the digitizer from taking any action on a positive peak. The entire digitizer ,Y controlled by two clocks, a 60-cycle clock and a 100-kc. clock. The 100-kc, clock controls the digitizing rate, the digital voltmeter, operates the digital comparator, the greater-than and less-than loglc gates, and the register. The 60-cycle clock is used to control the logic circuitry of the bystem and is the fundamental time base for decision making. Due to the widths of the ion peaks (minimum of 70 milliseconds within 2% of the top) no error in measurement occurs. The 60-cycle time base also provides inherent rejection to 60-cycle interference. If desired this clock speed can be increased to several thousands cycles per second. Installation and Startup. The mass spectrometer digitizer is quite easy t o install. There are no modifications to make t o the mass spectrometer amplifier. The amplifier output signal and a 0- t o IO-volt t a p of the acceleration voltage must he supplied t o the digitizer input. Both of these signals are filtered by a pi RC filter which reduces the t o t d noise level to less t h a n 1 mv. peak-to-peak. -1fter the digitizer has been connected and is operating properly, the next step
is to determine K factors for each magnet range that will be used. The equation for mass number is: M / e =
Rz
which can be simplified to J l l e = E where K = R2B2 and B is fised for a 2 particular range. Therefore, K = E X mass number. Since the digitizer always reads only the acceleration voltage from 9.999 down to a lesser value, it is necessary for the operator.to indicate which magnetic range is in use. The different K factors can be determined by averaging the K factors from known sample runs. Figure 3 illustrates the output data format and Table I illustrates tabular values of mass numbers us. voltage. AI?,
K
PERFORMANCE
The solid state digitizer has been in use for 11 months. In that time, the operation yielded the following results; per cent onstream, 98; per cent utility, 99+. A labor saving of one operator per shift and a reduction in cost per sample by 570/, were realized. The capacity of the lab, in samples per month, increased by 66y0 to 2000 samples. The
estimated annual savings, based upon the results thus far, should be $50,000. As shown by Table 11, the precision in analysis was improved by a factor of 4. Mass number reproducibilities were hO.1 mass number up to mass 125 and 3~0.25mass number up to mass 250. The instrument has a dynamic range of 104. Input signals may vary from 1to 9999 mv. Minimum detectability is 1 mv., and the system is linear across the complete span. The digitizer, a photograph of which is shown in Figure 4, is unaffected by large voltage swings from the mass spectrometer signals and can accept ranges of over 600y0. ACKNOWLEDGMENT
The author gratefully acknowledges the assistance of L. Y. Saunders and S. P. Scarcella, and full credit for the design of the digitizer goes to Henry Reinecke of Non-Linear Systems, Inc., Del Mar, Calif. RECEIVEDfor review June 24, 1963. Accepted August 29, 1963. Presented at the ASTM-E14 Meeting, San Francisco, Calif., May 1963.
UIt ra rnic roldete rmin atio n of Iodine by a Rapid Automatic Reaction-Rate Method H. V. MALMSTADT and T. P. HADJIIOANNOU’ Department of Chemistry and Chemical Engineering, University of Illinois, Urbana, 111.
b An automatic spectrophotometric reaction rate method for the ultramicrodetermination of iodide i s based on the Sandell-Kolthoff reaction, in which a trace of iodine or iodide acts as catalyst for the reduction of Ce(lV) in the presence of As(lll). The time required for the reaction to consume a small fixed amount of ceric ions, and therefore for the absxbance to decrease by a preselecteld amount (about 0.06 unit), is measured automatically and related directly to the iodide concentration. Ultramicro amounts of iodide in the range of 0.015 to 0.45 pg. were determined with relative errors of about 1 to 25% and measurement times of only about 10 to 100 seconds. The method s’1ould be adaptable for the determination of proteinbound iodine (PBI).
T
HE method presented here utilizes the Sandell-Kolthoff reaction (15), in which a trace of either iodine or iodide acts as a catalyst for the reduction of Ce(1V) in the presence of As(II1). This reaction has b3en widely investigated because of its great impor-
tance for the determination of proteinbound iodine (PBI) in serum (3). The overall reaction is summarized in Equation 1.
