ANALYTICAL CHEMISTRY
2036 mond point is exactly opposite the lower side of the meniscus. AS the 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 at 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.
V O L U M E 28, NO. 1 2 , D E C E M B E R 1 9 5 6 and operation of a scanner-camera n-hich has now found extensive use in the field of steroid research both in this and other laboratories. (A similar machine n-ill be manufactured commercially by Labline, Ine., Chicago, Ill.) Obviously, the usefulness of this apparatus is not limited to work with steroids. I n fact, the ultraviolet photography of paper chromatograms was proposed independently by Markham and Smith ( 4 ) , who employed the technique for the detection of purines, pyrimidines, and derivatives of these compounds.
CONSTRUCTION
Ultraviolet Scanner-Camera. The ultraviolet paper chromatogram scanner-camera consists of a lighttight box containing a light source, an adjustable shutter and its enclosure, a transmission filter, a fluorescent screen, and a pressure plate. The box, which is constructed of stainless steel to resist the corrosive vapors of laboratory atmospheres, is 20 X 7 X 14 inches (length x width x height). The open top of the box is surrounded with a ledge to hold an exposed filter in place. A 15-watt ultraviolet lamp (Germicidal Type WL-15 Sterilamp, Westinghouse) is centered in the bottom of the box. The top surfaces of the shutter and its enclosure are 6 inches above the box floor. The transmission filter consists of three 6.5-inch squares of Corning filter glass KO. 9863 (transmission characteristics: range, 230 to 420 mp; maximum, 320 mp; 507',, 250 to 390 mp). Three squares of filter glass are needed to cover the top of the box because a single piece of appropriate size is not obtainable. The top surface of the filter must be higher than the retaining edges of the box. The fluorescent screen consists of Du Pont zinc silicate phosphor S o . 609 (activated maximally by light of 254 mp wave length) deposited on double-strength window glass. The phosphor vehicle and adhesive, which must transmit ultraviolet light, is poly(ving1 alcohol) (Elvanol 71-24, Du Pont). The screen is encased in a metal band with a hinge along the rear. The screen must be large enough for the metal encasement to rest outside of the filter glass. This is necessary so that the screen ail1 press tightly against the paper. The pressure plate, used with the adjustable shutter when the apparatus is employed as a camera, is simply a piece of aluminum sheet stock, 20 X 7 X 0.5 inches, which has one evenly flat side and an attached handle on the other side. A schematic diagram of the scanner-camera is given in Figure 1. Visible-Light Camera. The ultraviolet scanner-camera can be modified for use as a camera to photograph spots made visible by color development on paper chromatograms. This adapted camera is useful not only as an aid in the preparation of permanent records, but also as a means to obtain photographs for comparison with ultraviolet photographs of the same paper chromatograms taken before color was developed. The visible-light camera can be constructed by the use of a 15-watt fluorescent lamp instead of the germicidal lamp, and plate glass instead of the Corning filter glass.
2037
If the paper chromatogram is cut with channels as described by Zaffaroni, Burton, and Keutmann ( 5 ) ,the use of a complementary mask, cut from thin cardboard and placed between the chromatogram and the filter, prevents an excessive amount of fluorescence on the screen and renders the ultraviolet-absorbing substances more readily detected. A photograph can be made of the chromatogram if the fluorescent screen is replaced by photographic paper (Kodagraph Contact Standard), weighted down by the pressure plate. Such photographs can be used for prolonged study and can be filed as permanent records. If the data pertinent to the chromatogram are written on the paper with a soft pencil (No. 1 lead), they appear on the photograph as well. The chromatographic record, therefore, is complete on the photograph. Ultraviolet-absorbing spots appear white on the black background on the photograph. Exposure time varies relative to the type and amount of solvent on the paper. Two to 4 seconds, standardized as a certain number of shutter strokes, is generally satisfactory. It is sometimes of value to over- or underexpose photographs to reveal features not observable under standard exposure conditions. Low concentrations of material are more readily detected by underexposure; two components, not completely resolved, may be revealed by overexposure.
OPERATION
Detection of Ultraviolet-Absorbing Compounds. Khen compounds having ultraviolet absorption are to be detected on chromatograms, the paper strip is placed on the filter glass, and the fluorescent screen is lowered to rest directly on the paper. The light source is turned on in a darkened room to view the fluorescent screen. The filter paper background appears as a uniform, light greenish fluorescence, whereas areas bearing ultraviolet-absorbing materials appear as distinct, dark shadows. These shadows are due to the absence of fluorescence in the regions where ultraviolet light is prevented from impingement upon the screen. Lengthy observations (for more than several minutes) of the chromatogram should be avoided because of the possible decomposition of compounds on the paper by ultraviolet light and as a precaution against possible damage to the screen by the chromatographic solvents. The chromatogram should be airdried for about 5 minutes or longer prior to viewing in order to allow the evaporation of volatile solvents which may otherwise render it overly moist or opaque to ultraviolet light.
Figure 1. Schematic diagram of scanner-camera Fluorescent screen Papergram Transmission filter D. i d j u s t a b l e shutter E . Ultraviolet light A. B. C.
