characterization of the mixtures in a minimum analysis time. The high degree of separation available through the capillary gas liquid chromatograph, the rapidity with which mass spectral data can be obtained, and the versatility of the system make this an ideal, and perhaps the most complete, method for analyzing nonroutine mixtures. LITERATURE CITED
(1) Am. Petroleum Inst., “Catalog of
Mass Spectral Data,” API Research Project 44. (2) Brunnke, C., Jenckel, L., Kronen-
berger, E.,2. Anal. C h . 189, 50
(1962). (3) Desty, D. H., Goldup, A., “GasCbrorntutagraphy 1960,” R. P. W. Scott, ed., p. 179, Butterwortha, London, 1960. (4) Durrett, L. R., S i D O n S M. C., Dvoretzky, I., Preprints, Dksion of
Petroleum Chemistry, American Cbemical Society, St. Louis, March 22-25,
( 5 ! 3 , , l , ~B-63. o ke, R. S., ANAL. CHEM. 31, 535 (1959). (6) Hall, L. G., Hines, C. K., Slay, J. E;$ “Advances in Mass Spectrometry, J. D. Waldron, ed., p. 266, Pergamon Press, London, 1959.
(7) Henneberg, D., “Gas Chromatography 1960,”R. P. W. Scott, ed., Butterwortha, London. 1960.
A Compact Radiochemical Gas Chromatograph A. T. JAMES’ and E. A. PIPER Nafional lnstifufe for Medical Research, Mill Hill, London,
b A compact gaschromatographisdescribed in which the column i s connected directly to a combustion tube in which all organic compounds ore burnt to COz and water. The water is reduced to hydrogen by iron filings. The gas stream passes to a miniature kathorometer in which the hydrogen is detected. After injection of 5% of COz, the mixed gases enter a simple proportional counter which can record the ionization radiation from either C“ or tritium with efficiencies of approximately 100% for C14 and 60% for tritium. The apparatus is sensitive, simple, and cheap ta construct.
T
only a limited number of ways of counting ionizing particles, vis., counting in tubes in the Geiger or proportional region, in ionizing cbambers, or in solid or liquid state scintillation systems. Application of these methods to the continuous following of the radioactivity of gas streams from gss chromatographs has been accomplished in two ways, either operating the counter at column temperature or at room temperature (for reviews see 2 and 3). The general approach has been to design the counting equipment to fit on to the vapor detector of an existing chromatograph. I n our experience this is a disadvantage unless the lahoratory is rich enough to provide a large number of chromatographs, because with an apparatus used both for fraction collecting and counting it is often EERE IS
1 Present address. Unilever Research Laboratory, Colworth House, Shambrook, Bedford, England.
N. W. 7.,
England
inconvenient to keep disconnecting the counting equipment. With this in view, we have designed a radiochemical gas chromatograph that is both cheap, simple to construct, e5cient, and sensitive. It can also be used without modification as an analytical chromatograph. The previous device we described (3) consisted of a combustion train attached to the outlet of a conventional gas chromatograph, the proportional counter being operated at room temperature with argon as the carrier gas and COa added externally to give a final concentration of 5%. (This apparatus is now available commercially, Pye Scientific Instruments, Cambridge, England). The new apparatus consists of a coiled metal or glass column wound on a heated aluminum former; the
Figure 1.
column is directly attached to the quartz Combustion train containing copper oxide and iron powder. The argon gas stream passes into the proportional counter after introduction of 5% COS and then into a small volume katbarometer of the type described by Stuve (4). The katharometer is particularly sensitive to hydrogen in argon and, because it uses the filaments of miniature light bulbs, is simple to construct. APPARATUS
Column Assembly. The column consists of a &foot length of ‘/,-inch i. d. copper tube flared at each end to take screwed connectors. The tube is packed in the conventional manner when straight, the packing being supported by small pads of woven
Exploded picture of column and housing Front face plate Coiled column on heated block C. Column housing
A.
B.
VOL. 35, NO. 4, APRIL 1963
515
5-1
Argon
I
A. 6.
1
Side Elevation
Figure 3. A.
Front Elevation
Combustion tube assembly Copper oxide powder
B. Iron filings C. Spherical ioint D. Furnace
Figure 4. A. 6.
C.
