having 100 scale divisions. The amplitude is directly proportional to the analyzer scale reading and is therefore smaller with less concentrated samples. Sample supplied to the analyzer is diluted with twice its volume of water in a proportioning system to prevent precipitation of gypsum. The instrument is calibrated by use of standard solutions, or alternatively, by comparison of the instrument reading with the plant analysis obtained simultaneously (Figure 7 ) . The different readings obtained for the same sample are due to the different methods of analysis. The Kno-zles method (6) was used for the plant analyses. Furthermore, the instrument’s sampling system removes most of the calcium sulfate from the sample before delivering i t to the instrument and this results in a lower reading. Spot checks made on the calibration over a 4-month period showed that it
Table 1.
Std. Solns.
Calibration Checks
of the analyzer to colorimetric analyses also appears attractive.
so4
Analyzer Readings, yo
LITERATURE CITED
3.33 1.67
9-7-55 11-10-55 1-16-56 3.25 3.45 3.42 1.67 1.72 1.72
(1) Eckman, D. P., “Industrial Instrumentation,” p. 16, Wiley, Yew York, 1950. ( 2 ) Knowles .bsociates, 19 Reactor St., S e w York, private communication.
=
Concn., yo
(3) Kobe, K. A., “Inorganic Process
remains essentially constant as indicated in Table I. The analyzer is readily applicable to other analytical and control problems. The concentration of barium sulfate sol in the reaction vessel for full scale instrument reading is less than 100 p.p.m., and the analyzer could be made more sensitive by increasing the depth of the measuring cell. If reagent and sample streams were reversed in flow ratio, the analyzer would easily perform turbidimetric analyses in the parts per million range Application
Industries,” p. 323, Macmillan, XeF York, 1958. ( 4 ) Ibid., p. 324. (5) Sandell, E. R., “Colorimetri:, Determination of Traces of Metals, p. 69 Interscience, New York, 1944. ( G ) Snell, F. D., Snell,.C;,T., “Colorimetric Xethods of Analysis, p. 88, Van Sostrand, Sew York; 1936( 7 ) Yoe, J. H., “Photometric Chemical .\nalysis,” p 91. Wiley, New York, 1928.
RECEIVED foi review November 9, 1956. .-iccepted February 25, 1959. Pittsburgh Conference on dnalytical Chemistry and hpplied Spectroscopy, Pittsburgh, Pa , Febriiary 27, 1956.
Simple Ultraviolet Photometer RALPH E. THIERS, MARVIN MARGOSHESI1 and BERT L. VALLEE Biophysics Research laboratory, Harvard Medical School and Pefer Bent Brigham Hospital, 72 7 Huntington Ave., Boston, Mass.
F A simple photometer for use in the near-ultraviolet region is described, and considerations of its design and performance are discussed. A monochromatic beam of light with wave length maximum a t 355 mp and a half-band width of 40 mp is obtained from a lamp made commercially for fluorescent displays. Photovoltaic cells are used as detectors, to operate a direct current microammeter. The instrument, because of its small size and sturdy, simple construction, is well suited to routine work, including clinical determinations of serum enzymes, which may even be performed outside the laboratory.
C
and photometers have been the instruments of choice for routine measurement of the absorbance of colored solutions. Spectrophotometers, because of their greater complexity and expense, have been used less for routine work at fixed wave lengths and more for research or development. However, in the ultraviolet region of the spectrum the absence of suitable photometers of reasonable simplicity and monochromatic properties has made i t necessary to use spectrophotometers for routine work. Many analysts have been unable to take
advantage of methods which would be well suited to simple ultraviolet photometry, if a n appropriate but inexpensive instrument were available. I n addition, certain enzymatic determinations have recently become important in clinical medicine, and these are best performed by photometry in the nearultraviolet, This paper describes a new ultraviolet photometer which allows measurements of absorbance in the neighborhood of 355 mp. Because i t is simple, small, and sturdy, i t is well suited to routine work, including clinical determinations of serum enzymes which may even be performed outside the laboratory.
