LITERATURE CITED
(1) Am. SOC. Testing Materials, Philadelphia, Pa., “Methods for Emission Spectrochemical Analysis,” Deaignation E-115-59T, p. 1, and E-116-59T, p. 12 (1960). (2) Baer, W. K., Hodge, E. S., A p p l . Spectroscopy 14, No. 6, 141 (1960). (3) Feldman, Cyrus, ANAL. CHEM. 21, 1041 (1949). (4) Lundell, G. E. F., Hoffmann, J. I., Bright, H. A., “Applied Inorganic An-
p,” second ed. revised pp. 205-7, iley, New York, 1953.
(5) Margoshes, M., Chem. Ens. N e w s 39, . No. 38; 94 (September 18, 1961). (6) McKaveney, J. P., ANAL.CHEM.33,
744 (1961). (7) McKaveney, J. P., Crucible Steel Co. of America, Research Project 110, Research Book No. 20, p. 99, August 5,
----.
1Q59
(8) Pagliassotti, J. P., ANAL.CHEM.28, 1774 (1956). (9) Pagliassotti, J. P., Porsche, F. W.,
Zbid., 23, 198 (1981). (10) Raber, W. J., Crucible Steel Co. of America, Final Report Test 6175, pp. __ 3-7 (September 1, i96l). (11) Zink, T. H., A p p l . Spectroscopy 13, No. 4, 94 (1959).
RECEIVEDfor review May 24, 1961. Accepted January 5, 1962. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., March 3, 1961.
Continuous Analysis by Measurement of the Rate of Enzyme Catalyzed Reactions Glucose Determination W. J. BLAEDEL and G. P. HICKS Chemistry Departmenf, University of Wisconsin, Madison, Wis.
b An instrument is described which permits the continuous measurement of the rates of many enzyme catalyzed reactions. The instrument is designed specifically for the routine assay of enzymes or substrates. Illustrative application is made to the determination of glucose by the glucose oxidaseperoxidase coupled system. Glucose in aqueous solutions up to 60 p.p.m. may b e determined with a standard deviation of 1 p.p.m. Samples may b e analyzed a t the rate of 15 per hour, with a readout time of 4 minutes per sample. Faster analyses are possible. Glucose is also determined in 0.2-ml. samples of blood plasma. Sample and reagent manipulation b y the technician is kept to a minimum through use of a flowing system. Calibration is performed with a standard sample to permit direct readout on unknown samples, with no calculation.
E
are extremely useful in clinical and analytical chemistry ( I ) . Since the analytical and clinical applications are increasing rapidly, the need for instrumentation is apparent. Some automated procedures have been developed, which minimize effort and manipulation on the part of the technician ( 2 , 3 ) . Other instruments have been developcd which automatically measure the rate of an enzyme reaction by measuring the time required for the systrm to change from one particular composition (measured by absorbance or electrode potential) t o another (4, 6). Since the change is small, and since measurements are made 388
very near initial velocity, the instrument readout is inversely proportional to the initial rate, and therefore also inversely proportional to the concentration of sought-for substance. In this paper, an instrument is presented which gives a continuous record of the rate of a chemical reaction, provided that it is accompanied by a change in absorbance. The principle of the method is shown in Figure 1. A sample stream containan ing the sought-for substance-Le., enzyme or substrate-flows at a constant rate t o meet and mix with a reagent stream also flowing at a constant rate. The reagent stream contains fixed concentrations of all of the other components necessary for the reaction,
NZYME-CATALYZED REACTIONS
a
ANALYTICAL CHEMISTRY
UPSTREAM DELAY UPSTREAM CELL INTERCELL DELAY DOWNSTREAM CELL WASTE Figure 1. Outline of continuous measurement of reaction rates
except the one in the sample stream. As the reaction occurs, the absorbance of the resultant stream changes continuously as it flows a\?ay from the mixing point. Since the flow rate is constant, the time interval betnecn the two cells is fixed, and the steady state absorbance difference between the photometer cells is proportional to the reaction rate. The absorbance difference is measured with a sensitive differential recording filter photometer. The delay betwecn the mixing point and the upstream cell is made long enough to overcome any nonlinear induction periods, if such exist. The intercell delay is made long enough to obtain an accurately measurable absorbance difference for the range of concentrations to be determined. Several benefits and advantages over existing methods are inherent in the scheme of Figure 1 and are summarized here : Through use of a sensitive differential photometer] the time interval over which the rate is measured can be made very small. Thirty seconds is typical for the glucose determination. The extent of reaction is kept small, and the measured rate is virtually an initial rate, with very small changes in reactant concentrations. Such circumstances give the best chance of achieving direct proportionality between the measured absorbance change and the concentration of the sought-for substance. bleasurement of the absorbance difference largely eliminates errors due to absorbing, but nonreactive, impurities in the sample and reagents. Separate blanks for each sample are not required.
MULTIPLIER
REAGENT RESERVOIR
r'"'~:~~ING
ROTATING PCLARIZERJ
Figure 3.
