Photoelectric Technique for Spectrophotometry between 194 and 225 MiIIimicrons RAYMOND E. HANSEN and MARY V. BUELL Departmenf o f Biochemistry, University o f Chicago, Chicago 37, 111.
b To meet the requirements for accuracy in measuring absorption of light in the far-ultraviolet, a spectrophotometer has been developed for use between 1 9 4 and 225 mp, with a constant half-intensity band width of 1.5 mp. An RCA developmental photomultiplier Type C7180 was employed as the photoelectric detecting device. As the effective threshold of the photocathode was determined to b e 2 4 0 m p in this system, stray light of longer wave lengths could not b e detected and stray light of shorter wave lengths was not found, The sensitivity of the instrument is sufficient to transmitdetect increments of 0.1 tance in solutions of absorbance approaching 2.0, when read with a water reference, and approaching 4.0, when read with a reference having an absorbance of about 2.0 with respect to water.
70
T
primary instrumental problem in spectrophotometry in the farultraviolet is concerned with the spectral purity of the incident light ( 3 ) . The approach to the desired end-elimination of stray light-used in instruments designed to solve this problem has been the spectral purification of the incident light by double monochromation and detection of the transmitted light with a photodevice employing a n 5-5, S-13, or S-19 surface such as the 1P28 photomultiplier ( 7 ) . The approach described is complete elimination from the measured light of stray light of wave lengths longer than the threshold value of the photocathode by the use of a solar-blind tube photomultiplier, with appropriate amplification of the photoelectric current. In this case double monochromation would only reduce the intensity of the monochroniated light. For certain purposes monochromation, although expensive, may be adequate; for others i t n ill not suffice. I n Figure 1 three independent curves have been plotted together for convenience in illustrating certain interrelationships 11hich are basic to the thesis of this paper. BE
A gives the points which show the relative intensities of the xenon arc, 150 watts, as a function of wave length, 878 *
ANALYTICAL CHEMISTRY
the intensity at 700 mp being plotted arbitrarily as 100% ( 5 ) . It is apparent that the relative intensity is minimal in the far-ultraviolet; the ratio of visible to far-ultraviolet light is very large. B shows the relative response of the 1P28 photomultiplier as a function of wave length observed when a xenon arc and a Leiss single monochromator were used; this tube is sensitive to a wide range of wave lengths and the response is much greater to visible than to ultraviolet light. I n fact, the far-ultraviolet lies a t the extreme limit of wave lengths detectable by the 1P28 photomultiplier. C shows the analogous relative response of the C7180 photomultiplier as a function of wave length. Two important considerations emerge: No light was debected having wave lengths longer than 240 mp, and maximal response was found a t 200 nip, in the optimal region for work in the far-ultraviolet. It is apparent from B that the 1P28 photomultiplier is not suitable for use in the far-ultraviolet 1%hen any measurable amount of stray white light is present in the monochromated incident beam. Stray visible light is not eliminated completely by double monochromation, although it is claimed that in certain instruments its intensity is cut by a factor of 103, with significant loss also in the intensity of the monochromated light. The incident light entering the cuvette is inevitably composed of two parts, monochromatic and stray light. In the far-ultraviolet the intensity of the monochromatic light decreases sharply with decreasing wave lengths (with instruments employing crystal quartz optics and double monochromation), but the intensity of the stray light remains essentially constant. A s the incident light is passed through a n ultravioletabsorbing chromophore, part of the monochromatic light, in a n absorption band, is absorbed, but none of the stray light. This means that the light emerging from the cuvette n-ill necessarily contain a larger percentage of stray light than did the light entcring the cuvette and the percentage of stray light in the measured light will vary with the concentration of the chromophore. Therefore errors in absorption data may become apparent a t short wave lengths, especially in solutions of high absorbance, such as are encountered when en-
I
l
A31e
Figure 1.
i
1
"
-e-?'i
-
'
1
-+
Basic interrelationships
A.
