-150703, 115-volt alternating current subtractive counters. Microswitches a‘ and b’ are attached to and activated by their respective counters, a and b, when the. latter have counted down to zero from the preset number of fractions. Double-pole double-throw push button switches and a 115-volt alternating current pilot light ( p , Figure 2; not shown in Figure 3) are convenient to determine whether the microswitches on the counters are open as they should be a t the start of a run. Plug-in receptacles are provided in the fraction counter for the fraction collector control, the liquid pump, the magnetic stopcock, and the microswitches activated by the turntable. Cords connect to 115-volt alternating current and the turntable outlet in the fraction collector control. Standard Mixtures. Five standard mixtures were prepared from crystalline L-arabinose (Pfanstiehl 104’), methyl 8-Larabinopyranoside (+243’),
+
F.C.conlrol turntable
supply
delay relays Figure 3.
Table 1.
Wiring diagram of fraction counter
Per Cent Compositions Found for Known Methyl Arabinoside Mixtures
a-Furanoside @-Furanoside &Pyranoside a-Pyranosldt L-Arabinose
A
B
C
D
E
29(27) 22(23) 25(25) 24(25)
44(45) 6 (5) 44( 6 45) (5)
6 (6) 45(44)
18(18) 21(21)
6 (6) 43(42) 42(42) 5 (5)
21(2l)
4 (5)
chromatographically pure sirups of methyl a-L-arabinofuranoside (- 128’) (I), and methyl 8-L-arabinofuranoside (+118’) (I), standardized by optical rotation, plus a crystalline mixture of methyl a-L-arabinopyranoside ( 17.3’) (3) and methyl 8-L-arabinopyranoside (f245.5’) (a), analyzing 95.3’3, of the a-isomer and 4.7% of the 8-isomer by both quantitative chromatographic analysis and rotation measurements. The methyl arabinosides were obtained from a mixture made by refluxing Larabinose in methanol with a strongly acidic ion exchange resin, Dowex 50 (6). A 4 5 X 48 inch starch column using ethyl acctaten-propyl alcohol-water-5:3:2 (volume)-was used to separate them and although the pyranosides were not separated by this solvent mixture, the a-isomer could be obtained in 95.3y0 purity after several recrystallizations from hot ethyl acetate. Samples of 100 pl. containing approximately 10 mg. of the carbohydrate mixtures were chromatographed on the analytical starch column.
+
The percentage of a given isomer in a chromatographed mixture was obtained by adding all absorbance values of fractions representing a given peak, dividing by the sum of absorbance vahies for all peaks, and multiplying by 100. Isomers were identified by adding more of one a t a time to a mixture already containing small amounts of each, and after each addition chromatographing a sample on the starch column.
4!(1;{
%{g{
The order of elution was found to be the same as for chromatography on paper. LITERATURE CITED
(1) Augestad, I., Berner, E., Acta Chem. Scand. 8, 251-6 (1954). (2) Gardell, S., Ibid., 7, 201-6 (1953). (3) Hudson, C. S., J . Am. C h m . Soc. 47, 2 6 5 8 (1925). (4) Mowery, D. F., Jr., ANAL.CHEM.29, 1451-3 (1957). (5) Mowery, D. F., Jr., Zbid., 77, 1667-9 (1955).
RECEIVEDfor review May 20, 1959. Accepted July 30, 1959. Division of Carbohydrate Chemistry, 136th Meeting, ACS, Atlantic City, N. J., September 1959. Work supported by National Science Foundation grant G-1720.
Chlorinated Insecticides in Surface WatersCorrection The article on “Chlorinated Insecticides in Surface Waters” [A. A. Rosen and F. M. Middleton, ANAL. CHEM. 31, 1729 (1959)] was presented before the Division of Water, Sewage, and Sanitation Chemistry, a t the 133rd Meeting of the American Chemical Society, San Francisco, Calif., April 1958, and should have carried a footnote reference to this presentation.
VOL. 31, NO. 11. NOVEMBER 1959
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