An Automatic Potentiometric Reaction Rate Method for Cystine Using

An Automatic Potentiometric Reaction Rate Method for Cystine Using the Azide-Iodine Reaction. ... Kinetics of Cystine Catalysis of the Iodine-Azide Re...
0 downloads 0 Views 547KB Size
Adjust the pH and complete the titration as instructed in the recommended procedure I

LITERATURE CITED

(1) Barnard, A. J., Jr., et ul., “The EDTA Titration,” p. 30, J. T. Baker

Chemical Co.. November 1957. (2) Bobtelsky, ’M., Rafailoff, R., Anal. Chim. -4cta 17, 308 (1957). (3) Fritz, J. S., Johnson. M., .~N.IL. CHEM.27, 1653 (1955). (4) Goldstein, G., Manning, D. L., Zittel, H. E., Zbid., 34, 358 (1962).

(5) Johnson, J. S., Kraus, K. A., J. Am. Chem. SOC.78,3937 (1956). ( 6 ) Kelley, M. T., Miller, H. H., ANAL. CHEM.24, 1895 (1952). (7) Lukyanov, V. F., Knyazeva, E. M., J . Anal. Chem. (U.S.S.R.) 15, 71 (1960). (8) M.anning, D. I,., Meyer, A. S., Jr., White, J. C., U S. Atomic Energy Comm. Rept. ORNL-1950 (Aug. 12, 1955). (9) Marsh, S. F., Maeck, W. J., Booman, G. L., Rein, J. E.: ANAL. CHEM.33, 870 (1961). (10) Morgan, L. O., Justus, N. L., J . Am. Chem. SOC.78, 38 (1956).

(11) Pecsok, R. L., Sawyer, D. T., Zbid., 78, 5496 (1956). (12) Ringbom, A,, Siitonen, S., Skrifvars, B., Acta Chem. Scund. 11,551 (1957). (13) Schwarzenbach, G., Sandera, J., Helu. Chim. Acta 36, 1089 (1953). (14) Zielen, A. J., Connick, R..E . J . Am. Chem. Soc. 78, 5785 (1956). RECEIVEDfor review July 26, 1962. Accepted October 29, 1962. Division of Analytical Chemistry, 141st Meeting, ACS, Washington, 13. C., September: 1962. Oak Ridge National Laboratory is operated by Union Carbide Corp. fGr the Atomic Energy Commission.

A n Automatic Potentiometric Reaction Rate Method for Cystine Using the Azide-iodine Reaction HARRY

L.

PARDUE and SANDRA SHEPHERD

Department o f Chemistry, Purdue University, West lafayette, Ind.

A rapid automatic method for the determination of micro amounts of cystine is described. The method i s based on the experimental observation that early in the reaction time the rate of reduction of iodine to iodide b y sodium azide i s proportional to cystine concentration. The rate of disappearance of iodine i s measured b y following continuously the rate of change of the e.m.f. of a concentration cell sensitive to iodine concentration. Commercial equipment i s easily modified to present automatically the rate data directly on a readout dial. Cystine is determined in 2-ml. samples in the concentration range from 0.25 to 25 p.p.m. The error of the method i s of the order of 0.005 pg. of cystine or 1% relative, whichever i s larger. The measurement time for a particular sample depends upon the sample concentration. It varies from about 20 seconds for high concentrations to about 200 seconds for low concentrations.

T

A x o c m s of bii-alent sulfur compounds catalyze the reduction of iodine by sodium azide. The rate of the reaction depends upon catalyst concentration and in many cases can be used for its quantitative determination. The importance of cystine in food and dairy products and in protein research has resulted in several applications of this reaction t o micro methods for cystine. Holter and Ldvtrup (1) based a method on the manometric measurement of the volume of nitrogen produced after a 4-hour reaction time. Recently Strickland, Mack, and Childs RACE

( 5 ) described a method for cystine in protein hydrolyzates based on the cxtraction and qpectrophotometric nieasurem m t of the iodine remaining unreacted after a 1-hour reaction time. These and other similar methods provide good sensitivity for cystine but the procedures are laborious and time consuming. I n the present work it has been observed that during the early part of the reaction the rate of reduction of iodine is a simple and reproducible function of cystine concentration. This property has been used to develop a rapid method for the quantitative determination of trace amounts of cystine. The method is based on the continuous potentiometric detection of the decrease in iodine concentration during the early seconds of reaction time. Automatic measurement equipment pre7 iously described for the enzymatic determination of glucose ( 3 ) is used t o provide direct readout of rate data. The reaction takes place in a small test tube which is immersed in the electrolyte of a stable reference electrode. An asbestos fiber sealed in the bottom of the test tube provides electrical contact between the reaction mixture and reference electrode without significant mixing. The potential of a bright platinum electrode immersed in the reaction mixture is compared with that of the reference electrode. Within seconds after the addition of the sample containing cystine to the rapidly stirred reagents in the sample compartment a uniform decrease in the potential of the sample electrode is observed. The time required for the cell voltage to change

over a small predetermined interval is measured and related t o cystine concentration. PRINCIPLES OF THE METHOD

