Recording Colorimeter for Microchemical Determinations A.
K. SOLOMON and
DAVID C. CATON
Biophysical laboratory, Harvard Medical School, Bosfon I 5, Mass.
b A recording colorimeter has been designed and built to make rapid and accurate determinations of light absorption in samples containing 30 cu. mm. of fluid. The accuracy obtainable with the instrument a t a wave length of 4760 A. is about 1% a t absorbances in the 0.1 region and 0.270 at absorbances in the 0.4 region. Five samples and a blank can be measured and recorded in less than 200 seconds.
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OPTICAL BENCH
PR. I" I.D. BEARINGS
T
present apparatus has been designed to make rapid and accurate determinations of light absorption in samples containing 30 cu. mm. of fluid. It consists of a point-light source, an alternating current modulated detector and amplifier, and a servo recorder. A special rigid carriage has been designed to position the samples accurately and reproducibly in the light beam; six samples can be accommodated on the carriage for successive measurements. HE
DESCRIPTION OF APPARATUS
Mechanical Equipment. The cuvettes, made according t o the specifications of Lov ry and Bessey ( 2 ) , have a 10-nim. light path and are 25 mni. high (Pyrocell Mfg. Co.). The sample is contained in a slot 1.5 mm. wide X 10 mm. long X 25 mm. high in the center of t,he cuvette. Because the light beam is about 1.2 mni. in radius a t the entrance to the cuvette, a sample containing 30 cu. mm. of fluid is ndequa te for a determination. The relationship betiyeen the beam diameter and the slit width of the cuvette determines the specification that the position of each cuvette be reproducible within 0.1 mm. I n order to achieve this repraducibility, the cuvettes are mounted near the circumference of a carriage shaped as a sector of a circle, as shown in Figure 1. The radius of the carriage measured from the center of the central bearing t o the center of the cuvette is 157/16inches and the angle subtended by the carriage is 18.5'. The central bearing is a pair of prestressed ball bearings 1 inch in internal diameter; the carriage is supported near the circumference by two ball bearings '/* inch in diameter which roll on a track machined in the base. The carriage provides a rigid support for the cuvettes so that each cuvette always
LIGHT
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LENS\
INTERFERENCE
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1
L L Figure 1. Arrangement of optical and mechanical components of recording microcolorimeter
travels along the same path. The carriage contains six cuvette positions, allowing the determination of a blank, a standard, and four unknowns. Each cuvette holder is equipped with screws R-hich allow translational adjustment along the circumference of the arc and motion in a vertical direction, in order t o adjust the cuvette so that the light path just clears the bottom of the slot Springs are used to position the cuvette against one corner of the cuvette holder, as described by Lowry and Bessex (a). Two steps are involved in locating the cuvette exactly in the light path. First, the carriage is arrested by a spring-loaded ball bearing n hich falls into a sector-shaped detent (Figure 2 ) . After the carriage has come to this click stop, final location i s made by release of a spring-loaded tool-steel taper pin which fits into one of six lapped holes on the bottom of the carriage. I n practice, the handle controlling the taper pin is pushed down, the carriage is moved until it comes to the click stop, and the handle is then released allowing the taper pin t o make the final location. The carriage base is supported on three legs with the usual adjustments for leveling. The whole device is equipped
-cc- i - Y L
n i w - CI- BE>= GS Figure 2. Details of location mechanism for positioning cuvette in light path
Arrows show direction of motion of ball bearing click Etop and of handle Yhich controls the tool-steel taper pin
with a light-tight cover with access holes for the entrance of the cuvettes and the redacement of the interference filters. Optics. The optical path is also illustrated in Figure 1, which shows the light source, Tens, and interference filter-shutter assembly, each mounted separately on a n optical bench which allows adjustment of the position VOL. 30, NO. 2, FEBRUARY 1958
291
