REPORT FOR ANALYTICAL
CHEMISTS
The Role of the Electroanalytical Chemist in Biochemical Research by William C. Purdy Department of Chemistry,
r
piIE
ANALYTICAL
University
CHEMIST
can
play an i m p o r t a n t role in bio chemical research. H e has a t his command m a n y tools which can be applied effectively to the solution of biochemical problems. To illus t r a t e the topic, two electroanalyti cal methods have been chosen, polarography and constant current coulometry. Examples will be t a k e n from the work of Brdicka, Otto Miiller, and our own labora tory. The analytical chemist is a queer breed of animal. E a r l y in his training he is t a u g h t t h a t accept able accuracy is within 0 . 1 % . H e works with "clean" solutions and tries to study systems which are uncomplicated. I t comes as a rude awakening when the analytical chemist is faced with his first bio logical sample. T h i s is a " m o m e n t of t r u t h " and if he is willing to accept the challenge, the entire field of analytical biochemistry is open to him. T h e a u t h o r speaks from experi ence on this subject. Several years ago he became associated with the Division of Biochemistry of one of the major medical research institu tions in this country. A t first he could not see how an analytical chemist (with no biochemical t r a i n ing) could be of any help to a group of biochemists. H e quickly learned, however, t h a t he could pro vide a fresh viewpoint to m a n y
of Maryland,
College Park,
Maryland.
problems and, more important, he was able to bring to bear analytical techniques which were virtually un known to the American biochemist. Two of these techniques are polar ography and constant current cou lometry. I t might be mentioned a t this t i m e t h a t t h e E u r o p e a n bio chemist has been made aware of these methods principally by the efforts of Brdicka and through the monograph entitled " P o l a r o g r a p h y in Medicine, Biochemistry, and P h a r m a c y " by Brezina and Zuman.
POLAROGRAPHY, TRACE METALS, AND METABOLISM
Biochemists are becoming in creasingly aware of the importance of t r a c e metals and the role these metals p l a y in various metabolic systems. To elucidate this role, however, it is first necessary to know the effective concentration limits for the metal ion. This poses a very difficult problem to the analytical chemist for he is often asked to determine submicrogram amounts of material· in the pres ence of equal or greater concentra tions of m a n y other metallic ions. Spectrophotometric methods often possess this sensitivity, b u t in m a n y cases, they do not have the spec ificity of the electroanalytical method, and, in particular, the polarographic method. For this
reason, m a n y metallic ions in bio chemical systems have been deter mined by classical polarography. These include cadmium, chromium, cobalt, copper, iron, manganese, nickel, selenium, tin, v a n a d i u m , and zinc. I n addition, polarography has been applied to the determina tion of m a n y toxic substances, such as lead (18) and thallium.
Trace Selenium and Vitamin Ε Utilization
Selenium has become a trace ele m e n t of increasing importance. In moderate concentrations it is the toxic agent in "loco weed" which causes trouble to the rancher in our own Southwest. Hartley (16) demonstrated the importance of this element in t h e prevention of "white muscle" disease in sheep and Schwarz (24) has suggested t h a t selenium is an essential trace ele m e n t for some animals. I n fact, Schwarz has found t h a t selenium is the " F a c t o r 3 " needed for the utili zation of vitamin E. White muscle disease can be caused by the re moval of either selenium or vitamin Ε from the diet. T h e restoration of the removed compound will reverse the disease if it has not been al lowed to proceed too far. This would suggest t h a t there is some in teraction between selenium and v i t a m i n E. Experiments have VOL. 36, NO. 4, APRIL 1964
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demonstrated t h a t selenium is necessary for the utilization of vitamin E. You might ask the question, why do we worry about white muscle disease in sheep? T h e answer is t h a t there is a similarity between white muscle disase in sheep and muscular dystrophy in humans. One more interesting fact should be mentioned a t this point. Brewers' yeast and torula yeast are two sources of selenium. Brewers' yeast is effective in reversing white muscle disease whereas torula yeast is not. This would suggest t h a t the selenium in the two yeasts exists in different oxidation states and t h a t one oxidation state is effective. In point of fact, the + 4 oxidation state, the oxidation state of the selenium in Brewers' yeast, is the effective form.
