The addition funnel, 8, can be of various sizes, depending upon the number of troughs to be fdled with solvent. The funnel most often used in our work delivers 150 ml. of solvent, which fills three 101/,-inch glass paper chromatography troughs. The over-all length of the addition funnel is 75 cm.: diameter of funnel section is 4 cm, The siphon tube is constructed of borosilicate glass tubin 0.7 cm. in outside diameter. The adtition funnel is closed a t the top with a borosilicate glass standard-taper 30/30 cover, 7, through which the stem of the plunger is passed, The inside diameter of the opening a t the top of this piece is 1.4 cm., large enough to allow free movement of the plunger stem yet sufficient to eliminate solvent evaporation. The plunger used with this addition funnel has an overall length of 21 cm. The stem length is 17 cm., diameter 1.2 cm.; plunger length is 4 cm., diameter 3 cm. The solenoid, 5 , length 5 cm., diameter 4 cm., is positioned with respect to the iron rod to give maximum downward displacement of the plunger into the addition funnel when the current is turned on. A timing device, 9, is connected to the solenoid SO that the electric current is turned on for 4 seconds and then off again, using a 115-volt timer (Haydon Manufacturing Co., Inc., Torrington, Conn.). A 24-hour timer, 10, is connected to the first timer in such a manner that current is turned on after the preset number of hours. This instrument is a 115-volt, 60-cycle timer, Model 2T-20A (Vocaline Co. of America, Old
Saybrook, Conn.). Other commercially available timers can be used. An irrigation system constructed of borosilicate glam tubing, 0.7 cm. in
outside diameter, leads from the addition funnel to the troughs through a hole in the top of the chromatography tank. Any number of T-outlets can be used on the irrigation system, so as to fill the required number of troughs. The openings on these outlets are constricted or widened to assure that equal volumes are delivered to all the troughs. Standard-taper 10/30 joints connect the addition funnel and irrigation system. OPERATION
The funnel is filled to just below the bend of the siphon with the solvent used in the chromatogram under examination and timer 10 is set for the desired number of hours. When the current is turned on, the plunger is forced down into the liquid, the liquid level rises in the additior. funnel, and siphoning commences.
Figure 1. Diagram of funnel 1.
Spring Collar 3. Iron rod 4. Sleeve 5. Solenoid 6. Plunger 7. Funnel cover 8. Addition funnel 9. Timing device 10. 24-hour tlmer 2.
The addition funnel and irrigation system described can be used for both ascending and descending chromatography. It is desirable to use one addition funnel and irrigation system for each chromatography tank. LITERATURE CITED
( I ) Van Duuren, B. L., J. Natl. Cancer Zmt. 21, l(1958). (2) Ibid., (3) Van h % n , B. L., Schmitt, F. L.,
to be published.
WORKaided by a grant from the American Cancer Society, Inc., New York, h'. Y.
Automatic Recording Elution Analyzer for Organic Acids Chie Yarnazaki, Nobuya Nagashima, and Tadao Takenishi, Central Research Laboratory, Ajinornoto Co., Inc,, Kawasaki Kanagawa, Japan
A
LTHOUGH elution chromatography is now commonly used in organic chemistry and biochemistry, it is troublesome and time-consuming to titrate the separated fractions with alkaline solution. Therefore it is desirable to determine instrumentally and to record successively the amount of component acids in the effluent solution as it flows out from the chromatographic column. An automatic recording device may be suitable for elution analysis with a single solvent, but the interfercmetric method is useless for partition chromatography, because the change of the solvent is more sensitively detected by interferometer than the change of solutes. Infrared absorption measurement is suitable for the determination of organic acids in effluent solution Almost every carboxylic acid gives a strong and broad absorption band in the 1730- to 1700-
cm.-' region. Therefore, by fixing the wave number drive a t about 1715 cm.-' and flowing effluent solution through the cell in the infrared beam, the amounts of the acids can be recorded continuously. I n this recording the abscissa scale of the recorded curve is the time of elution, t. This was converted into milliliters of the effluent, v, by determining Av/At from time to time. Infrared energy can be readily obtained by using a large slit aidth, as high resolution is not required. By this infrared method, acids can be determined without chemical changes of the solutes, and the sample, after analysis, can be used for other purposes. EXPERIMENTAL
