Chemical Microscopy with Silicone Rubber-Coated Microscope Slides Donald E. Laskowski, Department of Pathology, Cleveland Metropolitan General Hospital, Cleveland, Ohio
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for recrystallization or as reaction media in chemical microscopy is severely limited because of their pronounced tendency to spread over the surface of the microscope slide. This leads to rapid evaporation of solvent, poorly forined crystals, and an undesirable dispersion of the test sample over a broad area of the microscope slide. This communication describes a technique which largely eliminates these undesirable features and which enables the use of many of the common organic solvents in chemical microscopy. SE OF ORGANIC LIQUIDS
EXPERIMENTAL
Microscope slides are rendered nonwetting by application of a thin, transparent film of SE-30 (General Electric Co., Silicone Products Dept., Waterford, N. y.) silicone gum rubber. SE-30, 0.25 grams, are dissolved in 100 ml. of chloroform to provide the coating solution. Microscope slides are immersed in this solution, withdrawn, and wiped free of adhering solution. The process is repeated to ensure complete coverage of the slide. If the coating has been properly applied, the resulting slides are free of visible wipe marks and the coating is transparent and invisible. The slides are then stored in the oven a t 120" C. for a t least 24 hours. The remaining coating solution is stored in a stoppered bottle for future use. The following organic liquids have been used successfully on silicone rubber-coated slides: Methanol Ethanol 2-Propanol n-Butanol 2-Butanol Amyl alcohol Capric alcohol Benzene Toluene Xylene Petroleum ether Kerosine Chloroform Carbon tetrachloride 1,2-l>ichloroethane
Glacial acetic acid Acetic anhydride Tetrahydrofuran Ethylacetate Dioxane Ethylacetoacetate 977, Formic acid n',lV-dimethylformamide Acetonitrile Ethylenediamine Glycerol
Xi trobenxene Acetone
The alcohols have a slight tendency to spread and hence do not behave as well as the other liquids in the above list,. However, even with these, performance is much improved as compared to untreated slides. hlost droplets are approximately hemispherical with little or no tendency to spread. For best perforinance, droplet size should be less than 1 mm. in diameter. With highly volatile solvents larger drolilets are used unless operations are 174
ANALYTICAL CHEMISTRY
performed rapidly. I n all cases of organic solvents studies, evaporation rates were lower on the coated slides than on the uncoated slides. I n addition to the organic solvents listed, concentrated sulfuric, hydrochloric, nitric, and phosphoric acids have been used. Although the acids destroy the coating in the droplet area, these liquids have little tendency to spread even on mild heating. I t is possible, therefore, t'o conduct chemical reactions in strong acid media and still to obtain compact hemispherical dropl e k Strong aqueous ammonia, sodium hydroxide, potassium hydroxide, and barium hydroxide have been used with no apparent attack on the coating. -4 wide variety of dilute aqueous solutions have been used with excellent results. I t is frequently possible to clean the slides wit,h distilled water and to reuse them several times. Hecause these slides yield well formed droplets, they constitute an excellent medium for hanging drop reactions. Although the full range of application of SE-30 treated slides has not been explored, the following examples illustrate their potentialities. Recryst,allization with relatively nonvolatile solvents is conducted merely by adding 0.5 to 1 ~ 1 of. solvent to the solid and then allowing the solvent to evaporate spontaneously. With more volatile solvents it is necessary to cover the solution droplet with a cavity slide to decrease the rate of evaporation. I n some instances it is desirable to place one or more droplets of solvent adjacent to the solution droplet and then cover the group of droplets with a cavity slide to further retard evaporation. These techniques have been used to determine appropriate recrystallization solvents for preparative purposes \vith several substituted phenazines. They have also been used to determine allpropriate solvents for morphological ident'ification of uric acid, cholesterol, xanthine, cystine, and oxalic acid in urinary calculi. The coated slides are particularly useful for sequential solubility determination of an unknown solid in a series of solvents because the sample is not lost by spreading even when solution occurs. Decantations and extractions are quite easy to perform because of the nonwetting characteristics of the slide surface. Thus, a particle can be contacted with a given solvent for a period of time, the solvent can be drawn into a niicrocapillary and then redeposited on an adjacent area of the slide. This process can be repeat,ed with the same solvent or with a series of solvents. Decantations can also be performed by immersing a solid glass microrod into the droplet. The glass rod is wetted preferentially and may be used t,o draw a portion of the droplet to an