The net reaction on which the method is based is

The concentrations of azide and iodide are large compared to that of iodine and remain essentially constant during the measurement interval. Under these conditions and at low cystine concentrations the rate of the reaction is proportional to the instantaneous concentrations of cystine and iodine as shown in Equation 2 .

where k l is a rate constant depending upon solution conditions, C is the cystine concentration, and [ 1 2 I t is the iodine concentration a t time t. If cystine undergoes no permanent alteration during the measurement interval then for a given value of C the reaction follows pseudo first-order kinetics. -4ny time interval, A t = t z tl, and the iodine concentrations a t the beginning, [I2I1, and end, [IZI2,of the interval are related by Equation 3. (3)

The only variable in the concentration cell is the iodine concentration in the sample solution. The time dependent VOL. 35, NO. 1, JANUARY 1963

21

component of the cell voltage is given by Equation 4. El

=

k In

[121t

(4)

where k is a temperature dependent constant from the Nernst equation. The change in cell voltage which occurs when the iodine concentration changes from its value at ti to that a t tz is given in Equation 5 .

where AE = EZ - E1 is negative since the iodine concentration is decreasing. Combining Equations 3 and 5 and rearranging gives Equation 6.

This equation predicts that the cell voltage will decrease linearly with time. I n addition, it predicts, that the time interval At required for the voltage interval AE to be overcome js inversely proportional to cystine concentration. The minus sign in Equation 6 is compensated for by the fact that AE as defined is negative. I n practice, the proportionality constant in Equation 6 is determined using a standard cystine solution. This expression then reduces to (7)

where C,, C,, At8, and At,, are concentrations and measured time intervals for standard and unknown solutions. INSTRUMENTATION

The basic instrumental components are the same as those described for the enzymatic determination of glucose (3). Only modifications introduced for this work are discussed here. Other measurement devices reliable to 1 0 . 0 2 mv. could be used to follow the change in e.m.f. of the cell if desired. Measurement System. The quantity measured is the time required for the cell voltage to overcome a small opposing bias voltage inserted in series with the cell. I n the original description of the instrument, the bias voltage was obtained from the zero adjust source of the Model Q concentration comparator. I n this work an external source of bias voltage is used. This arrangement permits the zero adjust voltage of the concentration comparator to be used to control the length of the time interval (pretime) between mixing of sample and reagents and making the rate measurement. The external source is constructed from a mercury cell, precision resistors and a 10-turn Helipot to give a source variable between zero and 10 mv. A single modification of the relay system described earlier (3) is required for the utilization of this external source. The connections between pins B and H of the concentration comparator and relay 22

ANALYTICAL CHEMISTRY

contacts b and h of the auxiliary relay system (Figure 4 of Ref. 3) are broken. The latter are connected to a shielded two-conductor cable for external connection. Concentration Cell. Openings in the concentration cell cover around electrodes, isolation compartment, etc., are sealed with paraffin to avoid volatilization of iodine from the reference solution. The reference solution is stirred mildly with a magnetic stirrer to maintain temperature equilibrium and provide a stable reference electrode potential. The samde solution is stirred bv the stirring m k o r of Unit B of the cohcentration comparator. The stirring rod also serves as the sample electrode. It consists of a 3-inch length of 3-mm. glass tubing sealed onto a 6-inch length of 6-mm. tubing. Platinum wire (22gage) is sealed into the bottom of the 3-mm. tubing so that l/4 inch of the wire remains exposed. A copper wire dipping into mercury inside the rotating electrode provides electrical contact. This arrangement with a properly conditioned electrode (2) gives potentials reproducible to within 0.01 mv. and provides rapid mixing of reactants.

within a few millivolts of the sample electrode a t zero reaction time. CYSTINESOLUTIONS.Standard solutions were prepared by dilution of a stock solution prepared by dissolving 0.0500 gram of C.P. grade lrcystine (Mann Research Laboratories, Inc., New York 6, N. Y.) in water and diluting to 1liter. PROCEDURE

Preparation of Equipment.