Figure 2,
The flowing system
Calibration can be performed easily by running a standard sample, and adjusting the sensitivity to give a direct readout on the recorder in terms of the sought-for substance. No calculations or working curves are required. Since the system is a continuous one, little manipulation of the sample or reagents is required. I n principle, the method is simple. Not only is the equipmrnt inexpensive, but it is fairly easy to build and to operati.. Further simplifications of the equipmmt drscribed in this papi'r are uncicr I\ ay. THE FLOWING SYSTEM
Figure 2 is a more detailed schematic of the system. The various parts described below are mounted on a vertical plywood board which has a shelf to hold the photometer. Reagent Stream. For simplicity and economy, t h e reagent stream is introduced by gravity flow. The reagent is stored i n a 200-ml. reservoir, fitted with a rubber stopper a n d air-inlet tube. When t h e stopper is tight, so t h a t air can enter t h e reservoir only through t h e air-inlet tube, t h e reagent head is maintained a t level a, regardless of t h e liquid level in the rcscrvoir. The reagent flow rate is then eonstant, being determined principally by the length and geometry of the flowing system. Any rate may be obtained approximately by choosing the length of 1-mm. capillary, and more precisely by adjusting the level b of the outlet tubing. A typical rate for a 25-em. difference between a and b is 2 ml. per minute when the length of the 1-mm. capillary tubing is 75 em. and each delay line is about 65 em. This arrangement is satisfactory for delivering large ~ o i u m e sof reagent at a constant rate over long periods of time. Sample Stream. For proper testing of the equipment, i t is desirable to introduce small intermittent samples, so as to obtain a b r u p t changes. S o emphasis is placed on automated introduction of samples, or on analysis of a continuously flowing
stream. The three-way stopcock (1mm. bore) shown in Figure 2 permits switching from one sample t o another without introduction of air slugs, and with negligible interruption of the sample stream. The sample is metered with a single channel peristaltic pump, rather than with gravity feed. The peristaltic pulses do not adversely affect the measurement system, provided that the off periods are of short duration. Since calibration is performed easily with a standard sample, it is not necessary to reproduce particular or absolute flow rates; the flow rates need only be constant. -4 typical sample stream flow rate is 2 ml. per minute. Pulser. The pulser is a n ll/lB-inch nylon disk mounted 1/16 inch 08 center on the shaft of a Barcol stirring motor that rotates a t 300 r.p.m. The nylon disk rotates eccentrically, and depresses a short section of rubber tubing (2 X 3/16 inch i.d.) inserted a t the end of the 1-mm. reagent capillary. The rubber tubing is backed with a small plate whose position under the tubing may be raised or lowered to control pulse amplitude. To reduce friction, a Lucite plastic sheet is taped between the nylon disk and the rubber tubing. The pulser introduces periodic reversals in the flowing stream. The magnitude of the reversals may be observed by noting the oscillations of a small air bubble deliberately introduced into the stream. -4typical displacement in the 2-mm. capillary is 1 to 2 em. Without the pulser, severe laminar flow occurs throughout the whole system. Not only is mixing of the reagent and sample streams very poor, but displacement of one sample by another is very slow, m-hich gives a slow approach to steady state when a new sample is introduced. The pulser greatly reduces both of these difficulties. The effectiveness of the pulser is increased by inserting kinked 26-gage platinum wire in the two delay lines. Back pulsing into the reagent line leading from the reagent reservoir is observable, but its effect on the response is negligible. As the pulse amplitude is increased, a point is reached a t which the response time no longer decreases rapidly; this is around the optimum pulse amplitude.
INTERFERENCE FILTER LENS
I
r$$MTING
SLITS The optical system
Photometer Flow Cells. A 1-inch section of 3-mm. square bore borosilicate glass tubing is polished flat o n two opposing sides (Wilmad Glass Co., Buena, N. J.). T h e ball of a 12/2 borosilicate glass ball and socket joint is sealed t o each end of the cell. Two cells are made and installed vertically in the photometer so that the ball ends protrude. This arrangement permits easy interchange of delay lines of various lengths and shapes, giving a range of choice of reaction intervals. Tubing. Reagent stream tubing u p t o t h e pulser, and sample stream tubing u p t o t h e pump, is 1-mm. borosilicate capillary. All other tubing is 2-mm. capillary except for t h e Tygon tubing (15 X 0.081 inch i.d.) in t h e peristalic pump. The outlet tube ends in a section of Tygon tubing (25 X I/g inch i.d.), so that the outlet level may be easily adjusted. To prevent air from being pulled in by the pulsing stream, the outlet tip is submerged into a 10-ml. beaker that overflows to a drain. No provision is made for therniostating any part of the system, despite the high temperature coefficient that has been reported for the enzyme reaction. An air conditioned room prevents rapid drifts and fluctuations in temperature. Slow temperature drifts may be compensated for by occasional and easy calibration with a standard snmplc. THE FILTER PHOTOMETER
Theory of the Filter Photometer. A schematic of the differential filter photometer is shown in Figure 3 . Radiation from the tungsten source is rendered parallel by the collimating lens. T h e parallel light passes through a n interference filter and then through the 3-mm. f l o ~cells located directly before 2-mm. slits. After the slits, the parallel light passes through a rotating (24 revolutions per second) Polaroid sheet. and then through a fixed Polaroid sheet. The filed Polaroid sheet consists of two halves (uand d ) butted together, with the axes of polarization perpendicular to each other, so that light from one slit is polarized perpendicularly to light from the other slit. A second lens focuses the light upon a multiplier phototube. When the rotating polarizer is aligned with one half (u) of the fixed polarizer, the light transmitted through the upstream cell (u) t o the multiplier phototube is a maximum. The other half ( d ) of the fixed polarizer is a t extinction, and the light transmitted through the downstream cell (d) to the photoVOL. 34, NO. 3, MARCH 1962
e
389
Figure 4. A.
