AIDS FOR THE ANALYST Pipet-Co ntrolling and Aspirating Device with PressureRelieving Action Robert C. Backus, Virus Laboratory, University o f California, Berkeley, Calif.
microvolumetric pipet measP urements require the use of a manual aid that combines rapid filling RECISE
and discharge rate with fine control of the position of the meniscus. The use of modified hypodermic syringes for this purpose is, in the hands of many otherwise technically competent individuals, difficult and frustrating, occasionally leading to accidental displacement of the contents of the pipet. An aspirating device has been in use in this laboratory for routine micropipetting operations that is convenient to manipulate and is precise in the control of microvolumetric pipets. It also has been found useful for a number of other applications. Construction of the device is relatively simple, without critical dimensions or tolerances.
As shown in the figure, the aspirator is composed of a length of rubber tubing held within a rigid barrel by folding the tubing ends concentrically back over the ends of the barrel. A thumb-actuated wheel, guided by a slot in the barrel, rolls longitudinally over the rubber tubing. The nTheel is retained within the slot by a transverse bar. the ends of which extend on either side of the wheel into slideways milled in the facing sides of the slot. Pipets are attached by insertion of the pipet shank into either end of the aspirator.
aspirator serving to position the meniscus and discharge the fluid. Because of the interchangeability of filled pipets, one aspirator can be used for a number of operations involving sequential additions and the holding of measured samples until mixing is desired. It is quite feasible to make transfers from one pipet to another using an aspirator in each hand, thus facilitating measurement by difference of quantities for which microvolumetric pipets are not usually calibrated. Another use for which the aspirator is particularly well suited is the mixing of microvolumes of reactants by aspiration within silanized glass tubes of small bore. A controlled to-and-fro motion is induced in the liquid column by pressing and rolling the thumb wheel of the aspirator back and forth. The wheel can be set a t will to position the liquid column a t any desired place within the mixing tube, eliminating the problem occasioned in closed-system aspiration by the creeping of fluid along the tube during the mixing operation. The filling of pipets with capacities several times larger than that of a single stroke of the aspirator can be achieved by a series of displacement strokes, momentarily releasing pressure on the thumb wheel while returning the wheel for a succeeding stroke. In this way the aspirator is used as a hand pump. The pumping action can be extended to the filling of smaller macrovolumetric pipets by attaching a check valve to the open end of the aspirator to prevent excessive loss of fluid due to the return of stir during the release of
Thermostated Microcell Adapter for Perkin-Elmer Infrared Spectrophotometers S. T. Zenchelsky and J. S. Showell’, Ralph G. Wright Laboratory, School of Chemistry, Rutgers University, New Brunswick, N. J.
T
deuterium content of organic compounds can be determined in a number of ways, but most involve burning the compound and isolating water, which is then analyzed directly for deuterium content or converted to hydrogen for analysis. The former alternative is most attractive because of the development of an elegant infrared spectrophotometric technique by Trenner, Arison, and Kallrer ( 3 ) . HE
T t J m b wheel
SIDL VIEW Re’aining bar
CROSS
sEcrio,v
Figure 1.
ilir is controllably displaced in either direction in the tubing by siniultaneously pressing and rolling the wheel along the tube. I n the released position the !Theel does not compress the rubber tube enough to prevent passage of air through the tube (see cross section). Thumb pressure on the wheel may thus be released at any point in the displacement stroke to equalize the internal pressure with that of the atmosphere. This arrangement permits removing and reaffixing a filled micropipet n-ithout displacing the fluid contents. Advantage can be taken, if desired, of capillary action in filling pipets. the
the wheel between strokes. The aspirator obviously can be scaled up in size t o accommodate pipets of large capacity. The rubber tubing may be quickly replaced in the event that corrosive or radioactive materials are draKn into it.
Partial cutaway view
Thermostatinp fluid channel, thermometer well, hose connections. clamping screw, and microcell guide bare
ACKNOWLEDGMEN’I
Their technique involves the measurement of absorbance at the 0-D stretching frequency (3.98 microns) as a function of the deuterium concentration of water. -4s the stretching frequency was found to be temperature-dependent,
The author wishes to express his appreciation to George Lauterbach for the fabrication of several niodificatiom of this device.