Relotive intensity of Xe arc os o function of wave length 6 and C. Relative response of 1P28 and C7180 developmental photomultipliers used with o Leis single monochromator and Xe arc CIS light source
zymic activities are studied by means of difference spectra. PHOTOMULTIPLIER
The photoelectric equation indicates that the energy required to cause the emission of photoelectrons from the cathode of a photoelectric device will be equal to or greater than its threshold value. Several solar-blind ultraviolet multiplier phototubes have been described by Dunkelman (4) and solarblind photocells by Zworykin and Ramberg (?). The apparent threshold value for the RCA developmental photomultiplier as used in this system was found to be 240 mp (C, Figure 1); the ultimate value given by the manufacturer is about 280 mfi. A practical working limit on the long wave-length side of the spectral region under investigation is 225 to 220 mp. On the short wave length end the practical limit is about 194 mk, and is determined by such factors as the in-
for collimating the light and directing it into the exit slit, a rectangular slit (which may be opened to 3 mm., with a device for regulating the height of the image), an optical path 22.5 cm. from prism to slit, and no quartz windows. It was calibrated with the lines of mercury appearing in the region under investigation.
‘J 2
VI
Jh.8
s-2
r- \ ; s I -
Figure 2. 61, Bz.
Schematic of direct current amplifier
1.5-volt electrochem. cell
MI. 1 0-ma. d.c. meter M2, Ma. 2 0 . ~ 0 .d.c. meter
-
lo9 1 0 ” ohms Rz, Rs. 50 kilo-ohms, 2-watt var. Rd. 1 -kilo-ohm Helipot R& 1 -kilo-ohm Helipot R1.
500-kilo-ohm 2-watt var. 50-kilo-ohm, 2-watt var. Rs. 20-kilo-ohm, 0.5-watt var. R9. 1-4-megaohm, 0.5-watt var. 50-kilo-ohm, 2-watt Rlo, RII, Rlz, R13. Rb R7.
creasing opacity of the prism, envelopes, and cuvettes, and the absorption of light by the atmosphere. The C7180 developmental photomultiplier may be mounted in any position and does not require protection from ordinary room light. It cannot be installed n-ithout circuit changes in instruments using the 1P28 or 1P21 photomultiplier. High Voltage Power Supply. The line-operated power supply t o the dynodes of the photomultiplier supplied voltages ranging between 250 and 3500. Protection was provided for work with high voltages. Batteries lack sufficient versatility to be used in this system. Suitable commercial high voltage power supplies are available. Dark Current. Dark current has been discussed by Zworykin and Ramberg (7). It is increased as the amplification of the photomultiplier is increased. I n practice, the effect of dark current may be reduced by using a more intense light source, requiring less amplification, and eliminated by using alternating current amplification with a pulsed light source. The manufacturer indicates that the dark current of the C7180 developmental photomultiplier n ill not eweed 1 X 10-8 ampere a t room temperature and 2500 volts. The dark current of the tube in present use was 1 X IO-” ampere a t room temperature and 1500 volts. It is not known what variations may be expected among different tubes. CUVETTES
The data shown in Figure 3 were taken rT-ith crystal quartz cells having a 1-cm.
R14. R16.
1 .O-megaohm, 0.5-watt 1.1 -kilo-ohm, 2-watt
Rre, R17. 5-kilo-ohm, 0.5-watt RIB, Rls. 1 .5-megaohm, 0.5-watt R20.
500-kilo-ohn1, 0.5-watt
SI. Selector switch, 1 pole, 3 positions SZ,S3. SPST toggle switch TI.
r2.
Slit Width. The smaller the halfintensity band width the greater will be the resolving power of the instrument; hence more fine structure may be observed with small slits. I n taking absorption spectra the half-intensity band width was maintained nearly constant a t 1.5 mp by adjusting the slit width a t intervals of 5 mp. The theoretical slit width required was calculated from the known focal distance and data relating the rotation of the Littrow prism n-ith n-ave length.