Proportional counter
Screwed brass insert to take tungsten wire and gos inlet Polythene cap '/*-inch. i.d. copper tube, polished and bright nickel ploted
outlet pipe (Figure 1) and the inlet manifold (Figure 2). Glass insulation is then wound around the column and the assembly is pushed into the column housing (Figure 1). The housing is inserted into a rectangular box and insulated with Fibreglass. The column temperature is controlled by a variable transformer in the mains circuit. S o thermostatic control is employed as the system is operated as an equilibrium heater. The 65-watt heating element will give temperatures up to 300" C. Combustion Tube Assembly. The combustion tube (Figure 3) is a 5-inch length of 3/8-inch silica tube fitted a t one end with a 1-mm. i.d. capillary tube of the same outside diameter as the metal outlet pipe of the column. The two tubes are connected by Teflon
Column inlet manifold
Removable seal Screw coupling to column
C
A. 6. C.
D. E.
F. ANALYTICAL CHEMISTRY
C
1
B
B
glass yarn (Fibreglass Ltd., London). The packed tube is then wound on a former of the same diameter as the heating block. The latter bears a slot, just large enough to take a soldering iron heating element (65 watts) that is held in place by the rear plate (Figure 1). The column is then forced over the heater block and connected to the
516
A
1
p
Figure 2.
I
I
D
Argon inlet a t 100-p.s.i. pressure Reducing valve set a t on outlet pressure of 2 p.s.i. Magnetic shut-off volve Reducing valve set a t 15-p.s.1. outlet pressure Pressure g a g e Line to column
tubing held in place by silicone rubber tubing. The end of the combustion tube protrudes from the furnace and LY uninsulated, unlike the column outlet. In this way there is sufficient radiation and convective cooling of the bared silica tube to give a rising gradient of temperature from that of the column to that of the combustion system. The far end of the combustion tube terminates with a male spherical joint; this end section also protrudes from the furnace. The packings of the combustion tube are supported a t each end and separated a t the center by coarse quart5 powder. The first section of packing consists of 40- to 60-mesh copper oxide and the second of iron filings. The furnace consumes 100 watts and 1s supplied with current from a variable
E
EYT
PULSE AMPLIFIER
OUTPUT
+ Figure 6.
transformer. At about i 5 0 " C. the center of the furnace is a t a dull red heat. A quartz capillary T piece fitted with a female spherical joint and a two-way tap is attached to the outlet of the combustion tube, COZ from the supply line is introduced into the side arm, and the outlet is attached by silicone rubber tubing to the inlet of the counter tube (Figure 4). The other tap outlet leads to the COZ flow meter. Proportional Counter. The body of the counter is made from 1/2-inch i.d. copper tube as in our prwious publication ( 3 ) . Bare copper tube shows a large memory effect when tritium is being counted; this is overcome by polishing followed by bright nickel plating of the inbide of the tube. The counter is housed in a 1-inch-thick cylindrical lead housing to which is attached the amplifier. The outlet of the counter passes to the katharometer block, connection being made by silicone rubber tubing. All joints made with silicone rubber tubing are arranged so that only the minimum area of tubing comes into contact with the gas stream. Counting efficiency is of the order of 1 0 0 ~for o C14and 60% for TI. Katharometer. The gas stream from the counter passes into one side of the katharometer block, the argon stream from the gas control valve passes into the other side of the block, and both chambers are fitted with filaments and bases obtained from miniature light bulbs essentially as described by Stuve (4). The filaments are just out of the direct gas stream. The bridge circuit used is of conventional
Circuit of pulse amplifier
Transistor f lip-1 lop
63 Photo-transistor and light source mounted in Recorder
Figure 7. Flip-flop circuit to automatically short out the integrating condenser when the recorder pen moves fullscale
04 Figure 8. A. 8. C. D. E. F. G.
Schematic flow sheet for either
C14
or tritium counting
Column Combustion tube Two-way top for directing COz either to counter or to flow meter Proportional counter Kathorometer Two-way t a p for directing argon either to flow meter or to COSabsorption tower COz absorption tower
VOL. 35, NO. 4,
APRIL 1963
517
Figure 9.