Present address, Chemistry Department, Columbia University, New York, N. Y . VOL. 29, NO. 1, JANUARY 1957
167
b Figure 2. diagram
Construction
-
Microcell-housing cylinder Microcell guide bar Window for microcell housi ng 104. Sealing plate for fluid channel 107. Clamping screw 101. 102. 103.
thermostating was required. A further modification of the method by Trenner, Arison, and Walker ( 2 ) permitted the standard microcell to be used. In order to accomplish the necessary thermostating, modification of the standard microcell adapter was made within the spectrophotometer housing. This paper describes a thermostated microcell adapter suitable for use with the standard microcell. Its advantage lies in the fact that it slides easily into the externally located cell mount of the spectrophotometer; thus no modification of the spectrophotometer is required. The introduction of water lines into the spectrophotometer housing is also avoided. The adapter can be removed easily when not in use, and it may be operated from any standard laboratory circulating bath. Construction is simple and inexpensive. This adapter has been used for over 6 months with excellent results. A pictorial representation is shown in Construction details are Figure 1. given in Figures 2 and 3. Figure 4 is an assembly drawing. After the parts have been constructed according to Figures 2 and 3, parts 101 and 102 are silver-soldered to part 101. This is subassembly A. Next, the thermometer-well nut (part 106, Figure 4),is silver-soldered to part 105. This is subassembly B. Then subassembly A is inserted into subassembly B. and they are clamped and softsoldered together. Finally, the clamping screw, part 107, and the threaded hose connectors are inserted into part 105. The thermometer well nut (part 106) will accommodate a standard Bausch & Lomb refractometer-thermometer. The entire adapter is made of brass. For double-beam operation, a stainless steel screen is used in the reference beam. Kewark TT’ire Cloth Co. Type 304, 270 X 270 Tw mesh, 0.0016-inch wire diameter, has proved satisfactory. 168
ANALYTICAL CHEMISTRY
Figure 3.
Construction diagram
105.
Figure 4. Kote 1.
Adapter block
Assembly diagram
106. Thermometer well nut Solder t o ensure water-tightness. Assemble in order of part number
As the light beam passing through the sample converges, a loss of energy results when the microcell is used in the thermostated adapter. This loss was measured and found to he between 77 and 78% of the energy transmitted by the microcell in the standard adapter. Despite the loss of energy, instrument performance remained satisfactory because the signal to noise ratio is still sufficiently large. Data on the performance of the microcell adapter are given by Bennett (1). ACKNOWLEDGMENT
The authors wish to thank Monfred J. Weiss for construction of the cell and Richard Bennett for selection of the wire screen.
eter, equipped with the Model 85 microscope attachment (5). The entire optical path of the instrument was continuously flushed with a stream of air dried over activated alumina. A. Measurements on Self-Supporting Films, Suitable for Nonvolatile Liquids. A relatively nonvolatile liquid is measured as a thin film contained in a small hole drilled or cut in a thin sheet of suitable material. For example, a hole approximately 0.5 mm. in diameter was carefully drilled with a diamond-tipped pencil in a glass coverslip of 0.09-mm. thickness. After thorough cleaning of the glass surface, 4 p l . of a 0.4% solution of n,-decyl alcohol in carbon tetrachloride was delivered over this hole from a micropipet. The amount of solute actually delivered was 13 y (0.016 pl.). As the solvent evaporated, a thin film of the pure alcohol in hole nsed co1lect.e.d .._ . ..the ..... ....nnd w2.s _ . . ... to ..oh.. tain a satisfactory spectrum. I n like mann er, a satisfactory spectrum was obtafined from 20 y (0.018 ~ 1 of) benzyl beneslate. Support for this film was provnded by a Teflon sheet of 0.03-mm. thick]less with a hole 0.5 mm. in diameter. The end of a thin d a s s cauillarv was a metri,
in the top disk, B. The larger a-ire loop was used around the inner sample loop as a guide for refitting the cell together and to keep the stopcock grease, which was used to seal the cell, away from the sample loop. It was necessary to use insoluble stopcock greases whenever volatile solvents or solutes were placed in the cell. An esterification resin (7) and a glycerol-bentonite clay mixture were both satisfactory greases for this purpose. The cells were filled by touching the sample loop with the tip of a glass capillary containing the sample, and then covering it immediately with the top pellet.