OLORIMETERS
1 Present address, National Bureau of Standards, Washington, D. C.
1258
ANALYTICAL CHEMISTRY
DESIGN OF INSTRUMENT
Source and Monochromator. Ultraviolet radiation is provided by a 4watt B L B fluorescent lamp (General Electric Co. or Sylvania Electric Products Co.), which is a stock item used in fluorescent advertising. The envelope of t h e lamp is a special Corning Filter Glass, and t h e resulta n t spectral output of this source is a single band centered a t 355 m,u and with half-band width of about 40 mp (Figure 1). Detector. Barrier layer or “photovoltaic” photocells (General Electric Co. KO.PV-1, ultraviolet sensitive) form t h e light-sensitive element of t h e instrument. B y using a cylindrical
lens parallel t o the source, and a cylindrical absoi ption cell, light levels a l e reached nhich allow the photocell t o operate a direct current microammeter rather than a galvanometer, and in a simple, nonamplifying circuit. Absorption Cell. Ordinary 13-mm. borosilicate glass culture tubes are used as absorption cells. Optical and Photoelectric Design. Figure 2 s h o w a diagram of the photoelectrical circuit and optical arrangement of the instrument Light from the fluorescent tube, T , passes to the right through a waterfilled cylindrical lens, L , 13 mm. in diameter, then through the solution to be measured, S,in the cuvette, C, through a variable-aperture diaphragm, A , and strikes the photocell, P. Light from the other side of tube T passes to the left, through a Variable-aperture diaphragm, Ai, and strikes photocell PI. The two photocells are connected in series and are bridged by a 20-pa. Ion resistance, direct-current meter, JI so that current from the left-hand photocell deflects the needle downscale, and current from the right-hand photocell moves it upscale. The lens, L , is an essential feature of the design of this instrument and may be used to fill a number of roles. It focuses the light from T for maximum flux through cuvette C and on to photocell P . It physically separates the cuvette and sample from the lamp, thus minimizing variations in enzymatic reaction rates due to excessive heat
Figure 1. Emission spectrum of ultraviolet lamp and absorption spectra of pyridine nucleotides in reduced (DPNH) and oxidized (DPN) form
2 5
t z10-
’
$;
z
W + z
0 WAVE
transfer from lamp to sample. Solutions other than water may be placed in or circulated through tube L. Under these circumstances, it may beused as a n optical filter utilizing narrow bandpass liquids (along with the BLB fluorescent lamp or visible lamps available in the same size), thus changing the wave lengths at which the instrument operates and making it more adaptable. The instrument is controlled by optical means, using variable apertures A and AI. The electric components are not variable. Aperture A forms the “sensitivity adjustment” for setting full scale reading on the blank or sample, Aperture Al and photocell P1 form a ‘Lzero-suppression”feature which allows zero light transmittance to be suppressed below the zero of the meter scale. 0PERATION
of Transmittance. The instrument may be used to compare t h e transmittance (yo T ) in the near-ultraviolet of any solution with any “blank” or “zero’) solution. T h e meter is adjusted to read 100% T (full meter scale) with the blank solution in a cuvette. The blank solution is then replaced with the solution in question and the transmittance is read directly from the meter. Two or more prematched cuvettes may be used. Zero Suppression. The instrument can be used with one photocell only. Under these conditions, the scale of the meter reads from 0% to 100% T (infinite t o zero absorbance). However, higher sensitivity and precision in the region close t o 1 0 0 ~ T o may be obtained by using the second photocell, P I , t o cause definite amounts of zero suppression. On a meter marked from 0 to 100 units, it is convenient to move the 0% T to 100,200, or 300 units to the left or minus side of the zero of the meter. I n these instances, the meter scale reads from 50 to 100% T . 66 to 100% T , and 75 to 100% T , respectively. To transfer 0% T to 100 units below the meter zero, the following steps are used : Measurement
1. Aperture A , is closed completely. 2 . Aperture A is adjusted so that the
meter registers 100. 3. Aperture A I is opened until the meter registers zero. 4. Aperture A is then opened further with the blank in place and operated in
LENGTH
MILLIMICRONS
the usual way as a sensitivity or blank adjustment. 5 . The blank is replaced by the sample and the meter scale is read with the zero reading equal to 50% T and 100 equal to 100% T . To transfer 0% T to 200 units below the zero of the meter scale, the following steps are taken: 1. Aperture A1 is closed. 2. Aperture A is opened until the meter reads 100.