The schematic construction of the apparatus is shown in Figure 1. This
was used in conjunction with a PerkinElmer Model 21 infrared spectrophotometer equipped with an NaCl prism. A chromatographic column was prepared as usual. The outlet of the column was connected with a sample cell (1 mm. thick) by a Teflon tube. A cell with CaF2 windows was used, because the effluent solution contained a little water. The effluent solution was introduced into the cell through the lower inlet of the cell and led to collecting vessels through the upper outlet of the cell. The wave number drive was fixed a t 1715 cm.-', and only the chart drive gear was connected with the drive gear. The suitable chart speed was found to be 2 to 6 cm. per hour. Even the slowest chart speed of the Perkin-Elmer Model 21 spectrophotometer was too faat for the present purpose. A pulley system was therefore used to obtain a suitable speed. The slit aidth was fixed a t approximately 140 microns, and the source VOL. 32, NO. 6, M A Y 1960
733
Table I. Quantitative Data for Succinic and Malonic Acids Peak Heilrht Sample, (.lbsorbnn&), Half Width Area, JIg. I. (Ml.)) d o I X d hlg./hrea Succinic Acid 2.7 0,078 14.0 1.09 2.49 3.5 0.120 12.0 1 44 2.42 5.8 0.133 17.6 2.34 2.48 0.217 2.41 16.1 S.4 3.48 13.6 0,360 14.0 2.70 5.04 Mean 2.50 Std. dev. 4 . 2 %
JBE 'N
Malonic Acid 3.8 11.7 12.1 13.3
a
0.069 0.171 0.170 0.212
22.7 25.0 27.7 25.0
1.57 4.28 4.71 5,35
Mean Std. dev.
2.41 2.72 2.57 2.50 2.55 4.4%
TO EFFLUENT
Figure 1 . Schematic diagram of elution analyzer
Shown in Figure 3.
TIME. H O U R S
0.0
m
1.0 I
0
20 40
60
80 100
1.u 1 1 5 1
200
EFFLUENT, ml.
Figure 2. Chromatogram of mixture of acetic, fumaric, succinic, a-ketoglutaric, and malonic acids
Figure 3. Quantitative determination malonic acids
Flow rate changed after 4 hours of elution, as indicated by vertical line Column. Silica gel, 1 2 gromr saturated with 0.5N Solvents. Chloroform-butanol, 1 H~SOI Sample, Mg. Acetic 3.3 Fumaric Succinic 5.7 a-Ketoglutaric 5.0 Malonic 2.2
5y0,
-
current \vas adjusted to 0.3 to 0.4 ampere. Khen the elution was carried out with a solvent of constant composition, a suitable net screen was inserted into the reference beam to compensate for the loss of infrared energy through absorption by the solvent of the sample beam. When the composition of a developing solvent was changed continuously during elution, the base line balance was obtained by introducing the solvent into a reference cell through a separate reference column prepared in the same way as the sample column. The variation of the absorbance due to the eluent with time (fluctuation of the base line with temperature) is not 734
0
ANALYTICAL CHEMISTRY
serious, as may be seen in the flat part of the curve in Figure 2. A typical elution curve obtained by use of this apparatus is shown in Figure 2 To identify the acids, the effluent solution was collected in collecting vessels corresponding t o the different peaks recorded. The effluent solution need not be separated into so many factions as when a fraction collector is used. hfter the solvent in the individual fractions collected had been evaporated in vacuo, the residual acid was identified by observing the infrared spectrum in the usual n a y . Identification by infrared should be more reliable than paper chromatograph)-.
of succinic and
By measuring the area of separate peaks recorded from the half width (in milliliters) and the peak height (in absorbance), quantitative determination was possible. Quantitative data for the synthetic mixture of succinic and malonic acids are given in Table I. The methods of measurement of absorbance and half width are illustrated in Figure 3. These organic acids can be quantitatively determined from the elution curves recorded, by use of the factor 2 50 mg./l X d for succinic acid and 2.55 mg./l X d for malonic acid, with a n accuracy within &lo% (20). Path length may be critical, but displacement of eluent by solute does not affect linearity in the quantitativc determination of solute concentration, since solute concentration does not exceed 1%. More precise quantitative analysis is not so easy, because it is difficult to obtain the elution curves with constant flow rate. But routine quantitative and qualitative analysis of organic acids should be greatly facilitated through the use of the automatic recording apparatus.