Tailing of tritium produced b y brass connecting tubes from combustion tube
design. A T piece containing a twoway tap is joined to the outlet of the cstharometer so that flow can be directed either to the flow meter or to an absorption tower of soda lime to remove active COS. Gas Control Lines. The argon gas line enters the apparatus via a cylinder reducing valve a t a pressure of 100 pounds per square inch. It passes through the gas circuit shown in Figure 5. A magnetic valve in the main circuit is operated by a time clock; a t the shut-down time each night the valve closes and the major gas stream to the column is cut off. Argon then passes through a parallel circuit to the column via a reducjng valve (Flicka automatic expansion valves supplied by Refrigeration Spares, 31 Harrow Road, Leytonstone, London, E. l l ) , adjusted to give a pressure of 2 pounds per square inch a t the outlet. In this way a continuous slow stream of gas passes through the column and gas train. A similar arrangement is used on the C02 line (Figure 6) except that the cylinder is placed inside the apparatus giving a pressure of 35 pounds per square inch to the reducing valve which supplies CO, to a length of metal capillary tube acting as a throttle. At the shut down the COZsupply is cut off completely. The reducing valves controlling both argon and COz lines are displayed on the front panel of the instrument, to allow easy alteration of flow rates. Flow Meters. These are of the conventional soap bubble type, the argon meter being constructed from a 50-ml. buret and the COzmeter from a 1-ml, graduated pipet. Circuits. The circuit of the pulse amplifier is shown in Figure 6, and is of conventional design. It is placed in the same box as the counter so that the connecting leads can be easily screened. The amplifier has a gain of 200. and is suitable both for C14 and tritium. Rate Meter. The modifications to the ratemeter were given in our first paper. The recorder pen carriage is arranged to operate the “flip-flop” shorting out of the integrating capacitor (Figure 7), either by screening a photo-transistor from a light beam or by operating a microswitch, when full scale deflection occurs. Time Switch. The apparatus is controlled by a time switch that a t the set time switches off the following:
518
ANALYTICAL CHEMISTRY
ti
Figure 10.
F
- 0 -
>
Schematic flow sheet for simultaneous C14 and tritium counting A. Combustion tube B . Two-way tap for introduction of C 0 1 C. D. E. F. G.
Counter recording both C14 and tritium
COI absorption system Two-way tap for introduction of fresh COI Second counter recording tritium alone Kathorometer
/-9@ Figure 1 1.
Katharometer sensitivity
Relationship between peak area and mass of methyl palmitate at two flow rates and three sensitivity settings
[I
PALMITIC
/I
OLEIC
2
s! + u
8F 5 W Y
e, -I W
3
60
v)
4 4
Y =3
40, w z 0
a
a W
20
I
68
1 64
60
56
52
48
44
40
36
32
28
24
16
'20
12
4
8 minutes
TIME IN MINUTES
Figure 12.
8
Typical result showing both records, (A) plotting count rate and (5) Katharometer record Integrating time '/a second.
main argon stream, COz stream, katharometer bridge, chromatogram and radioactivity recorders, and rate meter. The following remain on continuously, subsidiary argon flow, column heater, combustion furnace, and pulse amplifier. In this way the apparatus is ready for use in a few minutes. RESULTS
A schematic diagram of the flow system applicable to either CI4 or T 2 counting is shown in Figure 8. The arrangement is dictated by the great readiness with which tritium absorbs on metal surfaces. When the brass katharometer is placed before the counter considerable tailing of tritium labeled peaks (but not C14) occurs (see Figure 9); this is avoided in this
Polyethylene glycol adipate stationary phase at 180'
arrangement. Metal connecting tubes should not be used as the absorption effect is increased. The counter itself shows practically no tailing even when large amounts of tritium pass through. The metal in the combustion tube is kept in the heated zone, the packed section of the tube protruding is filled with quartz powder. When simultaneous counting of C14 and Tz is required, the arrangement in Figure 10 could be used. A11 the COZ is absorbed between the counters so that the first counter measures both C14 and TZ and the second Tg alone. Solid absorbents such as soda lime cannot be used owing to exchange of Tz for Hzin the water present in the alkali. We have experimented with condensation of the C 0 2in a cold trap but have
C.
not done enough work to define finally the best system. Most of our work has been carried out with singly labeled substances. It is particularly important in labeling studies to be able either to place a known aliquot of the sample on the column or to load the whole of the sample. This is difficult by conventional methods so we have used loading tubes. The tube 1 inch long, is a close fit in the loading port of the chromatogram and it contains a short length of woven glass yarn. The tube can be used in three ways. h known aliquot of sample in a volatile solvent is pipetted on to the glass yarn and the solvent is evaporated in a stream of nitrogen. A solvent-free sample can be pipetted on to a weighed tube which is then
80 0 0 "8 W
1
-60
-
i l
Figure 13.