adjacent area of the slide. This operat'ion is particularly successful with aqueous solutions because a small fraction of the parent droplet) may be removed and manipulated across the surface of the slide almost as if it were a sphere rolling across a smooth surface. Chemical reactions in organic solvents have been studied only to a limited extent. The formation of molecular addition compounds between 2,4,7trinitroffuorenone and polycyclic hydrocarbons proceeds well on these slides. dilute solution of 2,4,7trinitrofluorenone in glacial acetic acid serves as the reagent and is applied directly to the solid hydrocarbon on the microscope slide. Hydrocarbon fragments estimated to be less than a microgram have yielded good crystals of addition compound either on contact or as the solvent evaporates. I t has also been possible to prepare amine picrates using a dilute solution of picric acid in either absolute ethanol or normal butanol as the reagent. The 2,4-dinitrophenylhydrazonesof several carbonyl compounds have been prepared on the microscope slide using a concent,rated hydrochloric acid-ethanol solution of 2,4-dinitrophenylhydrazine as the reagent. In t'he above reactions, the droplet's were hemispherical and compact and yielded good crystals. Because of the nonspreading charact'eristics of the slide surface limits of detection should be quite low, although this has not yet been investigated. Hanging drop reactions have been tried to a limited extent with encouraging results. A 0.5-p1. droplet of 2,4dinitrophenylhydrazine reagent inverted over a cavity slide and observed microscopically yields the 2,4-dinitrophenylhydrazone of acetone when a dilute aqueous acetone solution is added to the cavity. Licetone transfers from the aqueous solution in the cavity through the vapor phase to the reagent droplet where it reacts to yield cryst,alline 2,4-dinitrophenylhydrazone. Similar hanging drop experiments were conducted successfully in the formation of amine hydrochlorides and picrates. X variation of the hanging drop technique which works well with the coated slides involves determination of the solubility of an unknown particle in a series of solvents. A small fragment of the solid under study is deposited on a coated slide and inverted over a cavity slide. The fragment is viewed microscopically after a small quantity of solvent under study is pipetted into the cavity. If the solid has an appreciable solubility in the solvent, it, will deliquesce and gradually transform into a droplet of solution. Removal of the slide enables the solvent to evaporate and the solid to recrystallize in its original location on the coated slide. Solvents can be used
sequentially to determine solubility properties in a series of solvents. I t is possible to concentrat,e dissolved material from a larger volume of solution by repetitive deposition and evaporation of micro drops on the same area of the coated slide. Because spreading does not occur, the solid becomes concentrated in the area of solution deposit'ion. This technique is particularly useful in the microscopical
examination of chromatographic fractions. Refractive index determinations have been made on these slides without difficulty. They are especially useful when it is desired to change the refractive index oil on a given particle. I t is only necessary to decant the refractive index oil by one of the methods described previously. This is best done with the aid of an extracting solvent
such as toluene to remove the original oil prior to application of the next oil. I n our studies on urinary calculi constituents this technique has been used on uric acid, cystine hydrochloride, and osalic acid dihydrate. Melting points have been measured on t,he silicone coated slides and with the limited number of compounds observed there appears to be no influence on the observed melting points.