~ ~ 7 2 2
VI‘, 671 8 0 photomultiplier (only anode shown) Vi. C K 5 8 8 9 Va, V4. 6 A U 6 W A
light path. They were matched throughout the far-ultraviolet and shown to be free from borate, the impurity found in most fused silica cuvettes, which introduces errors into absorption curves due to its absorption and fluorescence (SIC). A “special silica type C” (Corning Glass TTorks) was demonstrated to be free from fluorescent impurities and to have significantly higher transmittance in the far-ultraviolet than crystal quartz. An unselected sample 1 em. thick transmitted 77% of the incident light a t 200 mp; four samples of commercial fused quartz of equal thickness transmitted 4,7, 14, and 34’%, respectively. Cells made of special silica type C: should be ideal. Care of Cuvettes. The matching of cells should be checked frequently, because scratches due t o improper handling or cleaning nil1 introduce error. The cells should not be allowed to stand when they contain any solution or solvent other than water. Alkalies and concentrated acids etch the cells sufficiently to reduce their transmittance in the far-ultrai-iolet and cause differences in matched sets. For cleaning, i t is recommended that cuvettes be scrubbed gently in 2 5 hydrochloric acid with a tightly n-ound cotton swab (Q tip). After rinsing, the outer surfaces are dried with a bird’s-eye cotton cloth nhich has been n ashed about nine times with a detergent. MONOCHROMATOR
The Leiss single monochromator was used (Carl Leiss, Berlin, Germany, through the Photovolt Go.). It has a Littrow crystal quartz prism, two compensating aluminized parabolic mirrors
AMPLIFICATION
Light Source. The amplification required for the anode current of the C7180 photomultiplier depends upon the light source and the dynode supply voltage. Any suitable source of ultraviolet light may be used, such as the xenon or mercury-xenon arcs or the hydrogen discharge lamp. Sources of ultraviolet light have been compared by Baum and Dunkelman (1). Care in handling xenon lamps is indicated because they are said to develop a pressure of 20 atm. when in use. Absorption spectra taken with the light sources mentioned are similar. The xenon arc was mounted in a housing having an internal liquid cooling system. Light and heat shields protect both instrument and operator. Schematic of Amplifier. The schematic of the amplifier in present use is given in Figure 2. It is based in principle upon the Wheatstone bridge, in which Rl1 and R12are arms with fixed resistance, Vsis the unknown resistance, Via the calibrated resistance, and Ma a null-indicating device. Regardless of the light source, a large anode load resistor, R1 (lo9 ohms or larger), is required. An electrometer tube, V,, is used in the first stage of direct or alternating current amplification and provides an input with high impedance. The housing of the photomultiplier was designed so that the leads from both the controlling grid of V 2and load resistor could be connected directly t o the anode pin of the photomultiplier, to minimize stray capacitance. Additional amplification is necessary in the form of matched pairs of transistors, TI and T2, and of tubes, V8and V4. Essential to the operation of the instrument are: a dark current control, R6;a sensitivity control, Rs; a shutter to cut the incident light from the optical path (not shown); a variable power supply for adjusting the amplification of the photomultiplier (not shown); a potentiometer, R4,for reading per cent transmittance; and a selector switch, SI, which selects the connection of the VOL. 31, NO. 5, MAY 1 9 5 9
879
grid voltage of V4 with the potentiometer. Sensitivity Control. Sensitivity is controlled by t h e variable currento used t o limiting resistor, Rg. R ~ is limit t h e current flowing through t h e null indicator for its protection. This method of sensitivity control is independent of the incident radiation. The sensitivity used should be no greater than required for accuracy in the region in which readings are made. Betrieen 100 and 25% transmittance minimal sensitivity may be used; below 10% maximal is required. Measuring Device. Neasurements as per cent transmittance are made on Rd with a G. W.Borg micropotentiometer, Model 205, with a directreading microdial, Model 1307, calibrated in 1000 divisions. With this equipment (Allied Radio Corp., 100 Korth Kestern Ave., Chicago, Ill.) measurements may be made directly to the nearest 0.1% transmittanceLe., they will not vary from the true value by more than f0.05% transmittance. This degree of precision has no significance in solutions of low absorbance, but becomes a factor which limits accuracy as the absorbance approaches 2.0.
Series A
Figure 3. Absorbance, referred to water, of a q u e o u s solutions of uridylic acid as a function of concentration, read at 194 mp, with a xenon arc as light source Points for curve B were read with water in reference cell; for curve A with a solution of absorbance 2.38 in reference cell
1
2
3 4 5 6 Relative Concentration
Readings on a linear scale as per cent transmittance were preferred to those on a logarithmic scale as absorbance because of the greater accuracy and availability of the linear scale and variable resistors. Test of Amplifier. Before the amplifier is used initially certain adjustments, tests, and calibrations should be made.
with test point E maintained a t 5.7 volts. The amplifier should be calibrated by determining the per cent transmittance on the microdial as a function of the potential applied to the controlling grid of Vz. The authors found the curve linear, with standard deviation 0.006.
