ANALYTICAL BIOCHEMISTRY 88, 334-337 (1978)

Multiple Fraction Collection Using a Peristaltic Pump and a Fraction Collector Modified for Multiple

Channel Operation

The fractionation of gradients in sedimentation analysis of proteins is a time-consuming operation. This operation can be performed more rapidly by using a multichannel peristaltic pump and two or more fraction col- lectors. Specifically designed fraction collectors are available for multiple fraction collection, notably the Slave Micro-Fractionator (Model SFC-8O)l from Gilson Medical Electronics, Inc. Use of multiple fraction collectors during the fractionation of gradients has the disadvantage that these units occupy considerable space and are expensive. In addition, the total number of fractions collected from one gradient is often considerably less than the capacity of most fraction col- lectors. To offset the space and expense disadvantages, we have devised a modification for the Gilson Micro-Fractionator (Model FC-SOH)’ which permits three sets of 25 fractions each to be collected simultaneously from 3 gradients using only 1 fraction collector. A multichannel peristaltic pump is employed, and an attachment which permits three drop tubes to be positioned above the rack of fraction tubes is secured to the drop detector head’ of the Micro-Fractionator. One drop tube is clamped in the normal manner in the drop detector head, and fraction size is determined by counting drops in the normal manner. Fractions from the other two drop tubes are not counted. Alternatively, fraction size can be determined by adjusting the pump speed and using the timer on the fraction collector.

CONSTRUCTION DETAILS

The Desaga peristaltic pump (Brinkman) has the capacity for pumping multiple channels at identical rates of flow and requires no modification. Other makes of peristaltic pump with this capability are also available.

The Gilson Micro-Fractionator is modified with the attachment shown in Fig. 1A. A Lucite bar, named the fractionator manifold, is secured at one end to an aluminum bracket fitted to the drop detector head and supported at the other end by a Lucite support block described in detail below. One drop tube is passed through a hole in the fractionator manifold and is clamped in the drop detector head in the same manner

’ The terms used are those applied by Gilson Medical Electronics in the description of their fraction collectors. Instruction manuals or advertising literature relating to the Micro- Fractionator fraction collectors should be consulted.

0003-2697/78/0881-0334$02.00/O Copyright 0 1978 by Academic Ress, Inc. All rights of reproduction in any form reserved.

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SHORT COMMUNICATIONS 335

FRACTIONP

fractionator momfold support ?2%Km ‘CHAIN BRACKET

DROP DETECl -OR

FIG. 1. Modification of Gilson Micro-Fractionator for multiple channel collection (A) The drop detector head is shown but the guide bars and drop tube clamp have been omitted. The chain bracket is attached to the drop detector head by two short screws (not shown). These screws are replaced with longer screws so that an aluminum bracket can he attached to the back of the chain bracket. The Lucite fractionator manifold is fastened with two small screws to the aluminum bracket as indicated and is supported by the Lucite support block. The support block rests on the guide bars (see B). The length of the fractionator manifold is 20 cm; other dimensions, for the aluminum bracket, fractionator manifold, and support block depend on the model number of the fraction collector being modified and should be determined by comparison with the fraction collector. The vertical and horizontal holes in the fractionator manifold are drilled as described in the text. The tubing from the outlet side of the peristaltic pump is passed through the horizontal hole and looped around so that the drop tube inserted in the end can be located in the vertical hole. (B) The support block is pictured as seen in end view from the left of the fraction collector, demonstrating how the block rests on the guide bars (indicated by circles) and supports the fractionator manifold. All other fea- tures of the fraction collector are omitted. (C) One tube assembly indicating the peristaltic pump and micropipette connected by Tygon tubing. Three assemblies are required for gradient fractionation.

as would be done for normal operation. Two additional drop tubes are inserted into holes drilled vertically through the fractionator manifold in the positions described in detail below. The vertical hole for the counted channel is made large enough to permit the drop tube to be clamped in the drop detector without breakage of the drop tube, whereas the vertical holes for the uncounted channels are just large enough to accomodate the drop tubes. The drop tubes are made by cutting 50-~1 disposable micropipets in half. The Tygon tubing coming from the peristaltic pump is passed through horizontal holes in the fractionator manifold, and the drop tubes are inserted into the end of this tubing before installation in the vertical holes. This prevents any stress being applied to the drop tubes during movement of the drop detector head.