E VI
c
a L
40
Typical radioactivity record using an integrating time of 2 seconds VOL. 35, NO. 4, APRIL 1963
519
A
32
28
14
20 16 12 TIME MINUTES
8
4
Figure 14. Typical integrated record (curve 6) and Katharometer record (curve A). Column conditions as in Figure 12.
reweighed to give the amount of sample. The sample can be evaporated to small volume at the bottom of a small conical centrifuge tube by heating the solvent to boiling and then immersing the tube in ice water. The solvent condenses on the walls and washes the material to the bottom of the tube. The sample can then be transferred with a micropipet to the loading tube, the solvent evaporated, and the process repeated by washing the centrifuge tube with fresh solvent. In this way a sample can be quantitatively transferred to the column. These methods are applicable only to relatively nonvolatile substances such as long-chain fatty acids and their esters and sterols. The apparatus is set in operation by measuring the argon f l o r rate at the outlet and adjusting the COa flow rate by means of the flow meter to the required level. The counter plateau is approximately 100 volts long, the operating voltage being 1850 volts. The katharometer is operated at a bridge voltage of 6 volts, the resistance of the two arms being of the order of 100 ohms. The katharometer has two arms
520
ANALYTICAL CHEMISTRY
since we have found a single filament to cause the bridge to drift slowly throughout the day; two filaments prevent this effect. I n loading, the argon gas stream to the column and the katharometer are switched off; after 30 seconds have elapsed, the seal on the column loading port is removed and the used loading tube is removed by inserting a metal rod (turned down to a long conical section at one end) into the tube. The new tube is dropped in while a slow stream of gas is passed in; the tube is pushed down until it touches the top of the packed section, the slow gas flow is turned off, the seal is replaced, and the full gas stream is turned on after 1 minute has elapsed to allow the sample to reach column temperature. The loading tube must not be packed tightly with glass yarn otherwise gas flow is restricted through the tube. When the gas stream is turned on, the katharometer is switched on and the recording is commenced. During the loading period, the counter is receiving only COz and ceases recording the background; this is resumed as soon as the argon front arrives.
Calibration of the katharometer is carried out at a variety of flow rates by using weighed samples of a pure compound, (see Figure 11). The relative response due to other compounds is easily calculated by working out how much COZ and Hz are produced by combustion of a 1-gram molecule. Operated in this way the katharometer is an absolute detector comparable to the gas density meter. Recording of radioactivity can be carried out either by plotting count rate or by integrating the pulses by a condenser. The first method gives the characteristic Gaussian shaped zones (Figure 12). The smoothness of the background is dependent on the time constant employed. We prefer to use a ’/n-second integration time when fast peaks are being studied (Figure 12) and a %second integration time for slower peaks (Figure 13). The integral record of accumulated charge on the integrating capacitor is preferred for quantitative work. Background radiation (at a level of 15 to 20 c.p.m.) gives a sloping base line, steps being recorded on the slope. By producing the background slope through the step, the number of counts can be directly read off (see Figure 14). From the known flow rate and counter volume the number of counts per minute can be calculated. The apparatus has been in continuous use for 11/* years, and provided the combustion tube is recharged every month, it is reliable and trouble free. Its chief recommendations are cheapness, simplicity, and ease of calibration. ACKNOWLEDGMENT
Figures 6 and 7 are reproduced from the Journal of Chromatography (Elsevier Publishing Co., Amsterdam) by permission of the Editor and Publishers. Figures 12, 13, and 14 are reproduced from papers in Biochimica et Biophysica Acta by permission of the Editor and Publishers. Thanks are due to John Coumbing for help in construction of the apparatus and to C. H. Hitchcock for collaboration with the katharometer. LITERATURE CITED
(1) Adloff, J. P., Chromatog. Rev. 4 , 19
(1962).
. ,(1961).
( 2 ) Cncace. F.. Nucleonics 19, No. 5, 45 I
,
(3) James, A. T., Piper, E. A., J . Chromatog. 5,265 (1961);( (4) Stuve, W., in Gas Chromatography 1958,” p. 178, Rutterworths, London,
1958.
RECEIVED for review November 19, 1962. Accepted Februar 8, 1963. Presented at the International &;impos~urnon Advances in Gas Chromatography, University of Houston, Houston, Texas, January 21-24, 1963.