P o l a r o g r a p h y of S e l e n i u m
We have recently investigated the polarography of selenium in hopes of resolving some of the confusion in the literature with regard to the polarographic behavior of + 4 selenium (8). There are three polarographic reduction waves for selenium (IV) depending upon the p H of the supporting electrolyte. The limiting currents of all these waves are diffusion controlled. Only the second wave is reversible. The first wave corresponds to a net four-electron reaction with the formation of mercuric selenide as the product. The selenite ion is reduced to selenide which in turn reacts with the mercury of the electrode to form mercuric selenide. There is also a slow reaction between the selenide and selenite ions to form metallic selenium. The second wave is due to the reduction of the mercuric selenide to mercury and hydrogen selenide, a two-electron change. In basic medium, the third wave corresponds to the reduction of selenium (IV) to the selenide state. For the purposes of analysis, the neutralized or slightly acid solution of the selenium (IV) sample is dissolved in the buffer electrolyte of p H 2.0 to 2.5. I n this medium, the first wave is a t a maximum height and no maximum suppressor is needed. A n y interference from
copper is easily eliminated by extraction of the buffered solution with dithizone in chloroform. Copper can act as an interference since the half-wave potential of the first selenium wave is about 0.030 volt vs. SCE. During this extraction about 2 % of the selenium is lost but this is of little consequence if a calibration curve is prepared under identical conditions. With this method we have been able to determine concentrations of selenium of the order of about 10~SM. A particular difficulty in this determination is the digestion of the sample, and we have been developing methods for recovering 100% of the selenium in the sample. W e have also been able to extend the sensitivity of the method for the determination of selenium (IV) into the submicrogram region, b u t our results to date are only semiquantitative. The method employed, however, does not come within the scope of this article. Cobalt-Catalyzed Protein Double
Waves
I n 1933 Brdicka (4) was studying the reduction of cobaltic salts in a supporting electrolyte of 0.1M a m m o n i a - 0 . 1 M ammonium chloride. He observed a maximum on the second wave which occurs a t a potential of about —1.1 volts and is due to the reduction of cobaltous ion to the metal. To suppress this maximum, Brdicka added serum. The new polarogram contained two more waves which Brdicka called the "protein double wave." The first of these waves is normal in a p pearance but the second had a peak. Since the protein double waves are not obtained in the absence of cobalt, the waves are cobalt catalyzed. The underlying reaction involves the catalytic reduction of hydrogen ion from the buffer solution. Brdicka investigated the factors which determined the magnitude of the double wave and found t h a t a plot of the height of the protein wave vs. the concentration of the protein was not a straight line but rather a curve approaching a limiting value. Boiling the serum in neutral ammonium chloride medium to coagulate the protein, gave a clear supernate which also exhib-
REPORT FOR ANALYTICAL CHEMISTS
Dropping mercury electrode apparatus is adjusted by University of Maryland chemist for studies of the reaction between zinc and certain antipyritic (fever reducing) substances such as aspirin and thioaspirin. Certain metal ions such as copper and zinc are known to be important coenzymes for oxidative processes which cause high fever in the body. Polarographic methods are valuable in determining dissociation constants and the nature of complexes formed between metal ions and biologically active sub stances
ited the catalytic wave. Brdicka concluded that some material other than protein was responsible for the wave. A study of a number of amino acids indicated that the sul fur-containing amino acids, cysteine and cystine, gave these waves whereas glycine, creatine, creati nine, leucine, and tyrosine, did not. He concluded that the double wave was caused by the sulfhydryl and disulfide groups in the protein. Cysteine and cystine only pro duce catalytic waves in solutions containing divalent cobalt. Pro teins produce double waves in either cobaltous or cobaltic solutions. The waves of cysteine and cystine are identical if the latter compound is present at half the molar concen tration of the former compound. This is because the reduction of the disulfide group at the dropping mercury electrode (DME) takes place at a potential more anodic than the double wave. Distinction
can be made between cysteine and cystine by the addition of potas sium iodoacetate to the solution. This compound in alkaline medium forms a stable complex with cys teine but leaves the cystine intact. Bonting (2) has used this reaction to study the percentage of disulfide bonds in the skin of adult and young rats. Attempts at relating wave heights to cysteine concentration proved futile. Brdicka (3) incor rectly assumed that the second wave was due to adsorption of divalent cobalt and he employed the Langmuir adsorption isotherm to obtain a concentration-wave height rela tionship. Other workers have tried a similar approach. Miiller (21) investigated the catalytic cysteine wave to see. if the wave heights were a function of the surface area of the mercury drop. This would be the expected rela tionship if one is dealing with a