Filter photometer layout
Light source, Beckman DU tungsten bulb, mounted on backplate assembly ('/*-inch brass) and base ('/z-inch aluminum) E . Aluminum block, contains collimating lens ( 1 44-mm. focal length, 19/16inch diameter) and interference fllter (2 X 2 X inch) C. Aluminum cell block, two vertical slits (not shown) 2 mm. wide by "4 inch long in front of each slot (CZ, CBI C1. Set screw. CZ, Ca. Slots, 1.25 cm., machined full length on back side of block Cq. Spring-loaded screw with needle point D. Aluminum block containing bearing assembly for rotating Polaroid 171. Back of pulley with collar, 2'/~-inch diameter with a 4-cm. hole in center. Dz. Fafnir MM91 OBWICR precision bearing (Wisconsin Bearing CO., Madison, Wis.)
tube is a minimum. Per revolution of the rotating polarizer, the light reaching the multiplier phototube from each ce!l. passes twice from maximum to minimum. At any instant, let I , be the intensity out of the slit from cell u,and incident upon the rotating polarizer, whose polarization axis is a t a n angle e with respect to the axis of half u of the fixed polarizer. Similarly, let I d be the intensity out of the slit from cell d, and incident upon the rotating polarizer, whose polarization axis is a t a n angle 90 - e with respect to the axis of half d of the fixed polarizer. If I t is the total intensity falling upon the multiplier phototube, It = I,, Sin28 I d Cos% If the intensities out of the two slits are equalized so that I , = I d = I , I t = I Sin% I C0s28 = I , a constant. Thus, in theory, a t equal slit intensities, the phototube sees a constant intensity, with no a x . component, regardless of the orientation of the rotating polarizer. On the other hand, any difference between the two slit intensities will cause an a.c. signal in the phototube. This ax. difference signal is amplified with a n a.c. amplifier, rectified, and recorded as a d.c. signal which measures the difference in intensities passed by the two cells. Small differences in intensities are easily measured. Under typical operating conditions, a difference of only 2% in intensities emergent from the cells can be made to give full scale deflection on the recorder. I n other words, full scale on the recorder represents the transmittance range from 98 to loo%, or a range of about 0.01 absorbance unit. The stability is such that a difference of about 1% of full scale is detectable, which is a transmittance difference of 0.02%, or a n absorbance difference of 0.0001 unit.
+
+
390
ANALYTICAL CHEMISTRY
E.
Bodine motor, 1 8 0 0 r.p.m. O-ring, used as pulley €2. Aluminum pulley, 2-inch diameter F. Aluminum block containing focusing lens (1 44-mm. focal length, lg/isinch diameter, not shown) G. Multiplier phototube, 9 3 1 A GI. Adjustable cylindrical base large enough to accommodate resistors and wiring around phototube socket H. Photometer base, 3/8-inch aluminum, has 1 '/Z-inch hole under G1 and appropriate holes to bring in power for motor and light source HI. Leveling screws H2. Thumb screws to secure photometer case El.
The above described method of chopping with Polaroids was selected after trying mechanical chopping with a rotating sector wheel. Mechanical chopping requires very careful machining and alignment of parts; considerable noise results unless the slit illumination is very uniform, and unless the slits are placed symmetrically about the center of rotation of the sector wheel. The principal limitation of the Polaroid sheets is the spectral cutoff around 400 mw. However, this limitation does not preclude the assay of systems involving the reduction of diphosphopyridine nucleotide (DPN) or triphosphopyridine nucleotide (TPK), since coupled enzyme reactions may be used. Such an application to the assay of isocitric dehydrogenase is under way in this laboratory. Construction of the Filter Photometer. The layout of the filter photometer is shown i n Figure 4. The light source ( A ) is operated from a 6-volt storage battery. The bulb is mounted on an adjustable backplate, which in turn is mounted on a movable base to permit focusing. Rigidity is essential; otherwise movement of the light source causes instability and drift. The collimating lens and interference filter are mounted in a n aluminum block (B). The lens is held in position by two diametrically placed drops of epoxy resin. The filter is held by two bronze spring clamps to permit easy interchange. The flow cells are held with setscrews (one shown, C,) in a slotted aluminum block (C). Half-inch holes (not shown) are drilled through the block and photometer base so &at the ball joints of the cells extend below the base for connection to the flowing system. The front side of the cell block has two vertical cell slits (not shown). A
needle point screw (C,) can be moved into or out of the path of one slit to make fine adjustments of the intensity. ii course adjustment on the other slit consists of a spring-loaded wedge (not shown) which can be screued into or out of the slit path. The rotating Polaroid is held in a machined aluminum block ( D ) . The Polaroid film (not shonn) is cut from a 2 x 2 inch sheet (E. H. Sargent, Chicago, Ill., Cat. KO. S-70944), and tacked mith epoxy resin onto the face of a pulley. The back of the pulley ( D l ) is machined to fit loosely into a n-here it is precision bearing (Dz), held nith two small drops of epoxy resin. I n turn, the bearing fits loosely into the block, n-here it is also hpld n-ith t n o small drops of epoxy resin. Press fits are not used because they may cause some binding in the bearing The rotating Polaroid should he niped clean before installation. and handled carefully; any imperfections or fingerprints arp a source of noise in the a x . signal. The rotating Polaroid is driven n i t h a n 1800 r.p.m. Bodine motor ( E ) . An O-ring (E1)is used as a pulley belt. The motor pulley gives a bearing pulley speed of 1440 r.p.m. or 24 revolutions per second for the rotating Polaroid, and a frequency of 48 cycles per second for the ax. signal. The focusing lens (not shonn) is held in a n aluminum block ( F ) , similar to that holding the collimating lens. The fixed Polaroids (1 X 2 inch, not shown) are each cut from different 2 x 2 inch sheets, and are mounted n ith masking tape on block F , n ith their polarization axes perpendicular to each other. Masking tape is used for easy adjustment. The multiplier phototube (G) is mounted in its socket on an adjustable cylindrical base (GI). A few drops of epoxy resin hold the tube firmly to the
B.