~
LITERATURE CITED
(1) Bennett, R. T., Ph.D. thesis, Rutgers
University, New Brunmick, PIT. J.,
1956. (2) Trenner, N. R., Arison, B., Walker, R. W.. ANAL. CHEM. 28, 530
(1956). ( 3 ) Trenner, N. R., Arison, B., Walker R. W., Appl. Spectroscopy 7, 166 (1953).
Sample Handling for Qualitative Infrared Microspectroscopy
E.
D. Black, J. D. Margerum, and G. M. Wyman Quartermaster Research 8. Development Center, Natick, Mass.
o IDENTIFY minute amounts of Tmaterials, a need arose for simple techniques for handling liquids and solutions in infrared microspectroscopy. While several ingenious methods have been described ( I ) , they usually require special equipment. For example, in one very elegant technique the samples are contained in capillary tubes made from silver chloride (2). These specially prepared capillaries were about 0.075 mm. in diameter and held only about 0.005 pl., although a somewhat larger volume was needed for manipulation of the sample. Another type of microcell in which the sample fills an etched-out space between polishedsalt flats has been described (4, 6 ) . These latter cells used minimum sample volumes ranging between about 6 p l . for a cell of 0.1-mm. thickness and about 0.2 ul. for a 0.01-mm. cell. APPARATUS AND PROCEDURE
All measurements were made using a Perkin-Elmer Model 112 Spectrom-
The effective film thickness of both samplcs was estimated to be 0.04 mm. by comparing their spectra with those obtained by conventional means in a 0.03-mm. cell. The films remained intact for a t least 1hour. Some liquids form satisfactory films more readily on one supporting material than on another. It is evident that such factors as the surface tension of the liquid and the wettability of the solid influence the formation of these films. Delivery of a liquid to the hole via a volatile solvent was preferred over direct delivery of the liquid from a micropipet or rod, for in the latter case the film thickness was apt to be too great. B. Measurements on Liquids Contained in Potassium Bromide “Sandwich” Cells, Suitable for Volatile Liquids and for Solutions. The sample cell consisted of a loop of thin platinum wire sandwiched between potassium bromide disks, and was very easily constructed with the aid of a press and a die used for making potassium bromide pellets (6). The cells were made in the following mannei : Sufficient potassium bromide powder for a thin disk was packed firmly with hand pressure into the pellet die. Ta.0 loops of wire, approximately 1 and 4 mm. in diameter, were formed from platinum wire 0,010 inch in diameter, placed on top of the packed powder and covered with a thin potassium bromide pellet. All this was pressed together (25,000pound total load) to form a unit, which was then separated at the interface with a razor blade and used as a demountable cell, as indicated in Figure 1. The wire was thus partially embedded in the bottom disk, A , and caused indentations
Figure 1. Potassium bromide infrared microcell
Because of its relatively high boiling point and good transparency in the 2to 13-micron region, bromoform (Eastman Kodak, spectrograde) was found to be the most convenient solvent for use in these cells. (On exposure to room light this diphenylamine-stabilized bromoform turned yellow. Homever, this color change did not cause a detectable change in its infrared spectrum.) A typical cell used only 0.075 p l . of bromoform to fill it, and gave a solvent thickness of approximately 0.08 0.01 mm. To check this met.hod, satisfactory spectra were obtained for solutions of ethyl propionate, isobutyraldehyde, and aaohenaene in bromoform. Each filling of the cell required about 5 to 20 y of solute, depending upon the concentration of the solution. Carbon tetrachloride can also he used as a solvent (demonstrated by obtaining a satisfactory spectrum of benzoic acid in this medium), but, because of its greater volatility, carbon
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VOL. 29, NO. I, JANUARY 1957
169