William C. Purdy is associate pro fessor of chemistry at the Univer sity of Maryland, College Park. He is the author or coauthor of some forty publications dealing with the fields of nonaqueous titrimetry, polarography, coulometry, separa tion techniques, and the applica tion of electroanalytical methods to biochemistry. Dr. Purdy was born in Brooklyn, Ν. Υ., in 1930. He received the B.A. from Am herst College in 1951 and the Ph.D. from Massachusetts Insti tute of Technology in 1955, where he worked with Professor David N. Hume. After spending three years as an instructor at the Uni versity of Connecticut, he joined the chemistry department at the University of Maryland as an as sistant professor in 1958. He was promoted to associate professor in 1960. Dr. Purdy has been con sultant to the Surgeon General's Office, U. S. Army, since 1959. He is a member of the American Chemical Society, the Society for Analytical Chemistry (London), and Sigma Xi.
VOL. 3 6 , N O . 4 , APRIL 1964
•
31
A
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Amperometric setup is used to follow reaction of uric acid with the enzyme, uricase, by chemist at the University of Maryland. The reaction forms hy drogen peroxide which reacts with iodide ion to form iodine. The for mation of iodine is followed amperometrically, and the concentration of reacted uric acid is calculated from the amount of iodine formed catalytic wave. For cysteine con centrations between 2 χ 10 7 Μ and 10~3M in O.liVf ammonia—ammo nium chloride, the catalytic current was proportional to the surface area of the mercury drop. The currents were not proportional to the con centration of the divalent cobalt. Miiller and Davis (23) suggested t h a t the only suitable method for reporting these data was to plot the current density vs. the molar con centration of cysteine. For pro teins, the method of reporting is a plot of current density vs. the loga rithm of the protein concentration in grams per 100 ml. I t is only through the use of master curves prepared in this way t h a t the data of different workers can be com pared. Cancer Diagnosis with the Protein Double W a v e
A further study of the catalytic cysteine wave and of proteins which on hydrolysis yielded either cys teine or cystine hydrolysates, led to an interesting discovery. There was a difference in the n a t u r e of the double wave for sera of normal and cancer patients (5). These differ ences could be exaggerated by denaturation of the proteins with po tassium hydroxide or by peptic di gestion or by t r e a t m e n t with sul-
REPORT FOR ANALYTICAL CHEMISTS
WE KNOW OF NO S U B S T I T U T E FOR PROVEN PERFORMANCE
LABORATORY ACCURACY IN A PORTABLE SALIN0METER fosalicylic acid (6). As so often is the case with a discovery of this sort, a number of people have tried to apply this method. I n m a n y cases the work was done by inves tigators who had little knowledge of polarography. As a result, the literature abounds with m a n y "im proved" methods, but there is no uniform method of expressing the results. Widespread though this application to cancer diagnosis is, this method is no better, nor worse, t h a n a n y other test. All cancer tests seem to measure similar blood proteins or their degradation products. T h e denaturation of plasma from normal and cancer patients causes an increase in bo~h parts of the double w a v e ; the increase is a func tion of the time of treatment. I n order t o give a measure for com parison between normal and cancer patients, a "protein index" was de fined (22). T h e protein index is determined a t the same D M E , tem perature, drop time, etc. and is the ratio of t h e polarographic wave heights of the two tests, the digest and the filtrate test. I n the digest test, the oxalated plasma is treated with potassium hydroxide and al lowed to stand a t room temperature for 30 minutes before it is added to
Brdicka's solution and the polarogram is run. A normal value for the digest t e s t i s 16.7 μ&. / m m . 2 The filtrate procedure begins in the same manner as the digest test. After allowing the oxalated plasma in t h e presence of potassium hy droxide to stand for 30 minutes at room temperature, the solution is treated with sulfosalicylic acid. T h e resulting precipitate is allowed to stand in contact with the filtrate for exactly 10 minutes. The mix ture is then filtered, and 0.5 ml. of the clear n i t r a t e is added to the cobaltic buffer solution (0.001Λ/ hexamminocobaltic chloride, 0.1Λ/ ammonium chloride, and 0.8.1f am monia) and the polarogram is run. Normal values for the filtrate test are 2.72 μα./mm.2 The protein index for normal in dividuals is a b o u t 2.4 ± 0.17 and is obtained by dividing the value for the filtrate test by the value for the digest test and multiplying by an arbitrary constant of 15. New born infants have a low protein in dex; otherwise, the protein index is independent of the age of the indi vidual. Cows and r a t s have about the same protein index as man, r a b bits have a lower index, and dogs, horses, and monkeys have a higher index t h a n man.