The filaments of V3 and V4 should be aged. Before the amplifier’s power supply is turned on, SSis closed, S3 opened; R3 is set a t ground potential, Rs a t maximal resistance, and R5a t halfmaximal resistance. Tests of voltage are made with a VTVPII a t test points A to F. The negative test probe is connected to the grounded point, D, with the exception of point A , where the polarity is reversed. ;MI,which measures the filament current of V S ,is regulated by Rz (current 6 ma). X 2 , which measures the plate current of V z , is adjusted 7vith R3 (current 7 pa.). SI should be set a t the less positive position. K i t h S3closed and R9set a t minimal resistance, Rg should be adjusted so that M 3 reads zero. For calibration a potentiometer is required as input source. With the positive pole connected to test point D and the negative to the controlling grid of Tiz, the reading on .V3should be null when the potentiometer is set a t -0.66 volt; if necessary, it is adjusted with R5. The authors use a differential of 0.34 volt across R1 as 100% transmittance because this value gives a satisfactory balance between light and amplifier. With SI at the position of more positive potential (100% transmittance) and the potentiometer set a t - 1.00 volt, the reading on M s should be null. If i t is not, Re and R7 should be adjusted,
The amplifier and power supply reach stability in 6 hours, the xenon or mercury-xenon arc in 1 hour. For each wave length the slit is adjusted to give the desired half-intensity band width. K i t h S1 in the more positive position (dark current), ;If3 is adjusted to null. K i t h SIin the less positive position (100% transmittance), the reference cell is brought into position, the shutter opened, and M3 adjusted to null by varying the gain of the photoniultiplier. TVith S1 connected to the arm of R4 (70 transmittance) the experimental cell is brought into position and R4 is varied until J f 3 reads null. The reading on the microdial is recorded as per cent transmittance.
880
ANALYTICAL CHEMISTRY
0
OPERATION
PERFORMANCE
Figure 3 s h o w the results of an experiment designed to test the performance of the spectrophotometer and its potentiality for use in taking difference spectra in the far-ultraviolet.
A stock aqueous solution of uridylic acid was prepared with absorbance 4.76 a t 194 mp (solution A). A second stock solution was made by dilution of the first with a n equal volume of water (solution B). A series of dilutions of each stock solution was made such that their theoretical relative concentrations
7
8
r e r e those indicated in Figure 3. The per cent transmittance of light with half-intensity band with 1.5 mp, centered a t 194 mp, was determined with a xenon arc as source. I n the B series the reference cell contained glassdistilled water; in A the reference was the dilute stock standard, absorbance 2.38. Stray light of nave length longer than 240 mg was not detectable by the photomultiplier (C, Figurc 1). I t remained to demonstrate n-hether stray light of wave length shorter than 240 nip was present in the measured light. I n the absence of all strap light the absorbances of the solutions illustrated in Figure 3 should be a linear function of their relative concentrations. The points indicated on 9 and B represent experimental values; the curves ivere drawn to conform with the least-square lam- for a straight line. The standard deviation for B was 0.01, for A 0.02. On B the observed conforniity of the last point is sonleu-hat fortuitous. Even nithout this point there is satisfactory correlation between absorbance and concentration (Beer’s lan) until the former approaches 2.0, with water in the reference cell. I n the reference cuvette of series A (absorbance 2.38) more than 99% of the incident selected monochromatic light was absorbed by the chromophore. Therefore in the experimental cuvette less than 1%could serve as incident light for measuring increase in absorption due to increase in concentration above that appearing in the reference cell. If there were present, in the measured light, any significant amount of stray light of wave lengths detectable by the pliotomulti-
plier, this fact should become apparent in these stringent circumstances and result in deviation from linearity. By this criterion no measurable amount of stray ultraviolet light \vas found in the system. The somewhat greater standard deviation found for A than for B is attributable largely to the fact that the extremely small photocurrents measured required maximal amplification, resulting in larger values for the dark current. Xerertheless, absorbances approaching 4.0 (referred back to water) have been determined with a fair degree of accuracy. The last point shown on 8.with a n expected absorbance of 1.2, is in serious error. and illustrates the limitation of the instrument, as used. It has not been
included in the calculations. This limitation may be minimized by reduction of the dark current ( 7 ) . SUMMARY
LITERATURE CITED
(1) Baum, IT. A, Dunkelman, L., J . Opt. SOC.Am. 11, 782 (1950). ( 2 ) Buell, 11.V., Hansen, R. E., J . Biol.
Chem., in press.
(31 Buell. M. V.. Hansen. R. E.. Science 126, 842 (1957). (4) Dunkelman, L., J . Opt. SOC.A m . 45, 134 (1955). \
The spectrophotometer described is capable of making accurate measurements of the absorption of ultraviolet light of wave length as short as 194 mp. This technique may be used to characterize compounds by their absorption spectra in the far-ultraviolet, and follow the course of reactions in this region by difference spectra. Absorption spectra in the far-ultraviolet of certain biologically important compounds are presented elsewhere ( 2 ) .
