Sample Injector for Ion Exchange Chromatography and Flow Cell for

Ion Exchange Chromatography. Harold. Walton. Analytical Chemistry 1966 38 (5), 79-86. Abstract | PDF | PDF w/ Links. Cover Image ...
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somewhat less than that of the best visual titrations, but it is considered good for a n automatic titrator. The principal limitation is that the titration reaction must be rapid, a condition which must be satisfied in any automatically recorded titration. Because the instrument is assembled largely from commercial components, the worker Iyho is unsophisticated in instrumentation or is not even primarily a chemist but who needs a titrator to

serve his own ends may readily make use of it. If a Model DU spectrophotometer is a t hand, the cost is modest by present standards; if both a DU and a recorder are available, the cost is small.

(3) Malmstadt, H. V., Roberta, C. B., Zbid., 27, 741 (1955). ( 4 ) Marple, T. L., Hume, D. K.,Ibzd., 28, 1116 (1956). ( 5 ) Mullen, P. \Y.,i2nton, A , , Ibzil 32, 103 (1960). (6) Underwood, A. L., Hove, L II.,111, Ibid., 34, 692 (1962).

LITERATURE CITED

WORKsupported by the Sntional xieIice Foundation through Research G r s n t SSF-G13514. Taken from a thesis presented by Thomas XI. Robertsun in partial fulfillment of the requirements for the 11S degree, Emory Cniversitp. 1962.

(1) -4nton, -4.1 hfulleni p. w.2 Talanta 8, 817 (1961). ( 2 ) Malmstadt, H. v., E. -kXAL. C H E h I . 26, 442 (19%).

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Sample Injector for Ion Exchange Chromatography and Flow Cell for Continuous Photometry at 210 Millimicrons Arthur M. Crestfield, The Rockefeller Institute, New York 2 1, N. Y.

often desirable to determine the I concentration of substances in the T IS

effluent from a fractionation column by measurement of absorbance in the ultraviolet. While automatic sampling photometers are available for use with fraction collectors, it is simpler to pass the effluent through a flow cell in a recording photometer. However, optimal separations are obtained only if the flowing stream is of sufficiently narrow cross-sectional area. The construction of a micro flow cell for use with a Zeiss PMQ I1 spectrophotometer and columns of about 1 cm. or larger in diameter is described here. This equipment has been used to advantage in studies on ribonuclease reported elsewhere (2, 3). I n that work, the sensitivity in detection of proteins was increased by use of 210 mp (4) rather than 280 inp as usually employed. To obtain a steady baseline, it was necessary to keep the flow rate constant a t all times, even during addition of the sample, since the ion exchange resin itself released materials mhich absorb a t 210 mp. The device developed for injection of the sample onto the column provides other advantages to make it of more general interest. Therefore, this sample injector is described here too. EXPERIMENTAL

Chromatographic Unit. I n the present work, a solution of S a C l is emSpring clamp

Teflon c o i l

Figure 1.

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Sample injector

ANALYTICAL CHEMISTRY

ployed as eluent. Only the stationary phase of the column (a carboxylic resin) acts as the buffer. The procedure for the preparation of such a column is described elsewhere (3) ; other solutions may be used provided the absorbance at 210 mp is sufficiently low. Columns with a n internal diameter of about 0.9 cm. were equipped with a supporting disk of porous Teflon (Grade 50-55, Fluoro-Plastics, Inc., Filbert and Cuthbert Sts., West of 36th, Philadelphia 1, Pa.). Disks of porous Teflon are added t o 50% aqueous acetic acid, deaerated with a water aspirator for 30 minutes, rinsed several times with a solution of SaC1, and then deaerated again, this time without acetic acid. The bottom of the column is a 12/1 semiball joint. -1 PVC connector of the type used on the Spinco amino acid analyzer (Beckman/Spinco swivel fitting 120-558, PS'C 120-561 connector, 12-mm. socket to 1 / 1 6 inch 0.d. Teflon tubing) provides convenient attachment of the 22-gauge Teflon tubing (Pennsylvania Fluorocarbon Co. Inc., 1115 N. 38th St., Philadelphia 4, Pa.) leading to the flow cell. The detection system is sensitive to air bubbles which appear in the effluent stream %-hen air pressure is used to drive the sample into the column at the beginning of a chromatogram. Some other means of adding the sample is needed, therefore. The photometer also detects any alteration in the small, steady concentration of ultraviolet absorbing material leeched from the resin and the other components. The concentration of the extraneous ultraviolet absorbing material in the effluent is dependent upon the rate of flow through the system. During operation, the flow rate, therefore, must be held constant by a suitable pump. A MiltonRoy minipump fitted with Teflon or polyethylene tubing, and a pressure gauge (200 p s i . , E. S. Gauge) is operated a t 30 ml. per hour (6). A section of silicone tubing of l / l a inch i.d. and length of 6 inches is inserted in the delivery line to provide a n elastic component to reduce the magnitude of

