edge of the paper. After air drying at room temperature, end zones of ahout a 1-sq. em. area (hatched, in Figure 1) were cut off and burned (9) and the resultant CO, was assayed for radioactivity by gas phase proportional counting (1).
____ ____
RESULTS
&.,
70
____. 4.0
____. 6.3 1.0 ____.
5.0
____. 3.0
101.11
Figure 1. Results of radiocarbon assay carried out after water transpiration concentration of monosaccharides separated from a mixture b y paper chromatography. The asterisks refer to C14 radioactivity most likely due to D glucuronolactone spontaneously formed from the D-glucuronic acid (-6-C14) component of the mixture opplied a t B
-
end as indicated in Figure 1 (the shaded areas were discarded). The segments were held in quasicylindrical shape (&xisperpendicular to direction of irrigation) by insertion into a hole cut through an acrylic plastic plate supported ahout 1 em. off the bottom of a 600-ml. beaker containing distilled water to depth of ahout 2 em. (see Figure 2). Left at room temperature overnight, transpired water deposited all solutes in a rime at the upper
Count data for the end zones of each segment are tabulated as part of Figure 1. Recovery was satisfactory as judged by counts predicted by prior assay of aliquok of the solutions of individual monosaccharides which were used in compounding the mixture applied to the paper sheets for chromatography. The asterisks call attention to radioactivity which was probably contributed hy n-glucuronolactone spontaneously formed from 0-glucuronic acid (-64Y4). According to C14 measurements, 3 monosaccharides were recovered quantitatively from a mixture after paper chromatographic resolution by concentration of the individual monosaccharides to a small end zone of paper by water transpiration. It is anticipated that this simple, effective means for sample eoncentration should he widely applicable to quantitation of C"-laheled compounds separahle by paper chromatography. LITERATURE CITED
( 1 ) Bernsteh, W., Ballantine, R., Rev. Sei. In&. 21, 158 (1950). (2) Davis, F., Dubbs, C . A,, Adams, W. S., ANAL.CHEM.34, 175 (1962).
(3) Dieckert, J. W., Morris, N. J., J . Agr. Food Chem. 6,930 (1958). (4) Dimler, R. J., Shaefer, W. C., Wise,
Figure 2. Arrangement for supporting segments of paper chromatograms for tip concentrotion of solutes b y water transpiration. The horizontal lines mark 1 -cm. intervals
, Rist, C. E., ANAL. CHEM.24,
j . "
\-""",.
Ionald, E. J., ANAL. CAEM.29, 157).
Ilenberger, -- R. S., Moore, R. G., Ibid., 29, 27 [ 1YSiJ. (9) Vsn Slyke, D. D., Plash, J., Weisiger, J. R.,J. B i d . Chem. 191, 299 (1951). ~~
SUPPORTED in art hv Grants E-2068 and 23-39, Nationd Institute of Allergy and Infectious Diseases, U. S. Public Health Service, Department of Health, Educe tion, and Welfare, Bethesda. 14, Md.
Automatic Attenuator for a Hydrogen Flame Gas Chromatograph id J.
Frt
me auvamages 01 gaJ chrois the wide concentration range that can he covered. Routinely, measurements are now made from the parts per million level to components that are almost 100% of the sample. To maintain sufficient accuracy over this wide range, i t is customary to use a range changer or attenuator to change the input signal to the recorder. Most chromatographs provide for this selection manually. Electronic stepping switches have been used to switch voltaee divider circuits automatically wit< thermal conductivity detectors [Darling, D. J., Miller, F. D., Bartsch, R. C., Trent, F. M., ANAL.CHEM.32, 144 (1960)l. A similar method employing a motordriven switch has been described NE 01
0 matography
F. Johnson,
California Research Corp., Richmond, Calif.
(Johnson, J. F.,Waver, R. F., Baumann, F., Beach, J. Y., Journtes Intern. Etudes Separation Immediate et de Chromatographie 1961, 136). These devices
are well worth their cost because of the saving of operator time. With automatic attenuators, one operator can handle many chromatographs lorated at diverse points. The Beckmaii hydrogen flame accessory employs two separate sttenuators to enable the amplifier to handle a wide dynamic range. One attenuator switches the output of the amdifier in the normal manner. The other switches feedback resistors in the amplifier. This arrangement is not cornpatihle with the normal automatic attenuators, as they are equipped to switch only one attenuator. These types also expose the output
attenuator to switch transients which are transmitted to the amplifier hy the feedback loop. On the more sensitive ranges, the resulting output of the amplifier is sufficient to drive the attenuator through another step. Thus, a vicious circle is established. It was evident that a different type of attenuator was needed because the feedback resistors, which are part of a high impedance circuit, could not he removed from the electrometer tube box. Therefore, it was decided to rotate the attenuator switch automaticallllv.
A part
Ledex (Ledex, Inc,, Dayton, Ohio, No, 215210-031) bidirectional
~ i ~ i + , , ~ t was ~ ~ geared to the shaft. The pulses required by the Digi-motor are generated by separate relays for the up and down directions. VOL. 34, NO. 10, SEPTEMBER 1962
1351
I
1
Figure 1.
Thev are actuated bv microswitches in the "recorder. The "stepping speed is determined by the 400-ohm resistors and the 500-~f.capacitors. The pulse relays are operated from a separate d.c. supply to avoid faulty operation due to variations in the Digi-motor d.c. supply. A diode network provides a minimum attenuation high enough to give a smooth base line when high sensitivity is not necessary. A circuit diagram is shown in Figure 1. Mechanically the automatic attenuator is mounted in a steel box to avoid trsnsients due to radiation or induction. This is placed on the %me ionization attachment as shown in Figure 2. A 1/4-inch steel base plate is used to prevent deflections in the high impedance box of the chromatograph, which would also cause transients. The
i
I
I
I
Automatic attenuator circuit
Digi-motor is geared to the attenuator shaft and the indicating dial. A 2 to 1 ratio is used on the dial, so it makes only one revolution to cover the entire attenuation range. The manual knob is attached to the dial shaft and allows normal manual operation with the automatic attenuator switch in the off position. All the positions normally encountered are utilized: the six steps (1, 2,5,10,20,50) of the output attenuator and the four ranges (IO6, lo', IO2, 1) of the input amplifier. Five minimum attenuation positions were chosen: 1, 5, 20, 2 X 103, and 20 X IO2. More could be added by increasing the number of positions on the switch, but these five values normally suffice.
Figure 3. Time
Figure 2. Auto attenuator mounted on Beckman h y d r o g e n name detector
1352
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ANALYTICAL CHEMISTRY
.6&,
Attenuated peak 4 inches per minute
d i c e this device is a mechanical knob turner, the response of the chromatograph is essentially unchanged. The linearity still depends on the .characteristics of the detector and the amplifier system. The response of the attenuator is fast enough to follow the recording of peaks rvhicli are normally encountered in gas chromatography. The range changes occur a t a maximum rate of three per second. A peak is shown in Figure 3, which. illustrates the operation of the attenuator. The attenuations are marked on the chart. Parts for the attenuator cost about $100, and 2 to 3 days are required for construction. The charts produced are regularly used with the chart integrator described by Johnson et al.
