V O L U M E 2 8 , NO. 12, D E C E M B E R 1 9 5 6
2035
prepwed, 01 are stored under a protective liquid layer, and allon-s the sample to be withdrawn from the main bulk of the solution. The essential features of this apparatus are the constant maintenance of the two liquid phases existing in the storage vessel a t all times during the transfer operations required in the titration and the elimination of the elaborate apparatus setups required by the other methods for accomplishing the same results.
liquid phase, indicated a precision of >99.77, when carried out with a 50-ml. buret.
-1piston is constructed by cutting a rubber cylinder, with a diameter slightly larger than the bore of the buret to be used, from a rubber stopper by means of a cork borer. This cylinder is drilled out to grip the flared end of the glass rod, C, which is slightly longer than the buret,B, and enables the pistonto be raised or lowered. The rubber cylinder is placed on a rotating glass rod held in the chuck of a stirrer motor and its diameter is decreased by holding it against a piece of fine emery cloth on a XT-ood block until it barely slides smoothly through the buret.
Sjoerd L. Bonting' and Gerald R. Walters, Department of Physiology and Physiology-Pharmacology Machine Shop, College of Medicine, State University of Iowa, Iowa City, Iowa
Micro Test Tube Marker
microliter scale, diluting to volume is usually I-avoided onbytheemploying known and reproducible volumes of c
WORK
sample and reagents. However, in some cases this is not possible-e.g., in determinations involving a digestion a t high temperature, in the course of which varying amounts of fluid may evaporate. A micro test tube marker has been constructed, which allows the marking of micro test tubes to contain volumes of from 20 to 500 pl. An exploded view of the apparatus is shown in Figure 1.
A
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2-51 /3/
S L O T F O P PEWCCIL 3 2" 0 E E P, , / 4 * , D L
7-
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In use, the air-sensitive solution, L), is prepared, or stored, under a protective layer of a less dense, immiscible liquid, E . The buret tip, F , and a port'ion of the buret barrel, G, are filled with liquid E or an equivalent and the stopcock is closed. The piston is pushed into the buret with sufficient force to cause the leakage of all air trapped above the surface of G around the piston, A . When the piston touches G, a tight' seal is formed hetween the piston and the inner wall of the buret. The tip of the buret is now placed beneath layer E in the liquid to be transferred, D, the stopcock is opened, and tQe buret is filled by slon.1~xvit'hdrawing the piston. When the desired volume ha3 heen-withdrawn, the stopcock is closed and the piston removed from the buret. The unstable liquid is now in the buret, still protected by a layer of E. I n performing a titration, the buret tip should be flushed for each sample removed by alloiving a small volume of solution to run out (assuring fresh solution in the tip). Any droplet remaining on the tip is removed, and the tip is immediately immersed in the liquid to be titrated. The titration is then carried out in the normal Fay. In this laboratory t,his method proved to be extremely advant ageous for the standardization of chromous chloride Eolutions. The measured volumes of chromous solution were run in under the surface of excess ferric ammonium sulfate solutions, which n-ere then back-titrated with standard dichromate using diphenylaminesulfonic acid indicator. A large number of chromous determinations by this method, using dodecane as the protective
D ' ? I
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i
0.52 PELC
Figure 1. Apparatus
D,E , and F are made 01 !)us ~, liiA k and c' ('on num to reduce the total \\--rightof the apparatus. B marking pencil (Fisher Srieiitific Co., S o . 8-675). d and C k)oth have a slot 3 / 3 2 inch deep and ' 1 4 iiich aide to hold the pencil. They have a V-shape cut on one side, which t angular stage E . The dimensions are given in Figure 1. iq permanently fastened to block C with two 10-32 flat-head machine screm. Screw F runs through the hole in plate D. Triangular stage E is lightly riveted to F . The niarking pencil is posit,ioiied in the slots in A a n d C'. and is then held in place by tightening the four 10-32 roiind-llrBad machine screw in A . -1volume of water, equal t o the volume a t wliich the tubes are to be marked, is added to each of the tithes by means of a Lang-Levy constriction pipet (3licrochemical Specialties Co., Berkeley 3, Calif.). The tube is placed on stage E' in the l--shape groove, and held there with the index finger of one hand. The whole assemhly is held at eye level against a well-lighted background. The test tube and diamond point can be observed through the space rcmaining betn-een A and C. The stage is lowered or raised until the dia-~ . . ~ _ _ I Present address, Department of Medicine, Presbyterian HoPpital. and Department of Biological Chemistry. 1Tniversity of Illinois College of hledicine, Chicago 12, Ill.
