Table V.
Values of X and
COBr,
x 10-3 Variable 8 00 x Variable 1 33 x 10-3a lrariablea 8.00 x 10-36 1 3
0.067 mg./ml. of
TI
CY
Obtained from Plots of Equation 15
P C l , M Variable 1 33 x 10-3 Variable 8 00 X Variable I 33 x 10-3 Variable ool violet 6 B S present.
as given previously> since the per cent error i. calculated on the basis of the total amount of bromide originally present in the solution. Very good straight linec are obtained when the data on the dependence of per cent error on original chloride are plotted in this manner, and a fair plot is obtained for the c a v where the chloride is added after the precipitation of variouf amounts of bromide (Table
V) .
Fairly consistent values for A are obtained under the various experimrntal conditions employed. .Us0 the values calculated for CY. which rcpresents the ratio of chloride to bromide on the surface of the colloid a t the point of maximum potential change. are between 0.1 and 10 in all c a w . The value of CY depend. markedly upon whether or not the surface active substance, ~vool violet 6BS, is preqent in solution. The larger values of CY obtained nhen wool ent in solution reflect the de end point errors observed in thiz. cafe and might be accounted for bv the follolving. aidsorbed wool violet limits the ewhange of radioactive bromide with solid +-er bromide
x 0 0 0 0 0 0 0
12 12 17 06
15 27 29
Vff 0 20 0 19 0 39 0 18 0 02 0 13 0 12
ff
0 GO 0 63 0 43 0 35 10
2 1 2 3
to the surface layer only (8.I I ) , and reprewe.. the gron t h of the 4 r e r bromide particles vis Ostnald ripening (8). The tendency for the extra chloride, trapped in the interior of thc crvqtals, to re-enter the qolution via diffusion or recry~tnllizationsn.ould thus be minimized in the prcvnce of n-ool violet and one n ould cxpect a ma.tiniurn error. Evchange of some of the trapped chloride n-ith bromide ion in qolution nould a190 account for the decreav in error IT ith decreaqing rate of silver nitrate addition nhich n-ac obcerrcd in the absence of wool violet kincc, a t s l o w r rates of addition. the colloidal particle. have more of an opportunity to expel the extra chloride and thereby more nearly approach an ideal homogeneous mixed crystal compoiition. To the author+ knon lcdge, no quantitative nieaw-e of the relativc adsorbabilities of chloridc and bromide ions on silver bromide ha. been made. Leden and coworkerq ( 1 , 2, 22) have given values for the formation conqtants of the mono-, di-, tri-, and trtrahalogeno argentate (I) compleuci [ I g +
c1-
C1-
+
A w l ~ " l . kIG1,:
e rigCl~-, kzcc1),etc.].
XaCl,,,,
+
The values
they give arc: k l c c l , = 1.1 x lo3, k l ( B r ) = 2.4 x 104, kr ci) = 102, k 2 ( B r ) = 9.1 X 102, k Z ( C 1 ) = 1.0, and Jig(&) = 4.6. The follom-ing ratios of aq>ociation constants for correy2onding qilver chloride and silver bromide sprcie. [ r , = k,(cl, ' k n ~ ~ rare j ] calculated from the above: r1 = 0.016. r? = 0.11, and 7-3 = 0.22. The similarity between t l w e and the value obtained for X is quite qtriking and probablr indicates that the degree of diffociation or covalence of the silver halide bond iq of paramount importance in predicting the adsorbability of a given halide ion on R silver halide q u i face. LITERATURE CITED
(1) Berne. E., Leden, I., Scerd; Kem. Tzdskr. 65, 88 (1953). (2) Berne, E., Leden, I., 2. Y a t i i t f o r s c h . 8a,
719 (1953).
( 3 ) Clark, IT.,J . Chem. SOC.(loridon)
1926, 768. (4) Doernw. H.
.I.. HoskinP. I\-. 11.. J . i l m . Chetrz. SOC.47, 662 (1928). ( 5 ) Flood, H., Z. anorg. u. a l i y ~ r i , .Chem. 229, 76 (1936). (6) Flood, H., Bruun, B., Ibid., 229, 85 (1936). ( T i Flood. H.. Sletten. E.. 2. anal. Chem. 115, 30 (1938). (8) Iiolthoff, I. 11.,Box-ers, R. C.. J . .-tn~. Cheni. SOC.76, 1503 (1954). (9) Kolthoff, I. M.,Eggertson. F. T., Ibid., 61, 1036 (1939). 10) Iiolthoff, I . X.> Furmari, S. H., "Potentiometric Titrat ion. '' in>. 154-8, \Tilev. S e n York, 1931. 11) Iiolthoff, I. 11.. O'Rrien. .I. S., .I. .ivi. Chern. SOC.61,3409 (1939). 12) Leden, I., SLerisk Kern. Tzi16jl.i. 64, 249 (1952). (13) Thirl, -I.,2. anorg. Chein. 24, 1 I I
(1 ROO).