Units

A and B of the concentration com-

parator are connected as described in the instruction manual for the instrument. Plugs P1, P2E, and P 3 of the auxiliary relay system (Figure 4 of Ref. 3) are connected to matching sockets in the back of unit A. The timer is connected to P5 of the auxiliarv relay system. The shielded leads from contacts b and h of the relay svstem are connected across the exiern"a1 bias voltage. The sample electrode and black input lead of the concentration comparator are connected to the negative and positive terminals, respectively, of the bias voltage. The white input lead of the comparator is connected to the reference electrode. With the reagent REAGENTS selector switch in position 2 the instrument provides automatic readout of rate data. All solutions are prepared in water The reference electrode solution is purified by passing over a mixed cationadded to the thermostated reference anion exchange resin bed. All solutions compartment through a hole in the containing iodine are stored in glasscompartment cover. The hole is then stoppered bottles to avoid loss of iodine sealed with a rubber stopper. The by volatilization. All materials are sodium azide, acidified iodine-iodide (in reagent grade unless otherwise specified, a glass-stoppered bottle) and sample SODIUMAZIDE (4M). This solution solutions are adjusted to the working is prepared by dissolving 130 grams of temperature by immersion in a water sodium azide (Fisher laboratory chemibath. cal, purified) in water and diluting to The characteristics of the cell a t zero 500 ml. After insoluble material settles, time are determined by adding 0.50 the solution is decanted through Reeve ml. each of sodium azide and acidified Angel No. 202 filter paper. iodine-iodide solutions and 1.00 ml. Somuix CHLORIDE(2M) : prepared of water to the sample compartment. by dissolving 117 grams of sodium chloWith both stirrers operating, the cell ride in water and diluting t o 1 liter. voltage is measured by setting the esHYDROCHLORIC ACID (0.3M): preternal bias voltage to zero and adjustpared by diluting 24 ml. of concentrated ing the zero adjust Helipot on the comreagent to 1 liter with water. parator until the meter reads zero. POTa4SSIChl I O D I D E (2.5 x 10-2A1!f) : The prebias is adjusted by turning the prepared by dissolving 4.14 grams of Helipot clockwise 0.5 turn for the 0.25Dotassium iodide in water and dilutingto 2.5-p.p.m. range and 3 turns for the to 1liter. 5- to 25-p.p.m. range (2.00 mv. per turn IODINE (2.5 x 10-3i%f~-POTASSIUhI in P N P =tO.Ol-mv. position). The esIODIDE (2.5 X 10-2M): prepared by ternal bias is then set to 1.00 or 8.00 dissolving 0.635 gram of iodine and 4.14 mv. for the lower or higher concentragrams of potassium iodide in about 5 tion range. ml. of water and diluting to 1 liter. Reagents and samples are handled ACIDIFIEDIODINE-IODIDE SOLCTION: with tuberculin-type hypodermic syprepared by mixing 400 ml. of the stock ringes fixed with glass tips. Solutions iodine-iodide solution with 100 ml. of are-removed from t h e sample compart0.3M hydrochloric acid solution. REFERENCEELECTRODE SOLUTION: ment by an aspirator tube. The automatic sample handling equipment deprepared by mixing 500 ml. of 2M soscribed earlier (4) could be used. dium chloride, 75 ml. of stock iodineAnalysis Step. The sample comiodide, and 175 ml. of 2.5 X 10-2M partment is rinsed with deionized potassium iodide solutions and diluting water. After the rinse is completed, to 1 liter. Sodium chloride is substi0.500 ml. each of the sodium azide and tuted for sodium azide in the reference acidified iodine-iodide solutions are solution to avoid a drifting reference added. Then 1.00 ml. of sample is electrode potential resulting from a added and the automatic switch on the slow reaction between iodine and azide concentration comparator is closed in the absence of any added catalyst. momentarily. From this point the The reference electrode potential is

100-

80-

5

60t

x

I 0 K

B

~

5

05

I O IS CYSTINE CONCENTRATION (RPM)

20

10 I5 CYSTINE CONCENTRATION (PPW

2 5

RESULTS

parent increase in reaction order on the analytical method is demonstrated by consideration of the data for the 5- to 25p.p.m. range. Plotting the reciprocals of the average readout times in Table I for this range gives an exponential curve. This indicates that Equation 2 should be rewritten in the form

where n

3

Table 1. Reproducibility of Automatic Results for Aqueous Cystine Solutions

Cystine concn.,

1.

tl

p.p.ni.

tz

c-

23

/O

0.25- t o 2.3-p.p.m. range 297 285 253 139 140 140 71.7 7 1 . 3 71.0 47.3 46.6 4 7 . 1 26.9 27.1 26.9

0.9 0.5 0.5 0.7 0.5

5- t o 25-p.p.m. range

Equation 6 then becomes (9)

LIS.