336 SHORT COMMUNICATIONS

Position of Support Block and Holes for Uncounted Channels

Install the fractionator manifold on the aluminum bracket attached to the drop detector head and place a rack of fraction tubes on the black plastic pan1 of the fractionator and secure them in position with the test tube retaining bar.’ Number the fraction tubes from 1 to 80 in the order in which the Gilson Micro-Fractionator collects fractions during normal operation. Position the drop detector head over fraction tube 56 using the manual advance switch.’ Now place the Lucite support block on the guide bars in contact with the left hand drop head positioning screw’ and allow the fractionator manifold to rest on support block (Fig. 1 B). Glue the support block to the fractionator manifold in this position. With the support block in position and the drop detector head still above fraction tube 56, mark positions for vertical holes above fraction tubes 6 and 35. Remove the fractionator manifold and drill the holes so that when the drop tube is inserted it is central over the fraction tube. Drill horizontal holes adjacent to each vertical hole.

OPERATION

A diagram of the tubing assembly between the centrifuge tube con- taining the gradient, the peristaltic pump, and the fraction collector is shown in Fig. 1C for one channel. All assemblies for the three channels must be identical to maintain equal flow rates through all channels. The 50- ~1 micropipets on the inlet side of the pump are inserted carefully to the bot- tom of three gradient tubes, and the Tygon tubes from the pump outlets are installed into the fractionator manifold with drop tubes. With the movable supports’ of the Micro-Fractionator pushed to the rear and held in place by the hook latch,’ the drop detector head is pushed to the left until the support block contacts the left hand drop head positioning screw. The hook latch is released, and the manual advance switch is op- erated to move the drop detector head forward to a position above fraction tube 56. The fraction size and pumping rate are set, and fractionation is started by turning on the pump. The peristaltic pump can be connected to the power outlet at the back of the Micro-Fractionator so that the pump is turned off when the last fraction has been collected.

RESULTS

The device described enables gradient fractionation to be performed in one third of the time normally required. The Desaga peristaltic pump maintains equal rates of flow on different channels, provided the tubing is flushed with distilled water after use to prevent the deposition of buffer salts and sucrose. Some variation in the inside diameter of the Tygon tub- ing may arise, which will lead to slight differences in the dead volumes of

SHORT COMMUNICATIONS 337

the three tubing assemblies. When the peristaltic pump passes solution into the tube assemblies at identical rates, the differences in dead volume result in the solution arriving at the drop tubes at slightly different times, a larger dead volume leading to later arrival. The first fraction collected may vary in size between the three channels, but succeeding fractions are identical until the last fraction is reached when, again, a slight difference in size may occur. In our experience these variations are never greater than 25% of the fraction volume during the collection of OSO-ml fractions.

The number of channels collected with this device can be changed simply by locating the drop tube holes differently in the fractionator mani- fold. For instance, when four channels are to be collected, the drop detec- tor head is positioned over fraction tube 61, and holes for uncounted channels are drilled above fraction tubes 1, 21, and 41 with the care described above. A similar modification can be installed on the Gilson Model FC/lOOK Micro-Fractionator. When this fractionator is modified it is possible to collect up to five sets of 20 fractions each on individual test tube racks.

ACKNOWLEDGMENTS

This work was carried out under the support of a National Research Council of Canada Operating Grant to D. G. Clark in the Department of Chemistry at the University of British Columbia. The author is a recipient of a Postgraduate Scholarship from the National Research Council of Canada.

GEOFFREY WEBB

Department of Chemistry, University of British Columbia, Vancouver, Canada, V6TI W5

Received November 3, 1977; accepted March 9. 1978