of each element. The light source is a Sylvania tungsten 2-watt concentrated arc lamp with a light source diameter of 0.13 mm. and a brilliance of 18 candles per mm. (Sylvania Electric Products, S o . C2PT). A lens (f:3.2, telephoto lens for 8-mm. movie camera, focal length 1.5 inches) is used to bring the image of the source to a focus in the center of the cuvette. Interference filters are used to limit the light transmission t o a selected wave-length band. Square filters, 2 X 2 inches, may be secured from the Geraetebau-Anstalt, Balzers, Liechtenstein, with a maximum transmittance of 40% of the incident light and a band width a t half intensity of 160 A. The secondary peak (for a typical filter with its primary maximum a t 4760 -4., the secondary peak occurs a t 3380 A,) is suppressed by a Kodak Wratten 2 X 2-inch gelatin filter, Type 2B. The total light incident upon the detector a t 4760 A. is sufficient to permit operating the Densichron amplifier well below (on the 2 scale) its maximum sensitivity, thus providing a stable output. A camera shutter has been mounted in the light beam, adjacent t o the interference filters, to make it possible to measure the dark current. I n practice, it has not proved necessary to use the shutter because the dark current is negligible. Electronics. The arrangement of the electronic components is shown in the block diagram, Figure 3. The light source is powered by a George W. Gates power unit for a 2-watt concentrated arc lamp (Type GZ2U). The light is detected by a Welsh Densichron. This device makes use of a photocell placed in a magnetic field to give a 60-cycle modulated output, which admits of stable alternating current amplification. Two interchangeable photo cells are supplied, one with an S-1 sensitivity (maximum sensitivity, 4000 A,), and one with an S-4 sensitivity (maximum sensitivity, 8000 A.). The output from the Densichron, after rectification, causes a deflection in a 0 to 1-ma. meter. A 10-ohm potentiometer is inserted in series with the meter, and a suitable signal is led t o the input of a Brown, 10-mv., Psecond recorder, running a t a chart speed of 0.5 inch per minute. A standard Brown chart (No. 5871) is available with graduations in absorbance units. However, the signal from the Densichron is in the opposite sense to the ruling on the chart. To reverse the recorder-pen motion, it has been necessary to reverse the motor leads, the slide-wire, and the direction of rotation of the potentiometer in the standardizing network (which can be done by a simple electrical connection). Because the Brown recorder is a servooperated voltmeter, which balances the input signal against a voltage developed across a potentiometer, it acts as a null detector and is, thus, much more accurate than a conventional meter. It periodically calibrates itself against a reference voltage derived from a standard cell within the recorder. Neither the Densichron nor the arclamp supply is adequately regulated against line-voltage variation. Con292
0
ANALYTICAL CHEMISTRY
POWER SUPPLY
AMPLIFIER
AC
Figure 3. Block diagram showing arrangement of electronic components
Thin lines represent path of alternating current
for which the machine was initially devised. Cobaltous chloride solution has been used instead of inulin to provide a stable color reference which does not fade. Figure 4 is a composite of recordings which show: a t the top, the stability of the machine in the face of line-voltage variations; the reproducibility when the same solution is put in different cuvettes in different positions on the carriage; the results when the same sample is measured successively in the same position (10 replications; standard deviation 0.4%); and, at the
EFFECT OF INPUT VOLTAGE VARIATIONS
SAME CONCENTRATION DIFFERENT
Figure 4. Recorded output of microcolorimeter, under varying conditions as described in the text
{ CUVETTES POSITIONS
CONCENTRATION SAME { C U V E T T E POSITION
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1 GRAMS PER LITER
0
.05
I.6
0.8 IO
15
.20
24 .30
.43
3.2
.50
4.0
I1
60
ABSORBANCE
