An Apparatus for linear Gradient Elution under Pressure N. E. Delfel, Agricultural Research Service, U. S. Department of Agriculture, Mayaguez, Puerto Rico N THE CHROMATQQRAPHY of
I sapogenins
plant
on silicic acid with
organic solvents in this laboratory, the general linear gradient elution technique of McGilvery (1) has been valuable in avoiding peak-splitting and other artifacts associated with stepwise elution. We experienced difficulty, however, in adapting the original apparatus for use with pressure: a t atmospheric pressure, satisfactory flow rates could not be obtained with 100- to %mesh silicic acid. After a number of modifications, it became apparent that only the elimination of the free air space in McGilvery's original apparatus would maintain constant volume in the mixing flasks, yet avoid complicated design and operational procedures. Ground-glass fib tings on the mixing flasks were eliminated to avoid contamination of the organic solvents with lubricant : the solvents contact only glass, polyethylene, or Teflon. The inlet tube on the mixing flasks was placed on the top of the flask to minimize back siphoning during adjustment of the air pressure and to promote better mixing than that given by a symmetrical arrangement. The apparatus described herein can be constructed with a minimum of glassblowing ability. CONSTRUCTION OF APPARATUS
The a paratus (Figure 1) consists of a standarl commercial column, two mixing flasks of equal volume, a reservoir flask, and a means of controlling the air pressure (and hence the liquid flow) within the system. The column (Fischer and Porter Co., Hatboro, Pa.) is composed of three separate sections; a top with a side-arm, a central portion 12 inches by inch i.d., and a bottom with a stopcock of Teflon. The sections are joined with threaded couplings and ring seals of Teflon. The absorbept is supported by a sintered glass disk. This column is sufficiently air-tight for use with pressure, but any column may be used. Figure 2 shows how the apparatus could be modified for use with a column lacking a side-ann. The mixing h k s are made from two 200-ml. round-bottom flasks with a 1inch Teflon-coated magnetic stirrine bar sealed inside. The ground-glass joint on the M)o-ml. reservoir flask is held together with rubber bands or springs. The arms on all flasks are made from Zmm. i.d. capillary tubing, with an outer diameter approximately equal to that of the inlet ann of the column. The tip should have an opening of a proximately 0.5 mm. or larger, and t e outside portion at the end must be gently tapered to provide a snug fit with the polyethylene tubing which connects the f h k s .
R
3
0
0
IO
IS
20 C I
FLCXIDLC PLASTIC IIUO
SoLvcni INLCl IUDC
-
2
SOURCC
Figure 1.
Apparatus for gradient-elution chromatography under pressure
The various components are connected as shown (Figure 1), using tubing of an appropriate she for the apparatus as constructed. For the original apparatus we used PE280 polyethylene tubing (Clay-Adams, Inc., New York) and 3/l&ch i.d. flexible plastic tubing. Between the last mixing flask and the column the pol ethylene inlet tube is threaded t h r o u d a length of the larger tubing which seals the air in the column and, in the modification shown in Figure 2, which serves as the air inlet to the column. Two stopcocks ( A and B ) regulate the flow of air in the system. Stopcock A is provided with a capillary tip which restricts the rate of air flow to the atmosphere when stopcock A is open, and which is protected from breakage by an outer glass tube. A ll/,-inchdiameter gless funnel and rubber stopper are placed in the line as a safety valve and as a means of relievin the pressure within the system when t%e liquid flow rate is to be regulated. A tank of compressed air or nitrogen (not oxygen) equipped with a standard reduction valve is a suitable source of pressure. The assembled apparatus was tested initially a t 15p.s.i., but less than 5 p.s.i. is used for laboratoly operation. PREPARATION OF THE APPARATUS FOR OPERATION
The general method of choosing the concentration of solvents to be added to the various flasks and the shape of the resulting CUNW has been presented by McGilvery (1). For most of our work with steroids, the same solvent mixture ( A ) was added to both mixing flasks, and a stronger eluting mixture ( B ) to the reservoir flask. This is done by remov-
COLUMN
+J
Figure 2. Modifications of apparatus for use with column lacking side-arm
ing the top of the column and dipping the inlet tube into the first solvent ( A ) , disassembling the ground-glass joint on the reservoir flask, and applying gentle suction to that flask until the mixing flasks are completely filled. A measured amount of the second solvent ( B ) is then added through the neck of the reservoir flask. To fill the mixing flasks individually, the tubing is disconnected from the top of each flask and filled by the application of suction to the top of the flask while the plastic tubing on the sideann is dipped into the appropriate solvent. The flasks may also be cleaned, rinsed, and dried in a similar manner. The absorbent-e.g., 8 grams of washed 100- to 200-mesh silicic acidand sample are added to the column by standard technique. Approximately 5 ml. of e x c w solvent are left above the absorbent bed. The apparatus is then reassembled according to Figure 1, and the magnetic stirrers under the mixing flasks are turned on. There are two difTerent methods of operating the column; by siphoning or by maintaining constant air volume in VOL 38, NO. 10, SEPTEMBER 1966
1429
the column. The first method is simpler, but the second method is entirely satisfactory and may be necessary if the tip opening of the tubing on the flasks is too small. OPERATION BY SIPHONING
For this procedure, the inlet tube extends below the liquid level in the column. Stopcock A is closed, stopcock B is opened, and pressure is applied to give the desired flow rate. If necessary, pressure within the system is reduced by closing the air tank valve the required amount, and momentarily opening the stopper on the funnel tube. To start siphoning, stopcock A is opened and stopcock B is closed until liquid from the mixing flasks enters the column; whereupon the stopcocks are returned to their initial position. The reservoir flask is raised or lowered to give the desired liquid level in the column,
and is raised periodically thereafter as its liquid level falls. If the tips of the tubing on the flasks are properly constructed, this method provides satisfactory operation of the equipment. If necessary or desired, the following procedure may be used : OPERATION BY CONSTANT VOLUME
In this method of operation the inlet tube is shortened to be above the liquid level in the column. The liquid is first forced into the column with stopcock A open and stopcock B closed, then stopcock A is also closed. Thereafter, the volume of air above the absorbent is fixed, and under equilibrium conditions (constant air pressure) the volume of eluent entering the column is equal to that leaving. The level of liquid in the column may be raised by briefly opening stopcock A to permit the escape of air. The level may be lowered by briefly
opening stopcock B to permit air to enter the column: in this connection, addition of a short capillary tube between stopcock B and the column to restrict the air flow rate might prove helpful. Raising or lowering the reservoir flask also permits some regulation of the liquid level in the column. Once the liquid level has been adjusted, only periodic inspection and adjustment are necessary thereafter. The above apparatus is simple and inexpensive to construct, and has been useful in 2 years of operation in this laboratory. LITERATURE CITED
(1)
McGilvery, R. W., Anal. Biochem.