Figure 2. Detail of T-connections shown in Figure 1

the pulse of pressure during the stroke of the piston. A section of Tygon tubing is slipped over this silicone tube to prevent it from bursting. Sediment and cations emerging from the pump are removed by a 6-cm. column of coarser IRC-50, equilibrated in the same manner used for the chromatographic column. The effluent from this column of coarser resin is directed to the chromatographic column. The sample is inserted into the pumped stream nhich feeds the chromatographic column by the use of the arrangement shown in Figure 1. The sample is taken up in a coil of about 10 feet of 22-gauge Teflon tubing. The coil has a capacity of about 1 ml. It is connected t o provide an optional parallel path between the pump and the column. The T-connections are constructed of Teflon and Nylon fittings as shon-n in Figure 2. Flow through the tubing is interrupted by spring clamps adjusted in tension for the pressure employed. The sections of tubing to mhich the clamps are applied are replaced from time to time as they become damaged. ,4t the beginning of a chromatogram, clamps 2, 3, 4, and 5 are closed. clamp 1 is opened, and the pump turned on. Eluent passes through the column and the sample coil is bypassed. After the base line registered by the recorder has become constant (about 1.5 hours), the coil is filled with the sample to be chromatographed. For this purpose clamps 4 and 5 are opened

and suction is app1:ed a t one end. The other dips into the sample which contains 10 to 1000 b.6. of protein per milliliter of the same solution employed as eluent. TT7hen the coil is full, clamps 4 and 5 are closed To inject the sample into the influent stream, clamps 2 and 3 are opened and clamp 1 is closed. Pumping is continued until the end of the chromatogram.

S l o t t i i d bross tube F l a r e d leflon tubing Waster Sillcone rubber disk Porous t e f l o n d i s k Resin Col u r i n

Figure 3. Cross sectional view of top of an ion exchange column lor use with sample injector

This method of injecting the sample into the pumped stream feeding the column requires that there be no dead space above the top surface of the resin column. This can be accomplished if the top of the column is arranged as shown in Figure 3. A disk of porous Teflon 50-55, cut with a cork borer and deaerated as described earlier, is forced onto the top of the resin surface. The column is filled with eluent during these manipulations to ensure that air is not driven into the column. Teflon tubing of 22 gauge is passed through a :$ection of a disk cut from 6/32-inch silicone rubber sheet and the end of the tubing is flared by a hot tool so that it will not pull out. The rubber disk is inserted into the column and is forced down until it is resting directly on ,he Teflon disk. An aluminum washer is added on top of the rubber disk, and a suitable length of brass tubing with a slot in the end is then placed on top of the stopper and clamped in a ring stand to prevent movement of the disk. The bottom of the column can be closed off by the same type of device shown in Figure 3 for closing off the top. I n this case the chromE,tograph tube is simply a piece of gla:,s tubing. Recording Photometric Unit. The construction of a micro flow cell with a light path of 14 mm. and a n internal diameter of 1.5 m r i . is shown in Figure 4. It is employed with a Zeiss PhlQ I1 spectrophotometer equipped with a microcell compartment and a 3.5-mm. mask and heam condenser. The instrument was selected because the optical system yields light of high spectral purity down to a t least 200 mp, About 359& of the emergent light passes through the narrow bore of the flow cell. Absorbance was proportional to the concentration of protein (ribonuclease) up to ran absorbance of