RECEIVEDfor rwiev- June 20, 1960. Accepted Sovember 8, 1960. Division of .Inal>-tical Chemistrr, 137th JIeeting, ACS, Cleveland, Ohio, April 1960.
Amperometric Titration of Sulfhydryl and Disulfide Groups with Organic Mercury Compounds at the Rotated Dropping Mercury Indicator Electrode WALTER STRICKS and S. K. CHAKRAVARTI Department o f Chemistry, Marquetfe University, Milwaukee 3, Wis.
b The rotated dropping mercury electrode (R.D.M.E.) has the same sensitivity as the rotated platinum electrode. Polarographic studies a t the R.D.M.E. of the reaction of organic mercury compounds with cysteine, reduced glutathione, cystine (in the presence of sulfite), and albumin in the undenatured and denatured states show that this reaction can b e made the basis of a titration of sulfhydryl in these ma194
ANALYTICAL CHEMISTRY
terials, using the R.D.M.E. as indicator electrode. Procedures are given for the accurate and rapid determination of traces of sulfhydryl in amino acids, pepticks, and proteins with ethylmercury chloride as titrant. The method allows the determination of 6 to 120 pg. of sulfhydryl in biological materials in aqueous solutions with an The reaction of accuracy of &O.s%. phenylmercury hydroxide with sulf-
hydryl and disulfide i s slow. Titrations with phenylmercury hydroxide are therefore inferior to those with ethylmercury chloride.
A
( 6 ) and mercurimetric (7') amperometric titrations of sulfhydryl and disulfide groups in amino acids, peptides, and proteins at the rotated platinum wire electrode as RGENTIMETRIC
iiidicator electrode have been reported. The argentimetric amperometric titration of glutathione (GSH) gives low results in aqueous ammoniacal medium (7'). Mercuric chloride is bifunctional and forms several complex compounds with c!-steine (RSH) and glutathione its observed a t the dropping mercury (14) and platinum electrodes ( 7 ) . This c a n give rise to more than one end point in niercurimetric t>itrationsa t these c~lectrotles. Xercurimttric titrations of rystc~intm d glutabhione are therefore limitc~lto certain conditions of p H and chloritle roncentrations ( 7 ) . Also the surf:iw cwitlitions of the platinum electrode U I Y often critical in these titrat'ions. In c.ontrast to mercuric chloride, alkj-1- 311d phenylmcrcury compounds arc' nionofunctional and do not, form iiiorcfi than one complex wit'h sulfhydryl compi~miels. I n view of this fact, alkyland ~jlic~ii~-lmercury compounds are consitlcwil to he the most specific rcagcnts for sulfliyrhyl groups. Thus p-chloromerrury bmzoate has been used as reagc'nt i i i indicator (8j,spectrophotomc4rica t I ) , polarometric (Q), and potentioniotric. t / 0 ) titrations, while methylnicrc~irynitrate \vas u s d for the nitro~~riissiclc~ titration of guanidine-denaturcd protein ( 2 ) . Polarograpliic ex-. pv1init3iits C J I ~ rractions between sulfhydryl and p-chloroniercury benzoate a t t h tmvc.ntional dropping mercury elcc~trotlc (D.1I.E.) v m r reported by €lata ('3) :md by Rolilig et nl. ( 1 1 ) . S o inwstigations were rcyorted about the hIi:i~-ior of alkyl- and plicnylnicr(wry compounds at, the rotated platinum c~lcctrode nor were any attmipt.s iii:itlc to use the rotatcd dropping n i ~ ~ r c welectrode ~y (25) (R.D.3I.E.) for sulfliy~lryltitrations with these compc)unds :IY titrants. In the present 1 ) : ~ p tlw ~ r rcmlts of ampcromctric titrations or sulfhydryl and disulfide groups with org:iniv mercury compounds a t the rotntc(l p1:itinuni c~lcetrodcand a t thc R . l l . l I . 1 ~:m ~ . reported. EXPERIMENTAL
Reagents. Cystt4ne hydrocliloride was :i I)i'o'luc.t from Sutritional Bioclic~mic*alC'orp. and glutatliione was a Pf ails t iclil protluc t . Both protiuc t s \wre 1wttt.r than 99% pure as found titration with cupric c o p p c ~ ( 5 ) . Frcsli d o c k solutions of cystcinv and glutntliionc, (10-3-11) n'erc prcpared chily i n air-free douhlt~tlistillrd n-at,er. tiill> (RSSR) n-as a C.P. product from l1wc.k. Thc stock solution was 10P:3.1/in c?-stine anti C.1.11 in hydroCrystallized bovine cliloric~ ncid. p1asni:t albumin was an Arniour product n-liirh vontained 3.07, water upon hwting a t 110" C. to constant weight. T L i c s albumin stock solution was about i% in albumin and was kept in a refrigcmtor when not in use. Under such conditions the sulfhydryl content was ( ~ m ~ t a for i i t 48 hours.