4
3.0-volt battery
CI,CB. 0.1 -pf. paper capacitor 400 volt d.c. C3. 1 .O-pf. ceramic capacitor 4 0 0 volt d.c. C4, Cg. c6. Di,
PM. RI. RP.
1.0-pf. 2 0 0 volt d.c. paper capacitor 16-pf. 150 volts electrolytic capacitor Dz. 1 N 3 4 A crystal diode 9 3 1 A multiplier phototube
1 megohm, 1 1 2 watt
2 0 0 ohm, 2 watt 39,000 ohms, 2 watt Rp. 1 megohm, 2 watt Rg. 3,000 ohms, 2 watt R6. 150,000 ohms, 112 watt R7. 8,000 ohms, 112 watt Rg. 500,000 ohms, 1 watt Rs. 20,000 ohms, 2-watt potentiometer Rm. -_ 50,000 . ohms. 2-watt. 1 0-turn Dotentiometer Rec. 10-mv. Varian recorder, Moddl G-1 1 A VI, Vz. 12AT7 R3.
Figure 5. Multiplier phototube and amplifier
base. The phototube output is brought by shielded cable to the amplifier through a hole in the base plate directly under the phototube base. All of the components are mounted on a base plate ( H ) ,on a common optical axis which is 2 3 / 4 inches above the floor of the base plate. I n general, metal components are spray painted flat black to reduce reflections. However, the bearing itself is not painted. The base plate is equipped with four levelling w e n s (on shown. H I ) , and has holes to bring in power for the motor and light source. A lid (not shown) covers all components, and is held to the base plate with four thumbscrews (one shown, H z ) . The lid is painted black inside and contains a baffle with a ll/rinch hole between blocks A and B. The lid also has a chimney (2 inches in diameter and 3 inches high) to cool the light source. Two small openings give access to the coarse and fine adjustments on the slits. Finally, two 3/4inch square holes above the cell block permit connection of the cell ball joints with the intercell delay line. Electronics. The multiplier phototube wiring, included in the dashed box shown in Figure 5 , is located under the multiplier phototube, with a shielded lead to the amplifier. The power for the phototube is supplied by 11 45-volt B batteries in series (Ray-0-Vac S o . 709), giving 495 volts. An additional battery to give 540 volts approximately doubles the output of the phototube, but there has been no need for this much sensitivity. In general, it is best to operate the phototube a t the lowest voltage that gives the desired gain, in order to minimize noise. A regulated d.c. power supply (Kepco Laboratories, Inc., Flushing, N. Y., Model 510B) is also satisfactory. The amplifier (Figure 5 ) provides a d.c. signal that will operate a recorder. A blocking capacitor (C,) passes only the a x . difference signal and blocks the large d.c. component of the multiplier phototube. The a.c. signal is amplified, passed through a cathode follower. rectified, and fed to a 10-mv. recorder ihlodel G-11-4, Varian Associates, Palo Alto, Calif.). The power for the filament and plate of the ampli-
fier is supplied currently by a bench power supply (Kepco, Model 510B). Any regulated power supply should suffice. There are two variable controls on the amplifier: Rjo is a sensitivity adjustment, and R9 is a zero control. Their setting is described below. Adjustment of Photometer. First, the parts of the optical system are aligned as shown in Figure 4. The photo cathode is placed a t the focal point of the focusing lens, and then the lamp position is adjusted to give the sharpest image of the filament on a white card held just before the multiplier phototube. In this adjustment, power is supplied only to the lamp, and the flowing cells and stationary Polaroids are out of the system. All parts are rigidly fixed to the base except the lamp, which is left loose a t the base to permit small adjustments later. The fixed Polaroids are positioned in a dark room, with the photometer housing removed. The phototube. is masked with black construction paper, so that its cathode is exposed only to light from the focusing lens. Also, the light source is shielded with black paper, so that reflections from the surroundings cannot reach the phototube by paths other than the optic axis. Power is supplied to the phototube, amplifier, lamp, and chopper motor. A quarter hour is allowed for warm-up. The flow cells are not yet installed, and the course and fine slit adjustments are out of the path. The amplifier is set for low gain (R9 minimum, and Rio about one-fifth of full scale). One of the fixed Polaroid films is taped to one half of the focusing lens block, so that its polarization axis is vertical. The other fixed Polaroid is placed on the other half of the lens block so that its polarization axis is horizontal, but it is taped only along its bottom. so that its orientation mav be adjusted later. The loose Polaroid is rotated until a minimum signal is obtained on the recorder. As t h e adjustment is improved, the amplifier gain is increased by increasing Rlo. The lamp position has some effect on the minimum signal obtainable, so when the first minimum has been obtained, the light source