iodine
2 Ce(1V)
+ As(II1) catalyst 2 Ce(II1)
+ As(V)
(1)
There have been many studies (11) to determine the reaction mechanism. Kolthoff and Sandell developed a quantitative procedure for iodide by measuring the time required to reduce all of the Ce(IV), as indicated by a visual color indicator. I t was found that the iodide concentration was inversely proportional to the measured time for total reduction. In subsequent procedures the time for the measurement was decreased by not waiting for complete reduction of cerium(IV1. I n one basic procedure ( I , 2, 5, 9) the Ce(1V) remaining after a fixed time (usually 20 minutes) is determined by an absorbance measurement a t a selected wavelength characteristic of Ce(IV), usually between 350 and 420 mp. Since the absorbance changes continuously, it is necessary to
read the absorbance value a t the exact preset time. A plot of absorbance us. iodide concentration provides a suitable working curve. I n another procedure (7, 8, 10) the reaction is stopped by adding a reagent that reacts immediately with Ce(1V) to reduce the remaining Ce(1V) quantitatively to Ce(II1). One suitable reagent is brucine, which is oxidized to a colored reaction product. A plot of absorbance of the colored reaction product against iodide concentration provides a working curve. I n another procedure (4) the change of absorbance of Ce(1V) was recorded continuously and the slope of the recorded curve was related to the iodine concentration. To simplify and automate the iodide determination, a new procedure provides a digital readout value within a few seconds after start of the reaction. For example, extremely low concentrations of iodide in the range of 0.015 to 0.45 pg. per 4.25 ml. of solution were Present address, Department of Chemistry, College of Pharmacy, University of Illinois, Chicago, Ill. VOL. 35, NO. 13, DECEMBER 1963
e
2157
determined with relative errors of about 1 to 2% and measurement times of only about 10 to 100 seconds. This method utilizes the type of automatic spectrophotometric reaction-rate system recently used for the determination of glucose (la),alcohol ( I S ) , and amino acids (14). The automatic reaction-rate measurement method was tested for the determination of PBI in digested serum samples, and the results indicated that the procedure should be readily adaptable for this important determination. The method used for releasing the iodine prior to measurement was similar to time-tested procedures; the serum proteins were precipitated with zinc hydroxide and the iodine was determined in the protein precipitate after incineration in the presence of potassium hydroxide. The results obtained were close to the assigned values of commercially available control sera. GENERAL CONSIDERATIONS
Reaction Mechanism. There is some disagreement about the mechanism of the ceric-arsenite-iodide reaction (6, 15). I n the absence of chloride ions and a t very low iodide concentrations there is no direct proportionality between catalytic activity and iodide concentration. I n the presence of chloride, the linear relation is fulfilled and the rate of the reaction is pseudofirst order with respect to iodide. I t is assumed that the arsenite is in large excess and the starting concentration of cerium(1V) is the same for each sample. The rate of reduction of ceric ions, and the resulting rate of decrease of absorbance of the solution, are therefore proportional to the iodide concentration. Interferences. Reducing or oxidizing agents should be eliminated prior to the measurements; silver or mercury in trace quantities which reduce the concentration of the iodide, and osmium and ruthenium, which catalyze the reaction in the same manner as iodide ( I @ , interfere seriously. Since the rate of the reaction may be affected by several reaction-stimulating or reaction-inhibiting substances (17), the composition of the standard iodide solutions should be similar to that of the samples. Contamination. Precautions should be taken to avoid contamination of reagents and glassware by iodine and mercury, since they provide the two most serious and frequent sources of error. Iodide adsorbed on glass is not easily removed by rinsing with water. All glassware should be thoroughly cleaned with sulfuric aciddichromate cleaning solution, followed by repeated rinsings with deionized water. Glassware used for the iodide determination should be reserved for 21 58
ANALYTICAL CHEMISTRY
this purpose only. It is advantageous to perform the iodine determination in a separate room away from mercury and iodine compounds to prevent contamination. All reagent chemicals should be tested for their iodine content and only lots with a minimum iodine content should be used. To keep the blank small, the concentration of reagents should be kept to a minimum for satirfactory reactions. For example, chloride ions should be added by using the purest available source (hydrochloric acid), and only the minimum amount should be added that is necessary for chloride to exert its full accelerating effect on the reduction of ceric. Optimal Concentration of Reagents. 