1.1. Stability of this single beam instrument is generally u-ithin an optical density of 0.002 for periods of 2 to S hours, often longer when the temperature of the laboratory is constant. Light transmission at the shorter wavelengths gradually decreases owing to fogging of the mirror in the lamp housing. Therefore, new mirrors are installed with each new hydrogen lamp. A new lamp is installed when a slit width greater than 1.0 mm. becomes necessary to work a t 210 mp. The half intensity band width is ah-ays less than 2 mp. The output from the photomultiplier circuit of the spectrophotometer is recorded by a Speedomax Recorder (Leeds &. Sorthrup, Model G, 10 mv.. equipped with a variable chart speed, to 30 inches per hour) to yield the familiar type of effluent curve. The flow cell is filled initially with a 5% solution of Brij 35 (Atlas Powder Co., Wilmington, Del.). Suction from a water aspirator is used for filling and to dislodge bubbles. Once the optical channel is free of bubbles, liquid is pumped through the cell until the ultraviolet absorbing detergent is removed. Air bubbles usually travel through the cell, but occasionally one may stick to one of the optical faces from which it may be dislodged by flicking the inlet or outlet tubes sharply with a finger. DISCUSSION

The equipment and techniques described in this communication were devised to increase the speed and sensitivity of the chromatographic procedures (2. 3) for the detection and estimation of ribonuclease and its derivatives. The method depends upon the maintainance of a constant and rapid rate of flow through the column. Columns of finely divided IRC-50 have already been shown to yield high resolution a t flow rates of 45 ml. per hour per sq. em. (2). The sample insertion device as well as the head of the column has been found to operate a t pressures as high as 100 p.s.i. By measurement a t 210 mp, with the aid of the present techniques and a 0.9 cm. X 6 cm. column of IRC-50, it is possible to measure in 1 or 2 hours’ time the quantity of enzyme -only a few micrograms of proteinactually present in 1 ml of the mixture used for determination of enzymic activity ( I ) . The course of the alkylation reaction leading to the formation of the inactive l-carboxymethylhistidine-1 19 and 3-carboxymethylhistidine12 derivatives of ribonuclease has also been determined conveniently in this manner (3). Since no buffers are needed in the flowing solution, it may be possible to gain an additional increase

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Figure 4. Assembly of one of two optical faces of micro Row cell A, holes for ‘/>*-inch Teflon tubing connectors (Beckman/Spinco swivel fitting 120-558). E, tapped holes for securing bross clamp. C, holes, 1.5-mm. i.d., are carefully drilled to avoid shreds. D, silicone washer, 0.5 mm. thick. Central, oblong hole is formed from two 1.5-mm. diameter holes connected by straight cuts made with a razor blade

in sensitivity by use of waveiengths shorter than 210 mp. Spectrophotometer cells with narrow channels have been described for use with small volumes by Kirk, Rosenfels, and Hanahan (6) and for continuous flow of very narrow strpams by Vllrich and Hampel ( 7 ) . The former cell is not adaptable for flow. The smaller cross-sectional area of the latter cell was not compatible with many light sources. The cell preqented here offers a combination of features which include small volume, long light path, narrow stream, ease of construction and ease of disassembly for cleaning. A cell of similar dimensions, but constructed of fused quartz, is now available from Pyrocell hfanufacturing Co., Kew York, ?;. Y. ACKNOWLEDGMENT

Apparatus was constructed by Kerner K. Krug and Nils A. Jernberg of this institute. LITERATURE CITED

(1) Crestfield, A.

M., Stein, Moore, S., Arch. Biochem. Supplement 1, p. 217 (1962). ( 2 ) Crestfield, A. M., Stein, Moore, S., J. Biol. Chem.

W. H.,

Biophys.

W. H., 238, 618

(1963).

(3) Ibid., p. 2421. (4).Goldfa&, R: A., Saidel, L. J., Mosovich, E., J. Bzol. Chem. 193,397(1951). (5) Kirk, P. L., Rosenfels, R. S., Hanahan, D. J., ANAL.CHEM.19, 355 (1947). (6) Spackman, D. H., Stein, W. H., Moore, S., Ibid., 30, 1190 (1958). (7) Ullrich, K. J., Hampel, A., Pjluger’s Archiv. 268, 177 (1958).

THISwork has been supported in part by a grant from the Lnited States Public Health Service.

VOL. 35,

NO. 1 1 , OCTOBER 1963

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