should be adjusted to give a further minimum, if possible. -4lternate adjustment of the lamp and loose Polaroid should be continued to give a minimum noise level, which should be recorded. At this time, the loose Polaroid is securely taped to the block, and the lamp base is tightened. To check the magnitude of the minimum noise level, a vacuum tube voltmeter is put in place of the recorder, and the downstream slit is blocked off to give a maximum output which is proportional to the total intensity through the slit. The magnitude of this signal is read, with all controls having the same values as were used to read the minimum noise level. The minimum noise level should be less than 0.5% of the maximum output. The flow cells are next installed so that the slits in the cell block are entirely within the rectangular parts of the cells. KO attempt is made to obtain matched cells, although this would be desirable. The cells are filled with water and that cell with the highest transmittance is placed in line with that slit having the course wedge adjustment. The wedge is then moved into the slit to obtain a minimum signal on the recorder. In this adjustment, power is supplied to the multiplier phototube, amplifier, lamp, and chopper motor; the amplifier gain is close t o maximum (R9 a t minimum, and Rlo close to maximum) ; and the work is done in a dark room. Sometimes, slight readjustment of the lamp after cell installation gives a lower minimum signal and helps to compensate for cell mismatch. After cell installation, it should be possible to move the fine adjustment needle entirely into its slit path, and to compensate for this with the wedge to re-achieve a minimum signal. Once made for a particular instrument, these three adjustments (of source, fixed Polaroids, and cells) are permanent, unless the optical system is disturbed. APPLICATION TO GLUCOSE DETERMINATION
Glucose Oxidase Reaction. The determination of glucose by the well known glucose oxidase reaction was chosen to test the equipment described above. To determine glucose, VOL. 34, NO. 3, MARCH 1 9 6 2
391
t h e sample is mixed n i t h a reagent t h a t contains controlled concentrations of glucose oxidase, peroxidase, and o-tolidine in buffered medium. Glucose is oxidized b y air to gluconic acid, with the simultaneous production of Hz02. I n the presence of peroxidase, the HzO, oxidizes the o-tolidine to a benzidine blue derivative (7), giving an absorbance change that has been measured around 635 mp (8). Under controlled conditions, the rate of change of absorbance is proportional t o the glucose concentration, provided that the change is not measured until an induction period has passed (4, 5). Fragmentary batch work in this laboratory indicated that the reaction rate is not limited by the concentrations of oxygen, peroxidase, or o-tolidine, and this conclusion is substantiated by the linear dependence of rate upon glucose concentration which is described below. The glucose oxidase-peroxidase procedure has recently been described on a batch basis (8),but there are many variations. Some difficulties with the batch procedure are day-to-day fluctuations of calibration curves, instability of the colored product, enzyme inhibitors, and reaction of the dye with interfering oxidizing or reducing substances (9). The first two of these difficulties are diminished by the continuous differential method because of its rapidity and ease of calibration. I n general, the latter two difficulties are also operative in the differential procedure, but in some cases, they might be diminished considerably by the procedure, depending on the mechanism of the interference. Reagents. BUFFER SOLUTIOK. Acetic acid (0.1M) is adjusted t o p H 4.1 t o 4.2 with 3 M KaOH. The solution is stable indefinitely. A high buffer capacity is not needed for diluted blood plasma samples, b u t may be needed for other applications. ENZYME-DYEREAGENT.One gram of glucose oxidase (Worthington Biochemical Corp., Freehold, N. J.) and 20 mg. of peroxidase (Grade D, Worthington Biochemical Corp.) are added to 400 ml. of buffer solution. Then 500 mg. of o-tolidine dihydrochloride (Eastman Chemical Co., Rochester, N. Y.) are dissolved in 100 ml. of buffer. The enzyme and dye solutions are mixed and filtered to give 500 ml. of enzymedye reagent. For the following work, i t was standard practice to prepare this reagent daily. On standing, the dye oxidizes very slowly. and the solution becomes slightly greenish. After 8 to 10 hours a t room temperature, the absorbance increases by only a few hundredths of a unit, which causes no error in the differential continuous procedure. KO further study was made of the stability of this reagent in this laboratory. However, a similar solution stored a t 3" to 4 " C. has been reported to be stable for more than 4 weeks (8). 392
0
ANALYTICAL CHEMISTRY
Figure 6.