sequently, the line voltage has been regulated by the use of two voltage regulators in series (Figure 3). Regulator A is a Sola 250-watt regulator, and regulator B is a Raytheon 60-watt regulator, loaded by a 40-watt lamp in addition to the Densichron and arclamp power supply. The selection of regulators and load has been chosen empirically to give the best performance. The characteristics obtained with a Sola regulator and a Raytheon regulator in series are considerably better than those obtained with either two Sola or two Raytheon regulators in series. As the shutter is not used in normal operation, the signal has been removed electrically from the recorder during the period when the carriage is being shifted to avoid recording. For this purpose a microswitch is activated by the handle (which is depressed whenever the carriage is moved) controlling the taper pin. The microswitch operates an alternating current relay, which removes the normal signal from the recorder and replaces it with a fixed direct current voltage, obtained from a mercury battery and a suitable resistance chain. The resistances have been chosen so that the recorder is driven to the left of zero and, hence, the artificial signal is not confused with the true zero signal obtained with a blank solution. EXPERIMENTAL RESULTS
The accuracy of the machine has been tested with solutions of cobaltous chloride which absorbs a t 4760 A., the wave length used in the inulin determinations
bottom, the effect of increasing the concentration of cobaltous chloride. Considerable care must be exercised in cleaning and polishing the cuvettes to obtain a recording such as the second from the top, because the smallest dust particle produces a large effect in such a small light beam. The slow recovery of the zero value in the bottom recording appears to be a normal characteristic of the Densichron detector, following a considerable change in light intensity. The response of the machine to increasing concentrations of cobaltous chloride is typical and linear. A second set of experiments has been carried out to compare the results obtained on the present machine with those obtained by Lowry and Bessey (2) on their modification of the Beckman DU spectrophotometer for microanalytical purposes. To make this comparison, the cuvettes (not specially selected) were cleaned and polished to give equal readings a t an arbitrary absorbance, and filled with the test solutions. Four sets of readings were taken on each of the five sample cuvettes, giving for 20 replications: a t an absorbance of 0.041, a standard deviation of 4.5y0; a t an absorbance of 0.129, a standard deviation of l.Oyo; and a t an absorbance of 0.382, a standard deviation of 0.2%. Lowry and Bessey (2) report the following standard deviations of measurements made a t 8700 A. : a t an absorbance of 0.042, 1.5%; at an absorbance of 0.166, 0.5%; and a t an
absorbance of 0.422, 0.3%. It is difficult t o make a quantitative comparison between the two machines, since the Beckman instrument in the authors' hands does not give as good results as those obtained by Lowry and Bessey (8). The results obtained with the present machine appear to be more reproducible, by a factor of 2 to 5, than those routinely obtained on the Beckman instrument under the conditions of operation. The larger part of the improved performance is ascribed to the accurate positioning of the cuvettes in the present instrument. However, the absence of dark current and the stability of the signal also contribute to the improved performance, particularly a t low absorbance. The machine has been in use for about a year, and has performed reliably over this period. The instrument has been used for the microanalysis of inulin by the Lowry
(1) modification of the method of Roe, Epstein, and Goldstein (8). I n one experiment, seven 0.37-pl. samples of inulin mere taken from a solution containing inulin in a concentration of 4 y per pl. The results of the colorimetric analysis had a standard deviation of o.9yO. A similar replicate analysis (seven 0.37-p1. samples) from a solution containing 1 y of inulin per microliter gave a standard deviation of 2.2%. I n addition to the excellent reproducibility, the new microcolorirneter has the advantages of freedom from dark current, the accommodation of a larger number of samples, the provision of a written record, and great rapidity of measurement (five samples and a blank can be measured and recorded in less than 200 seconds). Furthermore, the results obtained do not depend on the skill of the operator.
ACKNOWLEDGMENT
Lloyd Cail of Laboratory Associates, Inc., has provided welcome and necessary assistance in the design of the mechanical part of the apparatus and is entirely responsible for its construction. Joan Lord and Bradford D. Pearson have helped by providing the cobaltous chloride measurements. LITERATURE CITED
( 1 ) Lowry, 0. H., Washington University,
S t . Louis, Mo., personal eommunication.