,
(5) Hanovia Chemical and hlfg. Co., 100 Chestnut St., Newark, S . J., Brochure 127. (6) Xlehler, A. H., Bloom, B., Ahrendt, &I. E.. Stetten. De W.. Jr.. Science
RECEIVED for review ;iugust 11, 1958. Accepted December 15, 1958. Investigation supported by a grant h-646(C) from the Sational Institutes of Health.
Spectrophotometric Determination of Palladium with Phenyl-alpha-pyridyl Ketoxime BUDDHADEV SEN Coates Chemical Laboratories, Louisiana State University, Baton Rouge, La.
b Phenyl-a-pyridyl ketoxime was found to b e a highly selective and sensitive reagent for palladium. The palladium complex of the ketoxime has two characteristic absorption maxima, one a t 410 mp and the other a t 340 mp. At these wave lengths the photometric error is a minimum in the concentration ranges 2 to 10 and 1.5 to 8 p.p.m., respectively. The molar extinction coefficient of the palladium chelate is 3 X a t 410 mp and 5 X l o 4 a t 340 mp. The reagent also forms complexes with iron, cobalt, nickel, and copper, which are all soluble in organic solvents. The interference due to these ions can be eliminated b y using (ethylenedinitri1o)tetraacetic acid (EDTA). Gold and cyanide are the only interfering substances.
lo4
P
HEhTL-a-PYRIDTL
lietoxime(1) was
reported (6)as a
fi
A 1
1
n c ~chelating agent for a number of
cations. It was found to be highly selective for palladium(II), with which it reacted to form a yellow, n-ater-insoluble chelate of definite composition. An analysis of the palladium complex
showed that palladium and the reagent combine in the ratio of 1 mole to 2 moles. The various reagents and procedures for the spectrophotometric determination of palladium were discussed by Beamish and RlcBryde (1, 2 ) in t n o excellent review articles. West ( 7 ) and Rlellon and Boltz (4) summarized the reagents used for the spectrophotometric determination of platinum metals during the last few years. The principal difficulty encountered in many of the proposed methods for palladium is the interference of the bivalent ions of the transition metals, particularly the colored ones, and of other metals of the platinum group. LTsually the interference of the metals other than the platinum metals can be eliminated by using EDTA.. Phenyl-a-pyridyl ketoxime formed stable colored complexes n-ith nickel(I1) , cobalt(II), iron(II), copper(II), gold(111), and palladium(I1). All were precipitated a t a definite pH, from the aqueous phase and extracted with organic solvents such as chloroform and carbon tetrachloride. The efficiency of precipitation and extraction for most of the chelates from the aqueous phase was best between pH 6 and 11. Honever, the incipient precipitation started a t a much lower pH. The palladium complex began to precipitate a t pH 2. K i t h the exception of copper(II), the spectral characteristics of the complexes were the same in both aqueous and nonaqueous phases. Figures 1 and 2 shou-
the absorption spectra of a number of complexes in chloroform, and also that of copper in the aqueous phase. The present paper describes the procedure for the spectrophotometric determination of palladium, using phenyl-a-pyridj-1 ketoxime. EXPERIMENTAL
Instruments. Spectral studies and light adsorbance measurements \yere made with a Beckman Model DK I recording spectrophotometer. Matched silica cells of 1-em. light path were used. p H measurements were made with a Beckman RIodel G p H meter. Preparation of Phenyl- a-pyridyl Ketoxime. Hydroxylamine hydrochloride (50 grams) dissolved in a minimum volume of n ater was added t o 200 ml. of a 107, sodium hydroxide solution. T o this were added 20 grams of 2-benzoylpyridine dissolved in 300 nil. of 95% ethyl alcohol. The mixture rras refluxed for 8 hours and evaporated on a water bath until all the solid separated as a crust on the top and the liquid was brownish. Sometimes the oxime separated as an oil which solidified on cooling. The magenta solid was filtered and weighed (yield 30 grams). The crude product was crystallized several times from 95% ethyl alcohol until the oxime was obtained as a white crystalline substance melting at 161' C. Two or three more crops of crystals were obtained from the mother liquor. The combined yield was 70% of the theoretical. The VOL. 31, NO. 5, MAY 1959
881