Rel. std. - dev.,

Direct time readout, sec.

0.25 0.5 1.0 1.5 2.5

AND DISCUSSION

Cell Response. T h e potentiometric response of t h e cell is rapid. Reliable rate measurements can be made within a few seconds after addition of reagents a n d sample t o the sample compartment. Recorded curves of cell voltage time are linear as predicted by Equation 6. Quantitative Data. Table I shows typical d a t a for two concentration ranges as read directly from t h e timer of t h e automatic measurement system. T h e d a t a within each range were taken at random over s 3-hour period. They show relative standard deviation for the method well within 1%. Results agree to n-ithin 2% over periods of several days if the same conditions are repeated carefully. The reciprocals of the average readout times are plotted us. cystine concentration for the lower range in Figure 1. Below 1.5 p.p.m. the plot is linear with zero intercept as predicted by Equation 6. Within this range unknown concentrations are determined by comparison with a single standard using Equation 7 . Results computed on this basis for the data in Table I are shown in Table I1 where the 2-1g. (1 p.p.m.) sample is selected as the standard. TT'ithin the indicated range Equation 7 is valid to within about 17' relative. Above 2 p.p.m. cystine, the reaction order with respect t o cystine increases gradually. This accounts for the large positive error obtained in the 5-1g. sample. The effect of this ap-

25

Figure 2. Working curve for cystine determination in exponential range

Figure 1. Working curve for cystine determination in proportional range

instrument completes the measurement automatically. After the time interval is measured and recorded, the timer is reset t o zero and the system is ready for the nest sample. Gnknown cystine concentrations are calculated as shown in the nest section or are read directly from a working curve.

'1

20

which states that C is proportional to the n t h root of the reciprocal of the time interval. The value of n is evaluated as the slope of the line ob1 tained from a plot of log C us. log atUnder the conditions of this work n is 1.24 for the 5- to 25-p.p.m. range. Figure 2 demonstrates the agreement of the experimental data in Table I with Equation 9. The numerical agreement is shown in Table I11 where Equation 7 (modified t o account for the nth root of At, and Atu) has been used to compare the other standards with the 20-pg. (IO-p.p.m.) standard. Agreement is well within 2%. It should be emphasized that the value of n depends upon experimental conditions and increases gradually with cystine concentration. The value of 1.24 merely represents an average for the fivefold concentration range discussed here. For a given set of conditions, n is reproducible and the nth root of reciprocal time is obtained easily from a plot of &)n us. AL At present, insufficient kinetic data are available to explain the apparent increase in reaction order with cystine

5 10 15 20 25

120 122 121 52.6 53.1 5 3 . 4 32.2 3 2 . 6 32.2 2 2 . 1 22.4 2 2 . 1 16.4 16.5 1 6 . 5

0.5 0.8 0.7 0.6 0.4

Table 11. Automatic Cystine Results Based on a Single Standard (Proportional Range)

0

3 50

0.50

7.i4 14.0 21.3 37.2

2.00 3.00 5.00

i.00

0.0 2.0 0.0 1.3 6.5

0.50 1.02 3.04 5.34

Based on 2 - p g . sample as a standard.

Table 111. Automatic Cystine Results Based on a Single Standard (Exponential Range)

1.24th Root fig. Cystine in

reciprocal 2-ml. sample time x l o 3 Taken Founda 21.0 40.9 61 82 104

(

a

10.0 20.0 30.0 40.0 50.0

Rel. error, yo

10.2

...

29.8 40.1 50.8

2.0 0.0 0.6 0.3 1.6

Based on 20-pg. sample as a standard.

VOL. 35, NO. 1, JANUARY 1963

23

concentration. This problem is being investigated, and results will be reported a t a later date. Temperature Dependence. The dependence of the reaction upon temperature was investigated between 20" and 30" C. Within this range the reaction rate increases by about 6% per degree increase in temperature. T h e working temperature is controlled a t 25' + 0.05' C. to avoid errors resulting from temperature fluctuations. p H Dependence. The cystine catalyzed reaction shorn a marked dependence upon solution p H . It exhibits a sharp masiniuni in t h e range of 5.8 to 6.0. This is in agreement with t h e findings of Strickland, Mack, and Childs ( 5 ) . This work Ivas carried out a t a p H of 6.0.