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REPORT FOR ANALYTICAL CHEMISTS
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Cancer a n d certain other infec tious diseases cause a n increase in the protein index. Cirrhosis of the liver gives a lower t h a n normal pro tein index. W e still do not know what we are measuring, b u t we do know t h a t these diseases cause a deviation from t h e normal values for the protein index. Certainly this is a field full of possibilities and a systematic study might yield significant results which would have great medical importance.
Effect of Chromium(lll) on Diabetes
One of the interests of our labo ratory has been the role of trace metals in intermediary metabolism. We have found that certain electroanalytical techniques can be em ployed to shed light on the role played by these trace metals. One of the important techniques is, of course, polarography. As a n exam ple of the application of this tech nique, consider the problem of dia betes. There are two main types of this disorder, one where there is complete destruction of the func tion of t h e pancreas a n d the second where sufficient insulin is produced, but the system cannot utilize it. I t is this second type, or later-onset diabetes, t h a t will be used as an example. I t has been found by Schwarz and Mertz {25) t h a t the presence of trace quantities of chromium ( I I I ) greatly increases the glucose uptake of the animal. Mertz (19, 20) has postulated t h a t this is due to t h e ability of the chromium ( I I I ) to help in the laying down of the insulin on the cell membranes, thereby allowing the glucose to pass through the cell membrane. W e proposed to study this problem by seeing whether chromium ( I I I ) did form a complex with insulin and with cell membranes. T h e mito chondria were chosen as a model system for this study. T h e mito chondria arc those portions of the cell which contain the enzymes for the oxidative phosphoralation reac tions by the cell and they have been used to study many enzymatic processes. Evidently, t h e mito chondrial membrane exerts consid erable selectivity on the ions and
molecules t h a t will pass into t h e mitochondria. The mitochondria were prepared by ultracentrifugation of rat-liver cells. They were suspended in a solution of deionized sucrose a n d studied in a supporting electrolyte of molar potassium nitrate. A p o larogram of the suspension indi cated t h a t there was a wave a t a p proximately —0.290 volt vs. S C E {11). T h e height of t h e wave was directly proportional to the concen tration of the mitochondrial suspen sion, and t h e wave appeared to b e diffusion controlled. On standing a t room temperature for a prolonged period, a second minor wave a p peared which h a d a half-wave p o tential of —0.420 volt. T h e same waves were obtained for mitochon dria from r a t liver, kidney, a n d brain, and from dog liver. A polarogram of mitochondrial suspension in Brdicka's solution ex hibits a typical protein double wave. T h e height of the double wave was found to be proportional to the mitochondrial concentration. Upon the addition of potassium iodoacetate, there was no immedi ate change in the height of t h e double wave b u t on prolonged standing, the wave height was r e duced to one third its original value. Since the double wave is caused by t h e sulfhydryl group and since iodoacetate is specific for the sulfhydryl group, some of the double wave must be due to the presence of disulfide groups which are reduced a t potentials more anodic than t h e double wave. However, the major portion of the wave for the mitochondrial suspen sion is due to the sulfhydryl groups which form a mercury mercaptide with the electrode. The mitochondria were treated with ultrasonic radiation a t 10 kc. for 30 seconds and the mitochon drial membranes, or ghosts, were separated from the mitochondrial contents by centrifugation. T h e mitochondrial ghosts in deionized sucrose represented a 5 0 % prepara tion from r a t liver tissue. T h e su p e r n a t a n t component was divided into two fractions. One fraction was treated in a water bath a t 100° C. for 10 minutes to coagulate
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REPORT FOR ANALYTICAL CHEMISTS {12). The magnitude of this shift indicates t h a t the chromium ( I I I ) is quite strongly bound to the insulin. This is not surprising since insulin is a protein, and the interaction be tween chromium ( I I I ) and proteins has long been known by the chrome-tanning industry. This in teraction between chromium ( I I I ) and insulin occurs at the physiolog ical p H of 7.4, but does not occur in more acidic solutions. Certain other d a t a indicate t h a t t h e active site is the intrachain group of the insulin molecule. If mitochondrial suspension is added to the chromium ( I I I ) solu tion, the chromium wave is again shifted to more negative potentials. T h e point of interaction is, most probably, the sulfhydryl groups of the mitochondrial m e m b r a n e . Addition of either chromium ( I I I ) or insulin b y themselves to mitochondrial suspensions causes the