3. AI is opened until the meter reads
zero.
4. Aperture A is opened wider until the meter reads 100. 5. Aperture A I is opened wider until the meter reads zero. 6. Aperture A is used to adjust 1 0 0 ~ o T with the blank.
Repetition of steps 2 and 3 will depress 0% T 100 divisions further each time, until the apertures are fully open. The marked stability of the photoelectrical circuit makes this procedure both feasible and advantageous. Measurement of Serum Enzyme Activity. The rate of change of
absorbance in the near-ultraviolet of any solution may also be measured. The solution to be assayed is placed in a cuvette and the meter is adjusted t o any arbitrarily chosen reading at time zero. The change in transmittance us. time is observed and converted to absorbance (optical density) by the formula: absorbance = 2 - 1% (%TI. The instrument can be used to measure the rate of reactions catalyzed by enzymes which utilize pyridine nucleotides as coenzymes, because the latter absorb light maximally around 340 mp. Transaminases and lactic dehydrogenase (LDH) are such enzymes and the determination of the L D H activity of serum after myocardial infarction is given as an example of the method. Serum, free of hemolysis, is obtained from the patient. The following reagents are placed in the cuvette of the instrument. 1.5 ml. of 0.1M sodium pyrophosphate buffer at pH 8.8 0.3 ml. of 0.0521f diphosphopyridine nucleotide (DPN) adjusted to pH 7.5 with potassium h droxide 1.0 ml. of 0.1727; sodium lactate at pH 8.8
Figure 2. diagram
Optical and photoelectrical A, A I . Variable apertures
C.
Cuvette Cylindrical lens Microammeter P, PI. Photovoltaic cells S. Sample solution T. Ultraviolet lamp I. M.
With zero suppressed one full scale, the sensitivity diaphragm, A , is adjusted to give slightly over 100% T, and 0.2 ml. of serum is added to the cuvette and mixed with the reagents by swirling or inversion. The cuvette is replaced in the instrument and the %T or absorbance is noted a t an arbitrarv zero time and a t 30-second intervals thereafter for 3 or 4 minutes. The activity of the enzyme is expressed as the change in absorbance per minute per milliliter of serum. The activity can be expressed in molar units by reference to a calibration curve for the absorbing species, reduced diphosphopyridine nucleotide (DPNH) (Figure 3 ) . PERFORMANCE
The instrument described is simple, rugged, and extremely stable. Its stability is inherent in the performance of barrier-layer photocells operating in a completely closed circuit with no variable components, and it is augmented by the excellent stability of the fluorescent light source. When the voltage supplied to the lamp varies between 100 and 120 volts, the resultant variation of the measurement amounts to about lOyoof the meter reading. Because the photocell circuit used automatically cancels out the effect of lamp variations on that portion of the total scale which is suppressed below zero on the meter, the variation of the meter reading with lamp voltage is almost independent of the amount of zero suppression. Thus, VOL. 31, NO. 7, JULY 1959
1259
Table I.
(Measurements on one solution, against water as a blank) Readings, pa. ._ Zero Zero suppressed suppressed N o aero one full three full suppression scale scales 15 8 1.5 7 15.8 15.85 15 85 15,s 15 7 5 15.7.; 15 815.85 15.85 15.8
against the buffer as blank (0.1N sodium phosphate at p H 7.4) to obtain line DPNH. The near ultraviolet absorption of this compound in the oxidized and reduced forms is shown in Figure 1 and compared to the emission of th lamp. The calibration curve is linear to 0.3 absorbance (50% T) in spite of the slight difference betn-een the maxima of the absorption bands. Very small amounts of chromium in minerals or other samples are often determined by oxidation to chromate ion, which has a n absorption band a t 386 nip (11). Line Cr of Figure 3 shows the calibration curve obtained when standard solutions containing chromate ion are measured. Figure 3, line N , shows the data obtained when known solutions containing ammoniuni ion are treated with Nessler's reagent and read us. the reagent as a blank. I n this last case, the turbidity of the solution is bcing measured.