(1949):
RECEIVEDfor review July 31, 1957. Accepted September 21, 1957. Work supported in part by the Atomic Energy Commission.
Microdetermination of Vo ati e Aldehydes IRVING R. HUNTER and EARL
F. POTTER
Western Regional Research laboratory, Albany, Calif.
b In the microdetermination of volatile aldehydes, particularly those generated by and distilled from ninhydrin oxidations of amino acids, the aldehydes are absorbed in and made to react with an excess of sodium bisulfite to form stable complexes. Excess bisulfite is removed b y oxidation with iodine. A measured amount of iodine in excess of the aldehydebisulfite equivalent and an alkaline buffer are added to the residual aldehyde-bisulfite complex. The iodine quantitatively oxidizes the bisulfite and aldehydes. Excess iodine is then determined b y back-titration with standard thiosulfate. A blank is run a t the same time and the difference in titration values is used to calculate aldehyde equivalent.
C
of rice that may be related to its behavior under various processing treatments are under investigation a t this laboratory. One of the groups of compounds being studied is the free amino acids, 18 of which have been identified by paper chromatography ( 3 ) . I n developing a method for determining some of the free amino acids of parboiled and other forms of rice by oxidizing them with ninhydrin and separating the volatile aldehydes by gas-liquid chromatography (i?),it became necessary to know whether the system of generation and collection ONSTITUENTS
used yielded aldehydes quantitatively prior to the actual partition phase of the analysis. Although numerous methods are available for determining aldehydes, one involving an initial reaction between aldehydes and sodium bisulfite was considered the most suitable for this purpose. This reagent reacts rapidly with aldehydes to form stable complexes. Other reagents-e.g., hydroxylamine and phenylhydrazine derivatives -react completely with aldehydes only after prolonged contact and would be difficult to use for absorbing very volatile aldehydes-e.g., acetaldehydefrom a gas stream. The method adopted proved more satisfactory from the standpoint of accuracy and precision than other procedures (1, 4, 5 ) . It is currently in use a t this laboratory as an aid in developing a method for determining amino acids. Aldehydes generated from amino acids are collected in an excess of 1% bisulfite solution. The solution is treated with iodine t o oxidize excess bisulfite. A measured amount of standard iodine and an alkaline buffer, added t o the solution, quantitatively oxidize both the bisulfite and aldehyde moieties of the complex. Excess iodine is determined by titration with standard thiosulfate. A blank is run a t the same time and the difference in titration values is used t o determine aldehyde equivalents.
APPARATUS A N D REAGENTS
Oxidation apparatus shown in Figure 1 is custom-made with standard borosilicate laboratory glassware. All chemicals are reagent grade unless otherwise specified. Ninhydrin, Eastman Kodak Go. white label. Sodium bisulfite, 1%, freshly prepared. Iodine, 0.1N and 0.02N in water (standardized). Soluble starch indicator, 0.5y0 protected from deterioration with elemental mercury. Sodium thiosulfate, 0.02N, freshly preDared from 0.1N reagent bv dilution. * Sodium carbonate;2N. " Sulfuric acid, 1N. Xitrogen, compressed, water-pumped. Potassium dihydrogen phosphate. Sodium chloride. Amino acids, commercial samples. All showed single ninhydrin reacting spots on a two-dimensional paper chromatogram, when the method and solvents described by Hunter, Ferrel, and Houston were used ( 3 ) . Aqueous solutions were prepared and protected with 0.01% sodium ethyl mercurithiosalicylate. Silicone oils, Dow Corning 703 and 550. PROCEDURE
Amino acids were oxidized to aldehydes by ninhydrin essentially by the method of Virtanen and Rautanen (6). Ten milliliters of an amino acid solution containing approximately 3 mg. of amino nitrogen were pipetted into the flask and 1 gram of potassium dihydrogen phosphate and 2.1 grams of sodium chloride were added, either as solids or as saturated solution. VOL. 30, NO. 2, FEBRUARY 1958
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