Record of standard glucose samples
GLUCOSE STOCK SOLUTION. One gram of glucose is dissolved in 1 liter of water to give a stock solution COIItaining 1.00 mg. per ml. A stock solution n-itli benzoic acid as a prrservative is stable for several months when stored in the refrigerator ( 9 ) . GLUCOSESTANDARD SOLUTIOV.The glucose stock solution was diluted with buffer to give solutions ranging up to 60 p.p.m. of glucose. Preparation of Equipment for the Glucose Method. Some adjustments must be made to prepare the equipment for a particular method of analysis, a n d for t h e range of sample sizes to be determined. Once made, these adjustments need not be repeated or changed from d a y to day. They need to be checked only infrequently, and changed only when t h e method of analysis or range of sample sizes changes. A bluish interference filter is installed (595-mp maximum, 9-mp half band width, Farrand Optical Co., New York, N. Y.). A filter with a 6 3 5 - m ~maximum was not available when the measurements were made. Because the absorption band of the blue product is rather broad, the choice of filter is not critical. The photometer housing is fastened, and power is delivered to the lamp, multiplier phototube, amplifier, and chopper motor. The photometer is connected to the flowing system by means of the ball and socket joints, which should be greased very lightly. T h a t cell whose slit has the needle adjustment should be made the upstream cell, so that its absorbance at steady state will be less than the absorbance of the downstream cell. The two delay lines are constructed of 2-mm. i.d. tubing, to provide a delay of a half minute each, a t a flow rate of 4 ml. per minute. A half minute for the upstream delay is sufficient to bypass a n induction period during which the rate is not constant, but increases slowly to its steady-state value (4). The half minute intercell delay gives sufficient sensitivity for the determination of glucose in blood plasma. The sensitivity may be increased by increasing the intercell delay, but then the readout time is also increased. To clean the system, 1% Alconox is passed into both the reagent and sample inlets, and light suction is applied with a n aspirator to the outlet, t o speed the flow of detergent and to remove air
bubbles. After the system is free of bubbles, the Alconox solutions are replaced with water to flush the system for 10 to 15 minutes. Khile the system is being flushed with water, the pulser is adjusted to give a 1 t o 2 em. pulse in the reaction stream. The outlet level is adjusted to give a total flow rate of 4 ml. per minute. (Since the sample pumping rate is 1.7 ml. per minute, the reagent stream flows around 2.3 ml. per minute.) Because of frequent calibration, it is not necessary to achieve particular flow rates, but only to maintain constant ones. If the instrument is operated close to the minimum noise signal, with the two cell intensities close to balance, there is deviation from linearity between the recorder reading and the glucose concentration. This deviation decreases and the sensitivity increases as the magnitude of the signal increases, that is as the difference between the two cell intensities increases. For differences above 3'35, good linearity is obtained. It is therefore necessary to operate the instrument in a slightly unbalanced condition, with the upstream cell intensity about 3% higher than the downstream cell intensity. Achievement of the unbalanced condition is described below. While water is flowing, and with t h e amplifier set near maximum sensitivity, the slit needle and slit wedge are adjusted to give the minimum noise signal with the slit needle inserted to about one-half of its range of travel. The noise signal is then nulled on the recorder by adjusting ,Rs. Kext, the recorder is replaced with the vacuum tube voltmeter, the downstream slit is temporarily blocked with a n inserted strip of black paper, and Rlois adjusted to give a maximum output signal of 1 volt. This operation amounts to measuring the total intensity through the slit. The paper blocking strip is then removed. The slit needle is adjusted to make the upstream cell lighter until a 10 mv. (1%) difference is noted on the recorder, after which this signal is nulled on the recorder by further adjustment of Rg. This operation is repeated twice more, until a difference signal of 30 mv. (3%) has been built up between the two cells, the upstream cell being the lighter one. Any difference in absorbance between the two cells due to reaction adds to the 3% difference, and good linearity always
diminished. It is good practice to flush the system with acetone for a few minutes every week or two.