Simple Electrical Divider for Analytical Applications H a r r y L. Pardue and S. P. Perone, Department of Chemistry, Purdue University, Lafayette, Ind:
H E R E is increasing interest in the 'complete automation of analytical methods with direct read-out of quantitative data in digital form. Often the final presentation of data in its most useful form requires the division and/or multiplication of one quantity by another. Landee, Davis, and hlbrecht ( 2 ) have suggested the use of operational amplifiers with a servo-controlled feedback resistor as a multiplier and with a servo-controlled input resistor as a divider. Although such systems are simple in principle and practice, they are not in common usage in analytical instruments. This communication describes a simple system emliloying servo-controlled resistors in both the input and feed-back circuits of an operational amplifier which provides the quotient of two variable signals. Data are liresented to demonstrate the characteristics of the system. The system is represented in Figure 1. Resistors R , and R, are transfer potentiometers whose shafts are coupled to the shafts of the balancing potentiometers in servos 1 and 2 , respectively. Test signals El and En connected to the inputs of the servos determine their balance positions and therefore the values of R, and R,. The resistance of each potentiometer is zero when the
corresponding test signal is zero and increases linearly with the signal. Under these conditions Rf = klEl and R, = kyE.2where k , and kn depend upon the resistance per turn of each potentiometer and the servo sensitivity. The output from the amplifier is given by Equation 1:
The result is a voltage proportional to the quotient E 1 / E 2 . If E , , is 1.00 volt and lil/k2 is equal to 1.00, the output voltage is exactly equal to the quotient, E I / E 2 . Alternatively, a voltage divider may be used to select that portion of the output equal to the e\act quotient. EXPERIMENTAL
The system for which data are presented is constructed using one Heath Operational Amplifier System EUW19-\ and two Servo-Recorders EUW-20.k (Heath Co., Benton Harbor, Mich.). One transfer potentiometer is mounted in each recorder. A built-in evtension of the shaft which drives the balance potentiometer in each servo permits easy coupling of the transfer potentiometers. R, and R, are 10-K and 100-K potentiometers, respectively, with spec-
& Figure 1.
Block diagram of divider
ifications of +0.5Tolinearity and 2 5 % accuracy (Helipot Division, I3eckman Instruments, Inc., Fullerton, Calif.). The slidewire terminal of each potentiometer is connected to the clockwise terminal. Esternal access to the slidewire and counterclockwise terminals is provided by female banana jacks mounted on the side of each recorder. R, and Ri are selected to have a ratio of 1 to 10 to avoid excessively high amplifications when E2 is small compared to E,. The actual resist,ance of R, was observed to be 0.104 Ri. This deviation from a ratio of exactly 1 to 10 was compensated for by adjusting the sensitivity of servo 1 to be 0.962 times that of servo 2. This adjustment would not be necessary if potentiometers with the accuracy required in the output were used. The input voltage, Ei,, to the amglifier is maintained constant, a t 1.344 volts using a mercury battery. Response characteristics for the system were evaluated both for static signals and for signals varying periodically with time. In the first case calibrated Heath Voltage Reference Sources were used to vary El and E2 stepwise. The distance from balance and the direction of approach to balance was varied randonily for both servos in collecting the data reported here. Output voltages representing the quotients were read on a digital voltmeter (Digitek, United Systems Corp., Dayton, Ohio). .I triangular wave (Hewlett-Packard LOW Frequency Function Generator, Model 202,\, Hewlett-Packard Instrument Co., Palo Alto, Calif.) was used to evaluate t'he response of the system for periodic signals. The same signal was fed t o the inputs of the servos so that, each had the same peak t o peak amplitude with a phase difference of 180". In addition bucking voltages were inserted a t each input so that the d.c. levels could be adjusted independently. A dual-channel recorder (FVestronics Dual Pen Recorder, Model L D l l A / K I Z / D V I O H , West,ronics, Inc., F t . Worth, Texas) was used to observe the output from the divider on the same time scale as the input signal. VOL. 37, NO. 1, JANUARY 1965
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