measurement of activities of serum enzymes for the diagnosis of a variety of clinical conditions, among which is myocardial infarction ( I S , IC), and to the measurement of various components of triphosphopyridine nucleotide, and diphosphopyridine nucleotide-dependent enzyme systems, it can be used for determining the alcohol level of blood (6) and other biochemical Compounds including glucose (Q),aspartic acid ( I O ) , lactic acid ( 8 ) , choline (1, 5 ) , and more than 20 different enzymes ( 2 ) . Other materials which lend themselves well to measurement in the near-ultraviolet include bromide ( 7 ) , copper (12) sulfate ( d ) , and uranium ( 3 ) . The instrunlent described is stable, reproducible, and free from drift. Effects of stray light have not been observed. It consists of t\To inherently stable components, the fluorescent lamp and the photovoltaic cell. Under usual conditions, the reproducibility of these components exceeds that of the meter. Zero suppression may, therefore, be used to increase the sensitivity of the meter and hence of the instrument as a whole (Table I). Figure 3 shows a linear relationship betxveen the known concentration and measured absorbance of standard solutions. This indicates that the solutions obey Beer's law at low absorbances and that the current output of the photovoltaic cell, P , is linearly related to the light falling upon it. If standard curves are measured under the same conditions as the sample, nonlinearity a t high absorbance imposes no limitation. The temperature coefficients of absorption of colored systems and of enzyme reactions are easily observed as short-term changes in readings on samples placed in this instrument (as in some visible colorimeters). Water a t constant temperature may be passed through the thermostat-lens-filter to observe such phenomena accurately. Under these conditions, the precision of this device equals that of conventional spectrophotometers, and the stability and ease of handling of the simpler inqtrumcnt are far superior.
DISCUSSION
ACKNOWLEDGMENT
The device described here demonstrates that nionochromatic filter photometry need not, in principle, be limited to the visible region. The valuable features of the simple colorimeter have been applied in the near-ultraviolet region, obviating the necessity of an expensive spectrophotometer for routine analyses in this part of the spectrum. Suitable light sources should permit extension of the usefulness of the instrument to even lower wave lengths. However, the near-ultraviolet is a t present a n important and useful range for measurement. I n addition to the
Thanks are due to Larry Cooke, General Electric Co., Needham, Mass., ior assistance in obtaining samples of various kinds of ultraviolet lamps, and also to A. S. Macalaster, hIacalaster Bicknell Co., 243 BroadIvay, Camhridge, Mass., which manufactures this instrument for sale.
Repeatability of Instrument
11 3 11 2.; - " ii.2 11.2 11.2 11.25 11.25 11.2 11.2 11.3 11.2 11.3
2 5 2 5 2.6 2 7 2.i
0.3 w
0
2 02 E 0
m m
a
0.I
2.75 2.75 2.7.; 2.65 2.7 2.75 2.6
0
DPNH
0 0
5 2
IOpgCr 4 ~ g N
CONCENTRATION
Table
I!.
Variation of a Single Setting with Time
(Zero suppressed, 60.0 pa. J Reading, Time, Reading, hlin. pa. Nin. pa. 0 62.65 8 62 65 0 6 15 62.70 62.65 1 62 i o 30 62 65 2 62.65 60 62.70 1 62.68 120 62.65
Figure 3. Calibration curves obtained on instrument
errors due t o variations in lamp intensity during measurement are reduced by one half when zero suppression amounting to one full scale is used, by two thirds when zero suppression amounting to two full scales is used, and so on. The repeatability of the instrunlent was tested by making 12 successive readings on a single sample solution. A11 controls were reset after each reading and a blank tube of water was read after each sample. The readings were made on a meter scale 4.5 inches long, which was divided into 40 units and labeled from 0 to 20 pa. Replicate readings were made Kith no zero suppression, with 20 p,a. zero suppression, and with 60 pa. zero suppression (Table I). If absorbances are calculated from these data, readings ranging from 0.101 to 0.106 absorbance unit are obtained in the first case, from 0.106 to 0.108 in the second, and 0.106 to 0.107 in the third, illustrating the improvement to be gained by zero suppression. The stability and freedom from drift of the instrument are illustrated by Table 11. The settings mere left unaltered and the meter was observed at the times shown. I n 2 hours, the readings varied by less than 0.1 Ma., which corresponded to an absorbance change of less than 0.0006. Figure 3 shows typical calibration curves obtained with this photometer. Replicate known samples of buffered solutions of diphosphopyridine nucleotide in its reduced form were measured 1260
ANALYTICAL CHEMISTRY
as chromate ( 7 7 ) DPNH. Reduced diphosphopyridine nucleotide Ammonia nitrogen, measured by nesslerization
Cr. Chromium, measured
Timc,
N.