I OO'
20 0
40
L60
GLUCOSE CONCENTRATION IN SAMPLE, PPM. Figure 7. Working curve for standard glucose samples
exists between the recorder reading and the glucose concentration. It is desirable to check this cell unbalance every few days or so, while water or a glucosefree sample flows in the sample stream, to ascertain that the difference signal is around 3% above the minimum noise level. Procedure for Analysis of Samples. Precision with Standard Samples. Before samples were run, the instrument was calibrated with a blank and a standard solution. All parts of the instrument were turned on. With the recorder input shorted, the recorder base line was arbitrarily set a t 10 divisions on the chart, using the recorder zero adjustment, so that drifts and fluctuations in the blank would be perceptible. Enzyme-dye reagent was put into the reagent reservoir, and water was put into the sample inlet. At steady state, the recorder was reset with Rg to 10 divisions on the chart. Then a blank (glucose-free buffer solution) was introduced, and a t steady state, the recorder was reset, if necessary, to a blank reading of 10 divisions on the chart. A standard 60-p.p.m. glucose sample was put into the sample inlet. At steady state, RIOwas adjusted so that the chart read 90 divisions above the blank reading. After calibration, five standard samples ranging from 10 to 50 p.p.m. were run a t 4-minute intervals, and the steady-state recorder reading was obtained for each. Figure 6 is a record (from right to left) of the calibration and samples, the 10-p.p.m. sample having been run twice. For shutdown a t the end of a day, mater is passed into the reagent and sample inlets for 15 minutes, and water is left in the system. After much extended use, dye gradually precipitates inside the cells and tubing. This causes no error in the differential method, providing that the transmittance is not greatly
The blank reading drifts for a number of reasons, among which are increase of the absorbance of the enzyme-dye reagent with time, and changes in room temperature. The drift is very small, and can best be corrected by running blanks occasionally. It is recommended that the blank reading be checked and reset if necessary by running a blank sample after every five samples. For similar reasons, the calibration with a standard glucose solution should be repeated after every 10 samples. Experience best determinw the frequency with which blanks and standards should be run. Figure 7 is a plot of the data from Figure 6. The recorder response is directly proportional to the glucose concentration in the sample stream. The precision is very high, the standard deviation of the points from a straight line being only 0.3 p.p.m. This is the order of precision that could be expected if a working curve were prepared and used in the analysis of samples. However, one-point calibration with direct readout is much simpler in practice, and the sacrifice in accuracy is tolerable for glucose analysis. Thus, in obtaining the data of Figure 7 , the chart mas calibrated to read 90 divisions above the blank reading for a standard solution containing 60 p.p.m. of glucose. If a linear working curve is assumed to pass through zero (see dotted curve, Figure 7 ) , then each chart division corresponds to z/3 p.p.m. of glucoseLe., 15 divisions = 10 p.p.m. The glucose concentration in parts per million may be simply found by multiplying the chart divisions by z/3. At low glucose concentrations, the error of direct readout becomes relatively important, because the true working curve does not extrapolate to zero. Thus, the relative error of direct readout at 10 p.p.m. is about 670, according to Figure 7 . These errors may be alleviated by any of several procedures which are more troublesome or restricted than direct readout, but still less troublesome than the determination and checking of a multipoint working curve. A few of these procedures are given categorically. Calibration for direct readout may be performed a t a value close t o the sample sizes that are considered most important. Direct readout gives no error for samples that have the same value as the standardizing solution. Direct readout on a low sample may be accepted as only approximate. Such a sample might be run again a t higher sensitivity. Or, if it has been prepared by dilution, as is the case for blood samples, it might be rerun at a higher concentration.
40 THEORETICAL
I I PTS. 3 PTS.
@ -oo
20 40 60 GLUCOSE CONCENTRATION IN SAMPLE, PPM.
Figure 8. Repetitive determinations on standard glucose samples
Since the true working curve is a straight line, it could be determined with just two standard solutions, in which case the direct readout method would not be used a t all. It is suggested that a two-point working curve be made every few days as a check on the instrument. If the working curve so determined does not extrapolate fairly close to zero, the cell unbalance (30 my.) may not be in the right range Some idea of the precision of direct readout may be obtained from Figure 8, which represents analyses on 38 standard solutions performed over 3 days, with several shutdowns during the series. The standard error of these determinations is 1 p.p.m. of glucose, omitting one very high determination a t 40 p.p.m. This estimate is probably unfavorable for several reasons. The analyses were made with fewer blanks and standards than recommended. For example, a series of 19 determinations was run over a period of 80 minutes with only one blank and calibration a t the beginning. Calibration was not done with the same standard solution for all series of analyses. Lastly, temperature control in the air conditioned room was poor. Response Time. Figure 6 shows t h a t the response time from one steady state to a new steady state is about 3 minutes. B y running each sample for 4 minutes, steady state is maintained for about a minute, which permits reliable measurement. The response time may be shortened by increasing flow rates, shortening delay lines, or both. I n any case, efforts t o decrease response time require more reagents or an increased sensitivity of the instrument. Response time has been reduced to 13/4 minutes simply by increasing the total flow rate to 11 ml. per minute in the previously described equipment. If emphasis is put on rapid analysis, it should be possible to achieve readout times of 1 to 2 minutes, with sample repetition rates of 30 per hour, and VOL. 34, NO. 3, MARCH 1962
e
393
without great modification of the existing equipment. Analysis of Blood Plasma Samples. Several dozen blood plasma samples were analyzed directly by the continuous enzyme procedure by diluting 0.2 ml. of plasma to 10 ml. 1%-ithbuffer solution, and the values were compared with those obtained by a n automated ferricyanide procedure. Agreement was roughly in accord with previous comparisons ( 9 ) . No mechanical or chemical difficulties were observed with the plasma samples. The continuous enzyme procedure was also tested on standard commercial blood samples. Results on a standard blood plasma sample (Dade Reagents, Inc., Miami, Fla.) were 17% lower than the value of 118 mg. yo given on the label for the method of Folin and Wu. Three consecutive re-
coveries of 100 mg. % of glucose added to the same standard sample gave an average of 94.6% with a range of 92.6 to 97.2%. Unequivocal evaluation of the accuracy of the continuous enzyme procedure by comparison with other accepted methods of glucose analysis is difficult, because of considerable differences among the methods ( 9 ) . Investigation of these differences is under way in this laboratory.
REFERENCES
(1 ) Devlin, T. M., -4h.a~.CHEV. 31,
977 (1959).