mitochondrial wave to shift to more anodic potentials; this shift is not accompanied by a change of slope of the mitochondrial wave. When both chromium ( I I I ) and in sulin are added to the mitochondrial suspension, the mitochondrial wave is shifted to more anodic potentials by about 100 millivolts, a shift which is approximately 3-fold greater t h a n the sum of the shifts caused by the chromium ( I I I ) or in sulin alone. I n d e p e n d e n t investigations sug gest t h a t the reaction between insu lin and its receptor site goes via a sulfhydryl-disulfide linkage (7, 15). Our polarographic results would support this observation. F u r t h e r more, these results suggest t h a t the effect of chromium ( I I I ) on the insulin-mitochondrial reaction is to facilitate t h e formation of the sulf hydryl-disulfide linkage possibly through the formation of an inter m e d i a r y t e r n a r y complex involving chromium ( I I I ) , insulin, and the mitochondrial membrane. As a result of these studies, coupled with other biochemical data, chromium ( I I I ) is now being tested in lieu of insulin for lateronset diabetes. T h e results, to date, look promising. One other in teresting fact deserves mention. There are two populations in this world which seem to be entirely free
of diabetes. In the Philippines and in Thailand, diabetes is virtually unknown. Also, an analysis of t h e blood of these two populations indi cates t h a t the blood contains about 5 times the chromium ( I I I ) content of normal h u m a n blood. Complexation and Radiation Protection
The formation of complexes with i m p o r t a n t trace metals has been postulated by m a n y to be the mech anism for certain metabolic reac tions. I n most cases, however, no a t t e m p t has been m a d e to deter mine the n a t u r e of the complex nor its stability constant. W e have re cently become interested in the measurement of the stability con stants of complexes formed between certain cations and biologically-im p o r t a n t substances. T h e polaro graphic method for the determina tion of complexes is particularly useful for this type of study. A number of people have been in terested in the development of a drug which would protect an ani mal from a lethal dose of ionizing radiation (1, 17). There are cer tain substances which, when in jected into t h e animal, will protect it from a lethal dose of x-radiation. A t the present time, there are cer tain difficulties with these drugs. First, the effective dosages are very close to the toxic limits for the substance itself. Second, and more important, the drug to be effective must be injected before exposure to the radiation and then only within a b o u t 30 minutes of exposure. F i nally, the mechanism of the protec tion reaction is not known. Among the possible mechanisms for protection is one involving trace metals. This mechanism is based on the temporary removal of cer tain of these trace metals, thereby stopping the metabolic reactions they catalyze. W i t h these reac tions temporarily halted, the n a t ural repair within the body can t a k e place and possibly throw off the effects of the radiation insult. The removal of the trace metal would be accomplished through the formation of a complex. One of the first body systems to show t h a t an animal has received a
lethal radiation insult is the t y r o sine oxidase system. This oxidative system is controlled by a copper-containing coenzyme. I t was our thought t h a t there might be some correlation between the ability of the agent to protect and its ability to form complexes with copper. Those compounds chosen for this investigation were the amine m e r c a p t a n s where the functional groups were either alpha or beta to one another. A moment's reflection would indicate t h a t these compounds could form stable complexes with the cation which would result in either five- or six-membered rings. T h e n a t u r e and the stability of these complexes were determined polarographically in a supporting electrolyte of orthophosphate at p H 7.4. This medium was chosen since it is one of the chief buffering systems of the blood. F r o m the m a g nitude of the shift of the copper half-wave potcntia". with added agent, the stability constant for the complex was determined. We found t h a t there was a correlation between the stability constant and the effectiveness of the radiationprotective drug (18). Those drugs which had been found by animal experiments to be the most protective, were the same substances t h a t formed the most stable complexes with copper. T h e one place where I can see a possible application for this work is in the t r e a t m e n t of a widespread cancer. One t r e a t m e n t for cancer is x-radiation. I n the case of a widespread cancer, whole-body r a diation might be necessary. This could lead to a situation where the disease was cured but the patient died of the cure. I t might be possible, however, to expose the p a tient to whole-body radiation if he is first protected by a radiationprotective drug. A t this time no one knows whether this drug will protect both normal and cancer cells.