LITERATURE CITED
(1) Appleton, H. D., La Du, B. B., Levv, B. B., Steele, J. M., Brodie, B. B., J. Biol. Chem. 205, 803 (1953). (2) Colowick, S. P., Kaplan, N . O.,
“Methods in Enzymology,” Academic Press, New York, 1955. (3) Feinstein, H. I., U. S. .itomir Energy Comm., TEI-555(1955). (4) Kato, Takeshi, Nomiao, Y., Shinra, K., J . Chein. SOC.Japan, Purr C h m . Sect. 76, 373 (1955). (5) Kushner, D. J., Bioehim. e1 Hiophys. Aclo 20, 551 (1956). (6) Monnier, D.,Fasel, M., .lfitt. Lehmsm. Hyg. 47, 141 (19,561.
mination df Traces of Metals,” 3rd ed., Interscience, New York, 1!158.
RECEIVEDfor review October 31, 1958. Accepted March 16, 1959. Work s u p ported by the Howard Hughes Medical Institute.
Isomer Ratio Analyzer for Toluenediisocyanate Based on Dielectric Constant Measurements SAMUEL STEINGISER, W. C. DARR, and E. E. HARDY Research Department, Mabay Chemical Ca., New Martinsville, W. Va. .In the production of toluenediisacyanate, a rapid reasonably precise method far the determination of isamer content was needed. Freezing points and infrared spectroscopy had certain disadvantages. The dielectric isamer ratio analyzer was developed to fill the need for a continuous record of isomer ratios far the toluene-2,4and -2,6-diisocyanate mixtures. This instrument can be used in the laboratory ar in the line in production. Its accuracy i s to about *0.20/, isomer unit, better than methods previously used.
~o~UENEnIIsocYANATE is one of the basic ingredients in the manufacture of urethane polymers, principally urethane foams. A very important aspect in the creation of these foams is the interrelationship of the rates of reaction of the toluenediisocyanate isomers with hydroxyl resins in forming the polymer. Toluenediisocyanate exists in several isomeric forms, having different reaction rates in terms of the desired reactions. As manufactured in this country, it contains mainly two isomers, the 2,4and 2,6- structures. A trace of the 2,5- isomer may be present, which will affect all the methods of analysis used. The main consideration has been given t o the reaction rates of the 2,4and 2 , 6 isomers and their mixture in thetoluenediisocyanates. Normalproducts sold in this country consist of nominal 100% 2,4- isomer (actually nreater t h a n 97.5%); nominal SOYo 2,4- and 20% 2,6- isomer mixture (actually 80 + 2%); and nominal 65% 2,4- and 35% 2,6-isomer mixture (actually 66 =t2%). For trouble-free production of urethane polymers, especially the very sensitive foams, careful control of isomer content is important,
The first step, then, is measurement of the isomer content of the toluenediisocyanate. Previous methods of measurement consisted of determining the freezing point of the mixture and comparing it with a calibration chart based on the values of the pure components ( I ) , or using the infrared absorption peaks a t 12.35 and 12.80 microns for each of the pure components and the ratio of the relative heights of each peak (3). I n each method, a sample of diisocyanate must be removed and analyzed for either the freezing point or infrared absorption with its consequent
Toble 1. Dielectric Constant as a Function of Frequency for Pure Toluene-2.4diisocyanate and Toluene-2,6-diisocyanate (Boonton Q-Meter, temp., 23.1”C.)
FIP
quency, Kc. 90 500
Dielectric Constant,p 100% 2,4-
100%
8.15 8.48
5.li
Figure 1. FoxbaroCapacitance Dynalog analyzer
VOL. 31. NO. 7, JULY 1
2,h
5.36
