Build Your Own Air-Displacement Pipetter Lawrence C. Davis Department of Biochemistry, Kansas State University, Manhattan, KS 66506 More and more of the procedures used in chemistry, hiorhemistry,and molerul~biologylaboratoriesdependonuse of sterile disposable pipet tips or repetitive pipetting procedures that a;e most Eoiveniently done with an air-displacement pipetter. In part this is because there has been a change in emnhasis in analvtical techniaues. hut i t is also aresponse toconcernsabour hazards ofdirict pipetting. Simple r"bber bulbs are awkward to use even for the experienced operator, and more versatile pipette controllers cbst about $i5 each. Air displacement pipetters are convenient hut present a considerable financial problem in the teaching laboratory because the instruments cost in the range of $100 each. I t is far beyond the means of most universities or students to s u ~ p l vevervone with their own. Commercially available pipe& are prerision instruments if operated properly and can be damaged by careless use with corrosive solutions. This is an unfortunately common occurrence in teaching laboratories. There is a simple and economical alternative to the pipetters, namely disposable plastic syringes. So long as one understands their limitations, they will work quite adequately. Their biggest defect which they share with even the most expensive pipetter, is that they are sensitive to differences in air temperature. The molecular biologists that I know simplv this problem and seem able to get away with it, . . ienore .. work one must nut ignore the problem. but for Solutions should be at room temperature, and the pipetter must be kept a t room temperature also. Otherwise the volume taken up and delivered may vary, either erratically or systematically. A second defect of syringes is the inherent inaccuracy of the minted scale that is eenerallv marked along the side in -1-6m increments. hisi is a problem that the; share with even ex~ensiverlass svrinaes, and calibration will take care of the &ohlem"for eethe;tGe syringe. Precision is quite eood in either case, and is mainly a function of how well one can read the scale markings and align the plunger to them. An error of 1 mm in a 5-cm scale is typical of the maximum likely reading imprecision. This is ahout 2% of full scale, hut represents a laraer percentage for smaller increments. i have devisd some simple ways to convert disposable plastic syringes into repetitive dispensers. The ones I describe are low cost in terms of material and only moderately labor intensive (figure). First, consider the case where one wants to d i s ~ e n s ere~eatedlvan exact volume, sav 1mL of a reagent. In this cas'a 3-mc syringe, the cheapest possible, costing about 12 cents each, is used.' A piece of polyvinyl chloride tubing (Tygon) is attached to the luer tip and to that is added a hlue pipetter tip, meant to deliver 1mL with a Pipetteman-type pipetter. The tip is cut off to about 1cm to ~ r o v i d ea tapered tip onto which hlue tips can he pressed. he syringe piunger is driven in the upward direction by a spring (type 9657K34 zinc plated compression spring of 7/16 in. outside diameter, 17/s in. long, witha tension of 3 lhs, from McMaster-Carr). The spring is cut in half with the cut end bent in very slightly t o let it fit into the recessed rim a t the ~~~
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top of the syringe. The plunger is driven in the downward direction by the thumb of the operator, so the spring tension should be enough to move the plunger up reliably but not so strong that i t is hard t o push down. T o give precision t o the plunger motion, a stop is provided to check the upward motion. This can be easily done by making two cuts in the harrel with a razor blade as shown in the fipureand then pressinpin the plasticofthe harrelat the cut flap. The first cut is about 1%c& along the barreland the second a few millimeters a t right angles to the first. The exact position for the horizontal cut that provides the stop action must be determined by trial and error. I have observed that i t needs to be high enough so that the lower end of the plunger is slightly above the marked line on the syringe. This kill vary with brand and perhaps lot number of syringe. Syringes are calibrated hy weighing water delivered to a small container on a precision balance. More than one set of cuts can be made on a single syringe so that, if the first is in error, a second cut can be made a t the appropriate position on the opposite side of the syringe from the initial trial. Remember that if you try t o set the volume before making the stop, you must do i t without the spring in place hut alwavs drawine the svrinee . is some slack in - - u ~.w a r d There the plunger system, and i t is the upward stroke that is pertinent for ~ositioninethe s t o ~I. have calibrated svrinees to a precision of ~PL/&Lwith bnly a few minutes effok. This would he a good experience in use of a precision balance for more advanced students and would allow them to make their own set of fixed volume pipetters. Of course it is possible to calibrate the syringes spectrophotometrically by diluting colored solutions of known concentration into precisely measured volumes of a diluent. The spring i* the most expensive component of theapparatus, costingabout 17 cents per half, whik the syringe isoily 12 cents, and the tubing and disposable tips add up t o about 10 cents more. This larger syringe
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Fisher Sclentlflc catalog 1989-90. Unless otherwise noted, all prices from mis source.