This is the
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COULOMETRIC METHODS FOR BLOOD ANALYSIS
Let us consider briefly the second topic, coulometry, as this illustrates another of our research interests.
HEERBRUGG Circle No. 186 on Readers' Service Card VOL. 36, NO. 4, APRIL 1964
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REPORT
The standard methods for blood analysis usually involve the devel opment of a color, the intensity of which is determined in a colorim eter or spectrophotometer. Al though these methods are fairly sensitive, they all require a number of reagents and, more important, a number of clinical standards. The storage of standards and the shelflife of these materials becomes a problem. In addition, many of the clinical analyses require samples of blood ranging up to 5 ml. I t would be desirable to develop clinical methods which do not require any standards, which can be run on 100 microliters or less of sample, and which will give the answer in con centration units directly. The advantages of a method of this type are obvious. The chance for technician error would be re duced to a minimum for the only important measurement that would have to be made would be of the sample volume. All standards could be dispensed with. Finally, working with such small volumes, the field of pediatric work would be open. A method such as is described is available. Cotlove (14) devised the coulometric determination of blood chloride by titration with generated silver ion. The blood sample was placed in a buffer solu tion and two generator electrodes were immersed in the solution. The anode was a silver wire. At the anode, silver ion was generated at a constant current. The product of the current and the generation time gives coulombs which are re lated to the equivalents of silver generated, and to the equivalents of chloride in the sample, by Fara day's law. Two indicator elec trodes immersed in the sample solu tion signalled the end point amperometrically. The amperometric signal automatically stopped the generation of the titrant and also the timer. In this apparatus, mar keted either by the American In strument Co. or the Buchler In strument Co., the currents were chosen such that the division of the coulombs by 96,500 was automati cally carried out so that the timer read directly in microequivalents of chloride in the sample.
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ANALYTICAL CHEMISTRY
REPORT FOR ANALYTICAL CHEMISTS T h e coulometric titration method employs the universal titrant, elec trons. B y proper choice of condi tions it is possible ;o cause an elec trode reaction to proceed with 100% current efficiency. T h e n u m ber of coulombs associated with t h a t electrode reaction is a meas ure of the concentration of the m a terial produced at the electrode. This material can then be employed as a t i t r a n t to determine other sub stances in solution which do not react a t the electrode surface. T h e sensitivity of the method depends on the accuracy of the constant cur rent and the accuracy of the time measurement. I n our laboratory we have been developing new coulometric meth ods for blood analysis. The re quirements have been two in num ber: (1) the methods m u s t possess the same or greater sensitivity and accuracy as the accepted clinical methods and (2) the sample volume should not exceed 100 microliters. Our present phosphate method, in volving the precipitation of silver phosphate from a water-ethanol supporting electrolyte, does not meet these requirements (9). Ammonia has been determined in our laboratory by direct titra tion with generated hypobromite (JO). I n alkaline medium, gener ated bromine disproportionates into bromide and hypobromite ions. The reaction between ammonia and hypobromite yields nitrogen and bromide ions. The first excess of the hypobromite can be detected amperometrically. We have em ployed this titration for the deter mination of as little as 1.4 micro grams of ammonia. Of course, any substance which can be converted quantitatively into ammonia can be determined in this manner. I hope t h a t I have been able to indicate the important role t h a t an analytical chemist can play in bio chemical research. Although I have chosen but two techniques to illustrate the point, equally good examples exist in conductivity, potentiometry, amperometry, etc. The systems t h a t one encounters are intriguing and they present a real challenge. However, once the analytical chemist is willing to bridge the gap between the usual
;
' c l e a n " samples and biochemical samples, the whole field of analyti cal biochemistry is open to him. This, I can attest, can be a very re warding experience.