1-arious initial ceric concentrations can be used. For the procedure described the concentration of ceric in the measured solution is 0.0006N. With the 16-mm. i.d. cylindrical cell and a navelength of about 390 mp only about 5y0 of the incident light is transmitted through the cell to the detector. At higher ceric concentrations the rate of reaction is increased but the decrease of transmitted light causes a decrease of signal-to-noise ratio a t the output of the photoconductive detector. Although various instrument modifications could improve the magnitude of the output signal for higher concentrations of ceric. it was not comidered worthwhile for this application. The accelerating effect of chloride ions (16) on the catall tic reaction for iodide is more pronounced a t low iodide concentrations (5 to '25 p.p.b.). It increases with increasing concentration of chloride, as chloride concentration rises from zero to 0.3S, but further increase of chloride concentration has but slight effect in accelerating the reduction of Ce(1V). Hydrochloric acid is the best source for chloride ions, because other sources often contain cations that inhibit the reaction and are usually more contaminated with iodide than HC1. The final acidity of the Ce(1V)arsenite mixture should be larger than 0.5.V to prevent precipitation of reduced cerium salts. Final acid concentrations up to 2.5N have been recommended (16). However, such concentrated solutions present mixing and heat problems. The final concentration of sulfuric acid in the proposed method is 0.6s. Order of Adding Reagents. I n the usual procedures using the SandellKolthoff reaction the ceric solution is added t o a solution containing the iodide sample and arsenite reagent. I n the rate method described here the arsenite is added to a solution containing iodide sample and ceric reagent. I n this way the zero adjustment on the Spectro unit can be made before starting the reaction. Any
small change in initial absorbance, AI (before the injection of arsenite), is compensated by adjusting the balance control in the Spectro unit. As soon as the arsenite reagent is added, because of dilution, the absorbance changes to a new value. Az. The instrument measures only the time required for a small preset change in absorbance (A3 - A , equal to about 0.06) to occur during the early part of the reaction. It is only the absorbance change (A3 - Aq) and not the absolute values of A ) and A , that are important for the measurement. The time required for the absorbance to reach the value A3 is not measured and can vary from sample to sample. Temperature Control. The reaction rate has a large temperature coefficient and therefore the reaction takes place in a thermostated cell a t 25' =t 0.1' C. To ensure thermal equilibrium and dissipation of the heat liberated by mixing of the sulfuric acid with the iodide solution, the mixture is stirred for 1 minute prior to starting the reaction by the addition of the arsenite solution. Premeasurement Times. After initiation of the reaction, a minimum premeasurement time of 5 t o 10 seconds is desirable to ensure thorough mixing of the reagents. The premeasurement time is controlled by appropriate setting of the comparator zero adjust. For example, the zero adjust was set at 5.80 to 5.90 for the 5- t o 50-p.p.b. iodide range. A higher comparator setting, 6.30 to 6.40, waq necessary for the higher concentration range nhere the reaction is faster. Larger zero adjust settings for the same iodide concentration range result in larger consumption of ceric during the premeasurement time and consequently in longer measurement time, but they do not affect the accuracy and precision of the results. The zero adjust setting may be varied, but that decided upon must be duplicated carefully from sample to sample, t o secure starting of measurement time a t the same ceric concentration. Preparation of Standard Curve. Although the rate of the reaction under selected conditions is proportional t o the iodide concentration, the standard curves (straight lines) obtained when reciprocals of measurement times are plotted against iodide concentration d o not pass through zero because of iodide present a s contaminating agent in the reagents used. Therefore, standard curves are rapidly established by using three to four standards which were prepared using the same reagents as for the unknowns. P B I Determination. The automatic rate method was applied t o the determination of P B I . The dry-ashing procedure (9) was used for the destruc-
tion of protein and the separation of iodide, but the method could readily be adapted to any of the sample preparation procedures now wed for PBI d e termination (9). INSTRUMENTATION
The basic instrumental components are the same as those used for determination of glucose (12) and alcohol ( I S ) . Since in the ceric-arsenite reaction the absorbance of the solution decreases as the reaction proceeds, the leads to the 1.5-volt battery in the bridge circuit which provides the Spectro balance control should be in reverse position to that previously used ( I S ) . The Sargent Rlodel Q-RR automatic reaction rate adapter is used in combination with the Rfod1:l Q comparator as control unit. The temperature is controlled a t 25" i 0.1" C. A narrow third-ordc r transmittance band a t 390 mp is selected by dialing the nominal 575-mp second-order interference filter on the Spectro unit and inserting a Corning KO.5970 filter in the auxiliary holder. REAGENTS
All reagents are prepared in deionized water. The iodide solutions are kept in glass-stoppered amber bottles. Sodium hydroxide, 0.5N, 20 grams of NaOH per liter of solution. Zinc sulfate septahydrate, 10% solution in water. Ten milliliters of this solution are diluted with 50 to 70 ml. of water and titrated with the sodium hydroxide using phenolphthalein as indicator; 10.8 to 11.2 ml. of titrant should be needed. Potassium hydroxide, 2 N , 112.2 grams of KOH per liter of solution. SULFURIC-HYDROCHLORIC ACID REAGENT. Solution A. Concentrated H&04 (70 ml.) i: add(hd to 100 ml. of water in a I-liter volumetric flask. After the mixture is cooled, 125 ml. of concentrated ISCl ar3 added, and the solution is diluted to the mark with water and mixed. Thi: solution is 1.5-V in €IC1 and 2 . 5 5 in H2EO4. Solution U. Soluticn A is diluted with HnO (1 to 9). Sodium arsenite. 0.15-V. As909 (7.5 gram