( 2 ) Furness, F. K.,Easton, C.. eds., X. Y . Acad. Sci. 87 (Art. 2 ) , 609-951 (1960). (3) Hill, J. B., Kessler, G., J . Lab. Clzn. M e d . 57 (6), 970 (1961). ( 4 ) Malmstadt, H. V., Hicks, G. P., h A L . CHEhZ. 32, 394 (1960). (5) Malmstadt, H. V., Pardue, H. L., Ibzd., 33, 1040 (1961). (6) Middleton, J. E., Brit. X e d . J . 1, 824 (1959). (7) Noller, C. R., “Chemistrj- of Organic Compounds,” p. 553, R. B. Saunders,
Philadelphia, 1951.
(8) Salomon, L. L., Johnson, J. J., .$VAL. ACKNOWLEDGMENT
The authors thank Frank C. Larson, of the University Hospitals, Madison, Wis., for supplying blood plasma samples that had been analyzed by the automated Technicon ferricyanide method.
Titrimetric Determination of p-Phenylenediamines
CHEM.31. 453 (1959).
(9) SunderiAan, F. W., Jr., Sunderman, F. R., Tech. Bull. Registry X e d . Technologists 31 (B), 93 (1961). RECEIVED for review September 15, 1961. Accepted December 18, 1961. \Tork sup-
ported in part by a grant from the \Tisconsin Alumni Research Foundation, and in part by a grant from the C . S. Atomic Energy Commission.
N, ”-Disubstituted
OTTO LORENZ and C. R. PARKS Research Division, The Goodyear Tire & Rubber Co., Akron, Ohio
b A spot test is described for the detection of N,N’-di-sec-alkyl-, N-secalkyl-”-aryl-, and N,N’-diaryl-pphenylenediamines in rubber compounds. The test is based on the deep coloration of the corresponding Wurster’s salts. Titrimetric methods for the quantitative determination of these diamines are discussed, including N,N’diaryl-p-quinonediimines as well as mixtures of N,N’-diaryl-p-phenylenediamines and their corresponding diimines. The determinations are based on a neutralization with perchloric acid in nonaqueous solution. The N,N’diary1 derivatives were converted to the Wurster’s salts by the addition of chloranil or hydroquinone prior to the neutralization.
compounds. This test permits one to differentiate the three types of p phenylenediamines An investigation of the neutralization of p-phenylenediamines in nonaqueous solvents showed that N,N’-disec-alkyl- and N-sec-alkyl-N‘-aryl-pphenylenediamines could be determined readily in this way. The N,N’-diary1 derivatives did not give satisfactory titration curves because of their low basicity. By the addition of chloranil, however, diaryl-p-phenylenediamines were oxidized to the Wurster’s salts with the formation of one equivalent of base which could be neutralized by acid. A similar procedure was applicable to the determination of N,N‘diaryl-p-quinonediimines, or mixtures of these diamines and diimines. I
20 ml. of acetone for about 10 minutes at room temperature. After the addition of each drop of the diamine solution, the solvent was allowed to evaporate before the addition of the next drop. The amount of diamine placed on the filter paper in this way should be 10 to 100 fig. One drop of chloranil solution and, after drying, 1 drop of the acid mere added to develop the color which appeared at once. With dialkyl-p-phenylenediamines a reddish color was formed which deepened after the addition of acid but faded after several minutes. Alkyl-aryl-p-phenylenediamines developed a violet or blueviolet color which also usually faded after several minutes. The coloration formed by diaryl-p-phenylenediamines was blue or blue-green and was usually stable. Titration with Perchloric Acid.
T
of p-phenylenediamine derivatives used extensively as antioxidants and antiozonants in rubber compounds are N,N’-di-sec-alkyl-, N sec-alkyl-”-aryl-, and N,N‘-diaryl-pphenylenediamines. Available methods for these compounds are based mainly on a colorimetric determination of their colored oxidation products, the Wurster’s salts (2) or the p-quinonediimiines (1, 7). The deep coloration of the Wurster’s salts was used as the basis of a spot test for the rapid detection of these diamines in rubber HREE TYPES
394
ANALYTICAL CHEMISTRY
EXPERIMENTAL
Spot Test for p-Phenylenediamines.
REAGENTS. Hydrochloric acid, approximately 0.1N in isopropyl alcohol or perchloric acid in glacial acetic acid. Chloranil, 0.2y0solution in acetone. PROCEDURE. A solution containing 5 to 10 mg. of the diamine in 10 ml. of acetone was added dropwise onto filter paper with an eyedropper or small pipet. The acetone extract of a rubber compound can also be used, in which case about 1 gram of rubber was cut in small pieces and extracted with 10 to
AP-
p H Meter, Beckman Model 9600 Zeromatic with glass indicator and sleeve-type silver-silver chloride reference electrodes. Microburet, 2 ml. REAGENTS.0.1N Perchloric acid in glacial acetic acid, chloranil, hydroquinone. SOLVENTS.Acetone, glacial acetic acid, acetonitrile, isopropyl alcohol, ethylene glycol. All solvents m-ere purified by distillation except acetic acid which was reagent grade. All the p-phenylenediamine derivatives were purified materials and are listed in Table I. Most of the pphenylenediamines were prepared by R. B. Spacht and his associates. PARATUS.