HOW
INDUSTRY USES
LITERATURE CITED
(1) Baldini, G., Ferri, L., Brit. J Radiol., 30, 271 (1957). (2) Bonting, S. L., Jr., Biochim. Biophys. Acta, 6, 183 (1950). (3) Brdicka, R., Biochem. Z., 272, 104 (1934). (4) Brdicka, R., Coll. Czech. Chem. Commun., 5, 112 (1933). (5) Ibid., p. 238. (6) Brdicka, R., Nature, 139, 330, 1020 (1937). (7) Cadenas, Ε., Kaji, Η., Park, C. R., Rasmussen, H., J. Biol. Chem., 236, PC 63 (1961). (8) Christian, G. D., Knoblock, E. C ,
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P u r d y , W. C , A N A L . C H E M . , 35,
1128 (1963). (9) Ibid., p. 1869. (10) Ibid., p. 2217. (11) Christian, G. D., Knoblock, E. C , Purdv, W. C , Biochim. Biophys. Acta', 66, 415 (1963). (12) Christian, G. D., Knoblock, E, C , Purdv, W. C , Mertz, W., Ibid., 66, 420 (1963). (13) Colonv, J. Α., Knoblock, E. C , Purdv, W. C , Am. J. Clin. Path., 39,652 (1963). (14) Cotlove, E., "Standard Methods of Clinical Chemistry," Vol, I I , p. 81. D . Seligson, éd., Academic Press, Inc., New York, 1961. (15) Fong, C. T. O., Silver, L., Popenol, Ε. Α., Debons, A. F., Biochim,. Biophys. Acta. 56, 190 (1962). (16) Hartley, W. J., N. Z. J. Agric, 99, 259 (1959). (17) Jacobus, D. P., Federation Proc, 18,74 (1959). (18) Knoblock, E. C , Purdy, W. C , J. Electroanal. Chem., 2, 493 (1961). (19) Mertz, W., Roginski, E. E., J. Biol. Chem.. 238, 868 (1963). (20) Mertz, W., Roginski, Ε. Ε., Schwarz, K., Ibid., 236, 318 (1961). (21) Muller, Ο. Η., Federation Proc, S, 115 (1949). (22) Millier, Ο. Η., Davis, J. S., Jr., Arch. Biochem., 15, 39 (1947). (23) Millier, Ο. IL, Davis, J. S., Jr., ,/. Biol. Chem.. 157, 667 (1945). (24) Schwarz, K., "Symposium on Liver Function," Publ. No. 4, p. 509, Am. Inst. Biol. Sci., W a s h ington, D . C. (25) Schwarz, K., Mertz, W., Arch. Biochem. Biophys., 85, 292 (1959).
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CLASSIFYING laboratory quantities of fine materials for p r o d u c t or new m a t e r i a l research.
E J J CONTROLLING industrial processes using or producing fine materials.
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DETERMINING the separating efficiency of air cleaners.
j p j l PRE-TESTING dust collector requirements.
DETERMINE PARTICLE SIZE DISTRIBUTION below 60 microns,in materials such as: Beryllium Powder Grinding Sand Catalysts Metal Oxides Ceramic Powders Penicillin Chemicals Polymers Diatomaceous Earth Powdered Metals Drugs Milk Solids Dry Powders Standard Dust Fly Ash Samples
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END • Circle No. 127 on Readers' Service Card VOL. 3 6 , N O . 4 , APRIL 1 9 6 4
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