1,
141 (1960).
Mention of various commercial products or industrial companies does not imply their endorsement by the U. S. Department of Agriculture over others of a sim-
ilar nature.
Determination of Small Amounts of Oxygen Using a Hersch Cell as a Gas Chromatography Detector Geoffrey E. Hillman and John Lightwood, Central Laboratory, West Midlands Gas Board, Nechells, Birmingham 7, England
has hitherto been 0 determined using a constant volume or constant pressure gas analysis apparaXYGEN IN FUEL GAS
tus. These methods have a lower detection limit of 0.1% v./v. With the introduction of catalytic processes for gas manufacture and treatment, the determinat,ion of very small amounts of oxygen has become of paramount importance. Modern gas chromatw graphic methods of gas analysis have enabled the detection limit for oxygen to be improved to 0.01% v./v. The disadvantage with gas chromatography is that argon is almost always present in fuel gas, thereby necessitating a second determination with oxygen removed, because argon and oxygen are not seg arated by molecular sieve unless subzero temperatures are used. Continuous determination of oxygen using a Hersch cell (1, 3, 4, 6, 7) is a well established technique and, provided no interfering compounds are present in the gas, satisfactory results are obtained. Organic sulfur compounds are present in fuel gas and interfere with the Hersch cell, necessitating removal traps before the cell which must be chauged frequently. By using a chromatographic column and a Hersch cell the organic sulfur compounds are trapped by the column used for the separation of oxygen from the other gases. This method has two distinct advantages. First, only m a l l samples of gas are used, leading to longer periods of time before renewal of column material, and second, if other 1430
0
ANALYnCAL CHEMISTRY
components are required in analysis-for example, argon, carbon monoxide, methane, and nitrogen-then another chromatography detector may be placed in series with the Hersch cell. With this method the same sample of gas may be used for both oxygen and argon determinations. EXPERIMENTAL
Apparatus. The apparatus is housed in a 1- X 1- X 1-foot box made of sheet aluminum using Widney Dorlec angle brackets and ends, and this is thermally insulated throughout internally with 0.5-inch expanded polystyrene sheet. The thermostat unit is a Sunvic controller Type TS3 and the unit is heated by a 15-watt bulb. A small fan is used to circulate the air inside the cabinet. The carrier gas flow is controlled by a Negretti and Zambra flow regulator Type P17 with inlet column pressure measured by a standard 0- to 30-p.s.i. gauge. Sample injection is by a push-pull, 6-port, gas sample injection valve (2). This has been constructed at this laboratory and has excellent reproducibility. A sample loop of volume 0.301 ml. is connected to this valve: its volume has been carefully measured and includes dead space in the sample injection valve. All gases are sampled at atmospheric pressure. The column used is 1.5-foot x O.2binch 0.d. of Molecular Sieve Type 13X. Column material is copper and all flow lines are 0.125inch 0.d. annealed copper tubing connected with “Enots” compression fittings. “Enots” fittings are obtainable
from Benton and Stone Ltd., Aston Brook Street, Birmingham 6. Detector. This is the Hersch cell as described by Phillips, Johnson, and Woodward (9). The electrical circuit, however, is somewhat different and is shown in Figure 1. Apart from backing off the standing current,. provision is made for various sensitivities and also for varying the proportion of the output onto the recorder. A combination of both adjustments enables peak height to be set virtually as required-e.g., full scale deflection 0-100 p.p.m. to 0-50,OOO p.p.m. of oxygen. At higher sensitivities the backing off current is almost certain to be required and this may be battery, or mains operated, as shown in Figure 1. Carrier Gas. White spot nitrogen is used as carrier gas and this does contain a certain amount of oxygen It is, therefore, (10-30 p.p.m.). necessary to remove as much of this as possible. The oxygen is best removed by using a tube of manganous oxide 18-30 mesh in the carrier gas line, rn close to the inlet of the unit as possible. The manganous oxide is prepared by heating manganese dioxide in a stream of hydrogen and nitrogen a t 350° C. and ensuring that the reduction does not become violent. Because of diffusion of air through polyvinyl chloride or rubber tubing, it is advisable to use metal or glass connections for the carrier gas line to the unit. Column. The column is 1.5 feet of Linde Molecular Sieve, Type 13 X i 44-60 mesh. Reagent Solution. Approximately 5N sodium hydroxide is used in the