MdlflCBtiond three syringes to deliver fixed volumes. Volume 68 Number 4
April 1991
355
works well for volumes from about 0.5 to 1.5 mL, though for the largest volume i t may be necessary to stretch the spring before starting. T o deliver small volumes in the range of 0.1 mL, one should use a l-mL syringe and yellow tips. The l-mL syringes present a slight difficulty because the plunger does not have much clearance with the barrel, so that simple cutting of stops does not work. I have devised two solutions. The first of these, shown in the second drawing, makes an adjustable dispenser that works on the same principle as a Chaney modification of the Hamilton syringe. I t requires use of two l-mL syringes and some epoxy glue as well as two small screws about 1cm long. I t is quite precise and can be adjusted to the desired accuracy of delivery by setting the screws. The second solution is convenient for a fixed-volume dispenser that works exactly as the large-volume kind described above. It requires one l-mL and one 3-mL syringe, a half snrine and some eooxv. in addition to PVC tubine. I t is iliust;ate$in the third d r k i n g . In this case a singleul-ml syringe is cut in half. The lower end is inserted through the drilled out lower end of a 3-mL syringe and held in place by heaw-walled PVC tubine. This will slide into d a c e easilv but k t e r a few days wiil "set" into place and he near$ immovable. I t would be better, if possible, to prepare the syringe to this stage, calibrate it, and let i t rest a few days before regular use, so that the l-mL barrel has no chance to slip after calibration is done. The upper half of the l-mL syringe is glued t o the top end of the plunger, and the finger erins are cut off leavine iust enoueh to allow them to eo into ;he barrel of the 3 - m l & i n g e . ~ h ; rubber end piece ofthe 1mL plunger is fitted into the lower half of thesyringe, whilea spring is placedaround the upper half. A stop for the plunger is cut in just the way described above for a 3-mL syringe. In this case it stops the upward motion of the inverted portion of the l-mL barrel, catrhing it at the cut-off finger grips.
356
Journal of Chemical Education
I t should be obvious from these descriptions that it is possible to devise a range of other instruments for special applications in particular laboratory settings. The use of disposable syringes should help make transparent to the students the nature of the high precision pipetters that they may later use and should show them that acceptable technology need not always he costly. T o reach into long narrow tubes, the syringe may be attached to a plastic disposable pipet and the pipet used for liquid transfer, or a sterile tip mav be added onto this. An alternative approach that also uses syringes and that mav he suitable in manv cases is t o fit a shortened vellow tin directly onto the luer tip of a syringe and then use the syringe directly in place of apipet. Thisis very good for rapid addition of repeated aliquots of a reagent so long as it does not attack the rubber -plunger or the syringe itself. Most biochemistry laboratory reagents are acceptable over the course of a laboratory period, though reducing agents in the rubber will cause a slieht backmound in some reactions such as the copper bicincLoninate reagent.2 Direct uae of the syringe allows one to add larger volumes without massive heavy duty springs and giant syringes. Both precision and accuracy are usually sufficient for most laboratory courses except perhaps for quantitative analysis. For the price of one broken pipet i t is possible to buy 10-15 syringes, which should please both students and faculty. Acknowledgment Supported by the Kansas Agricultural Experiment Station. This is paper #90-2965 of the KAES. I thank Tom Manney for encouragement in this effort.
. 1987, 181, 152-156 2Davis, L.C.: Radke, G.A., ~ n a lBlochem.