Interferences. Reaction 1 is selective for sulfur-containing compounds and therefore can serve as a convenient means of differentiating between these and other types of compounds. For example, alanine, leucine, histidine, and glycine do not interfere when present in concentrations up to 0.5 gram per liter. I n many cases this reaction will not differentiate between two compounds containing sulfur. For example cysteine is slightly more active as a catalyst per unit of sulfur than is cystine and will interfere if present. Oxidants or reductants which react with iodine or iodide will interfere if present in concentrations exceeding about 10-4M. Methionine falls into this category since it reduces iodine rapidly but does not catalyze Reaction 1. Metal ions which form insoluble salts

with iodide interfere. The ions C a t 2 , Zn+Z,Co+?, and Cd+z do not interfeie when present at concentrations up to 10-3.11. More detailed investigations of interferences are left for future investigations of specific applications of tire method. LITERATURE CITED

(1) Holter, H., Llbvtrup, S., Compt. Rend. Trav. Lab. Carlsberg 27, 72 (1949). ( 2 ) Malmstadt, H. V., Pardue, H. L.,

ANAL.CHEY.32, 1034 (1960). (3) Ibid., 33, 1040 (1961). (4) Malmstadt, H. V., Pardue, H. L., Clin. Chem., in press. (5) Strickland, R. D., Mack, P. A., Childs, W. B., ANAL.CHEM.32,430 (1960). RECEIVEDfor review August 13, 1962. Accepted November 13, 1962. 15th Annual Summer Symposium on Analytical Chemistry, College Park, Md., J u n e 1962.

Effect of Temperature on Spectrophotometric Measurements of Molybdenum(V) Solutions S. Z. MlKHAlL and D. F. PADDLEFORD Department o f Nuclear Engineering, Kansas State University, Manhattan, Kan.

b

Beer's law holds for Mo(V) solutions below 0.0 1 N between room temperature and 55" C. The rate of change of absorbance with concentration increases linearly with temperature, but the position o f the absorption peaks and the mode of increase of absorbance with acidity are not affected. Temperature increases the absorption at every wavelength, but the rate of increase is wavelength-dependent. Acidity increases the temperature dependence of absorbance, at a rate linear with Mo(V) concentration. An equation relating absorbance of Mo(V) to temperature and concentration has been developed, which makes it possible to calculate the Mo(V) concentration at any temperature and concentration in the range investigated. Two methods of solving for the constants arising in the equation have been suggested.

T

effect of x-ray irradiation on quinquevalent molybdenum was investigated. Irradiated solutions were to be analyzed spectrophotometrically immediately after irradiation to avoid radiation aftereffects. During irradiation the temperature of the samples increased; hence, it was necessary to test the effect of temperature on the light absorbance of the Mo(V) solutions. Preliminary results showed that in HE

24

ANALYTICAL CHEMISTRY

some cases the light absorbance increased 50% when the temperature of the solution was raised 20" C. To the authors' knowledge no systematic work along this line has been reported. Hiskey and Meloche ( 2 ) studied the color phenomena associated with R4o(V) in HCl solutions a t room temnerature

Preparation and Standardization of

Mo(V) Solutions. Quinquevalent molybdenum solutions were prepared from ammonium molybdate according t o t h e method suggwted by Furman and Murray ( I ) , based on reduction by mercury in 3N HC1. The i\fo(V) solutions were mechanically shaken for 15 minutes in a mercury reductor, then diluted with 3N HC1 to approximately 0.02M. They were analyzed by titration with a standard solution of cerium (IV) sulfate (trisulfatocerate) using ferroin as indicator and sirupy phosnhoric acid as a catalvst. as suggested by Rao and SuryanarIayana (ST- The standard cerium(1V) sulfate solutions

were standardized against sodium osalate (4) supplied by the Xational Bureau of Standards. Spectrophotometry. A Beckman Type DC spectrophotometpr and 1- x 1- X 4.8-em. Corex cells were used. The light absorption of a solution a t different temperatures was nieawred in a n indirect way. Each l I o ( V ) solution t o be tested mas heated to

clock was started, the thermometir removed, and the cell compartment cover put in place. Absorbance data a t a fixed ivavelength were then collected and the exact corresponding cooling time was recorded. X typical curve obtained in this manner is shown in Figure 1. The procedure was repeated a t several wavelengths from 350 to 450 m p . This range was selected on the basis that it included the absorption peaks corresponding to the HC1 acid normalities of the XIo(V) solutions inyestigated (8). From the cooling curves and the time us. absorbance curves the corresponding absorbance us. temperature curves n-ere obtained. A