ANALYTICAL CHEMISTRY
2036 mond point is exactly opposite the lower side of the meniscus. AS t h e upper part of block C is just below the meniscus, the bottom of the meniscus appears dark, and can therefore be aligned with the diamond poirlt with great accuracy. test tube is then rotated once or more, while being pressed gently into the V-shape groove and onto the stage. This gives a very fine mark all around the tube. The reproducibility in actual use was determined for 20 tubes, calibrated to contain 100 ~ 1 . Each tube was first weighed dry to 0.02 mg., then placed in a holder on a rack and pinion stage, and filled t o the mark wit'h water from a vertically clamped capillary pipet, The addition of fluid from the pipet was controlled by means of a 1.0-nil. hypodermic syringe, connected with the pipet through plastic capillary tubing. Observing the tube through a magnifying glass facilitated the setting of the meniscus. The tube was weighed again, and the volume of water contained in the tllbe w a ~calciilated from the difference in \\-eight.
The average volume was 99.03 @I., with a standard deviation of 0.72 pl. The constriction pipet,, used to add t,he water prior to marking the tubes, had a calibrated volume of 99.0 pl. .is the inner diameter of these tubes was 4.0 mm., t,his reproducibilitJmeans that the meniscus can be set to nithin =kO.O5S mni. from the mark. Parallax can easil!. be avoided, as the mai~liextends completely around the tulle.
the small elevated tip of the calomel electrode (Figure 1,A). The wells are connected a t the bottom by a narrow channel drilled in from one end only. The portion of the channel not connecting the wells is plugged with a cemented-in rod of Plesiglas. The sample chamber is cleared and smoothed by flushing with Plexiglas solvent. Dimensions for this particular vessel may be obtained from Figure 2. The scale near the left edge of the vessel (Figure 2 4 ) indicatrs, from bottom to top, the levels reached in the wells by 0.25-, 0.50-, 0.75, and 1-ml. samples, respectively. Khen electrodes are inserted to the bottoms of the wells, the level of each of these various sized samples is raised and more electrode surface is bathed by a given sample than is indicated by the scale. Hundreds of check pH determinations with the indicated electrodes have shown that this vessel does not change the accuracy of measurement. For greatest accuracy the sample chamber and electrodes should be dry before adding sample. Also, rather serious eirors may be incurred in the measurement of thin lajers of poorly buffered solutions due to the solubilit? of the gl:1ss.
Plastic Vessel for pH Measurements of Small Samples Kenneth M. Richter, Department of Anatomy, University of Oklahoma School of Medicine, Oklahoma City, Okla.
ONE
principle which seems to have been followed in designing electrodes for p H and electromotive force measurements has been to make electrodes fit the amount of sample avai1at)Ie. Thus, electrodes are listed commercially for large samples, for samples from 3 to 5 ml., for 1-drop samples, anti foi those even smaller. A \
I \
B
Figure 2.
Orthographic projection of plastic pH vessel
This type of vessel has been used for 4 years to make p H measurements on supernatants of 2 ml. and less from roller tube cultures, as well as in the routine preparation of tissue culture media, buffer solutions. and the like. A plastic cell of small capacity has also been suggested by Dietz [Dietz, V. H., Science 108, 338 (1948)] but its shape doos not conform t o that of the electrodes as does the vessel described here. ACKNOWLEDGMENT
Figure 1. Plastic pH vessel A , B. Wells, I 2-inch diamA has b u t t i i n i eter. shoulder. C. Connecting channel D. Cemented plug
A vessel has been fabricated in 11hich the sample chamber closely reflects the size and shape of standard Beckman electrode tips, and which can accommodate samples as small as 0 25 ml The vessel illustrated (Figure 1) is designed for use with the No 4990-80 and S o 4970 Beckman standard electrodes measuring x 51/4 inches. It consists of a block of Plexiapproximatrlv 7 glas, 1 X 1 5 x 2 inches, in nhich two closely spaced wells One well has a shoulder a t the l / 2 inch in diameter are drilled. bottom to conform to and a t the same time prevent damage to
The author wishes to thank H. A. Shoemaker, Pharmacology Department, for preparing the vessels illustrated, and Ernest Hiser, Medical Art Department, for preparing the illustrations. This work was supported by grants-in-aid from the Helen Hay Whitney Foundation.
Ultraviolet Scanner-Camera for Paper Chromatography Norman A. Drake, William J. Haines,' Raymond E. Knauff,z and Eldon D. Nielson, The Upjohn Co., Kalamazao, Mich.
R I E F descriptions of scanner-cameras for the visual and photographic detection of ultraviolet-absorbing steroids on paper chromatograms have been published from this laboratory previously (1, 2 ) . Details are presented here for the construction 1 2
Present address, Armour Laboratories, Chicago, Ill. Present addrpss, G. D. Searle and Co., Chicago. Ill.