Should it be desirable to elute the compounds from channeled chromatograms for further study, the paper strips may be placed off-center over the photograph, and the area containing the compound accurately marked for subsequent segmentation. Marking for segmentation on nonchanneled chromatograms can be accomplished with a blunt scriber while viewing on the scanner. If color tests are run on channeled paper, the location of chromogenic compounds and ultraviolet-absorbing compounds can be compared in a fashion similar to that described for segment elution.
ANALYTICAL CHEMISTRY
2038 The amount of ultraviolet-absorbing steroid detectable by the visual and photographic methods is about 5 y per square cm. of filter paper. Photography of Chromogenic Compounds. The use of the visible-light modification of the scanner-camera derives its greatest utility in the preparation of permanent photographic records of developed paper chromatograms. This is especially true if the color developed on the paper fades rapidly after development. Contact paper is used as in the ultraviolct 1 2 3 4
Tray for Dipping Chromatograms Nelle J. Morris and Austin C. F. Mason, Southern Regional Research Branch, Agricultural Research Service, U. S. Deportment of Agriculture, New Orleons, Lo.
\PER
chromatograms, after development, generally require
P the application of reagents for locating the positions of the separated components. Depending on the reagent, the solvent in which the reagent is dissolved may be allowed to evaporate and the dried chromatogram heated or allowed to stand a t room temperature until reactions have produced colored spots on the paper. The reagents are usually sprayed on the chromatogram. However, it is often better to dip the chromatograms in solutions of the reagents in organic solvents (I-S'), in which the separated components are not readily soluble. ilmong the advantages of dipping are uniform application of the reagent to the chromatogram and the avoidance of mists. In order to dip the chromatograms conveniently, an apparatus is needed that will achieve positive and uniform wetting of either large or small papers, will not exert much drag on the paper (danger of tearing u et paper), will require only a small volume of solvent, and will be easy to clean, inert to corrosive reagents, and free from sources of contamination-e. g., metals.
1 9 3 4
Figure 2. Comparison of photographs of same papergram Papergram developed on Whatman Yo. 1 paper in toluene-propylene glycol (6).
UV. Ultraviolet photography T C . Vkible-light photography tetrazolium color development
after
Channels, top t o bottom: 1, 4. 11-Epihydrocortisone, hydrocortisone, cortisone, ll-desoryhydrocortisone 2 . Tetrahydrocortisone (TC), tetrahydro-1 1-desoxyhydrocortisone (TC), tetrahydro 11 -dehydrocorticosterone (TC) 3. Tetrahydrohydrocortisone (TC), pregnenetriolone (UV), 21-desoxyhydrocortisone (UV)
-
photography; however, a somewhat longer exposure is necessary in this case. I n the use of this device to photograph chromatograms with developed tetrazolium color ( 3 ) ,a pretreatment of the processed paper is desirable, This pretreatment consists of: (1) washing excess tetrazolium reagent and alkali from the paper with water (deionized water is used in this laboratory), and ( 2 ) moistening the washed paper with 50% (by volume) aqueous propyIene glycol. The irashing and moistening operations are performed while the chromatogram is supported on a sheet of stainless steel a t an angle of 45" in a sink. The use of propylene glycol enhances the transparency of the paper. Figure 2 s h o w photographs of the same paper chromatogram, first, by ultraviolet photography and, second, by visible-light photography after tetrazolium color has been developed on the paper. LITERATURE CITED
Haines, W. J., "Recent Progress in Hormone Research," vol. 7, pp. 255-305,Academic Press, New York, 1952. Haines, W. J., Drake, K . d..Federatwn Proc. 9,180-1 (1950). Knauff, R.E.,Xielson, E. D., Haines, W. J., J . A m . Chem. SOC. 75, 4868-9 (1953).
Markham, R., Smith, J. D., Biochem. J . 45, 294-8 (1949). Zaffaroni, A.,Burton, R. B., Keutmann, E. H., Science 111, 6-8 (1950).
A tray that meets these requirements and provides a convenient means for dipping large chromatograms is made from a longitudinally split section of borosilicate glass tubing. For a roller to hold the paper beneath the surface of the reagent solution, a glass tube of smaller diameter, sealed a t the ends, is fitted into fixed glass sleeves in such a manner that the tube rotates freely. A 24.5 X 2.75 inch tray d l accommodate chromatograms up to 22.5 inches in width. The exact dimensions of the tray (or roller) are not important; however, trays much smaller in diameter would not be convenient for use with large papers. The glass sleeves were prepared from glass tubing having an inside diameter slightly larger than the short pieces of glass rod that seal the ends of the roller and fit into the sleeves. The sleeves were slipped on the roller and then sealed permanently t o the tray. In use, a dried paper chromatogram is slipped under the roller tube and drawn quickly through the reagent solution (approximately 50 ml.). This device is probably suitable for other laboratory coating and impregnating processes. LITERATURE CITED
(1) Jepson, J. B., Smith, I., Nature 172, 1101 (1953). (2) Smith, Ivor, Ibid., 171, 43 (1953). (3) Trevelyan, W. E.,Proctor, D. P., Harrison, J. S., Ibid., 166,
444 (1950).