VOLT
VI.
I
(
t
Figure 1 . Current-voltage curves obtained titration of cysteine with EMC a t the R.D.M.E. A. B.
in a
40 ml. of 1 O-4M cysteine (0.04M borate, pH 9.2) after adding 1 ml.; C. 2 ml.; and D. 2.5 ml., of 2 X 1 O-3M EMC solution
Ethylmercury chloride (EblC), a product from Aldrich Chemical Co., contained a slight amount of mercuric chloride. For purification the product was suspended in hot distilled water, filteied n-ith suction in a sintered glass crucible, dried, and recrystallized from absolute alcohol. The purified dried material n-as found to be free of mercuric chloride by polarographic analysis. Five hundred milliliters of a st'ock solution of 2.00 x 10-3AlIEhIC was prepared by dissolving an appropriate amount in 3 to 4 ml. of warm I S sodium hydroxide to n-hich an equal volume of v-arm water n-as added. The resulting clear solution m s transferred to a 500ml. flask and filled to the mark with distilled water. Boiling of the solution should be avoidcd since E l t C is slightly volatile with boiling n-at'cr (12 ) . Phenylniercury hydroxide (PNOH) and p chloromercurybenzoic acid were C.P. products from Delta Chemical Korks, Inc., and n-crc not purified furbher. Solutions of these compounds were prepared in the same n-ay as described for ElIC. 3.11 the other materials used were comiiiercinl C.P. reagent grade products. Methods. Current-voltage curves were measured a t 25' + 0.1' C. with a Sargent manual Polarograph, Model 111, and with a self-recording Sargent Polarograph, lIoil(~1XXI. All potentials were measured against the saturated calomc~lcc.11 (S.C.E.). 911 titrations w r e performed with the manual Polarograph. Oxygen was removed from the solution with a streani of nitrogen (Linde nitrogen, 99.9967, pure). The pH of the solutions was nieasurcd with a Bcckman Zeromatic pH meter. F i w - and 10-nil. semimicroburets with 0.01-nil. divisions were uscd in the titrations. The platinum \vir(, elect'rode was rotatrd at' a spcr~lof 600 r.p.m. The new elcctrode n-as clrxaned mith concenbrated nitric acid and kept in distilled water when not in use. After each use the electrode \vas rinsed with distilled wa t er . The R.D.X.E. is essentially a n electrode in which the mercury is dislodged in an u p - a r d direction into the solution from the tip of a rotated U-tube, the drop time being controlled by the di-
mensions of the electrode and by the speed of rotation. The construction and characteristics of the electrode w r e describcld previously (15). The speed of rotation used for the prcscnt n-ork was 225 r.p.ni. Thr drop timci a t this speed was 3.9 seconds a t open circuit. The rate of flon of the rotated electrode was 15.28 mg. per second. The height of the mercury column n-as 52 cm. The substances investigated in this work, such as cysteine, glutathione, and albumin, arc' electrocapillary active, and the limiting current produced by these compounds is not reduced by a maximum suppressor (15). Therefore, no maximum suppressor n as addrtl to the titration niiyture. CURRENT-VOLTAGE CURVES
At Rotated Platinum Wire Electrode. Solutions of EBIC, phenylmercury hydroxide, and p-chloromercurybenzoic acid a t concentrations of 10-5to 10-4J/ in boras (pH 9.2) and phosphate (6.7) buffers w r r clcctrolyzed at the rotat'ed platinum wire electrode. Sone of t h t w compounds gives a cathodic current a t this electrode. A niisture of 10PdJI ElIC and 5 X 1O-jJI cj-stcJine in a boras buffer a t p H 9.2 is also not reduced a t thc rot'atcd platinum elcrctrode. The mechanism of the electrochemical reduction of organic mercury compounds is complicated. Either the overpotent,ial for the reduction is large or the electrode is coated with an insolublc intermediate product which prevclnts the appearance of a diffusion current a t the platinum electrode,. The plat,inum electrode cannot, thcrcfore. be usrd as indicator c~lectrodcin titrations of sulfhydryl Tvitli alkyl- or phonylmcrcury compounds as reagents. At R.D.M.E. PHENYL- A K D -~LI(I.LJIERCCRY
CUr-
CoMPOCKDS.
rent-voltage curves of phenylmercury chloride in t h e presence and absence of gclntin were discussed previously (15). Phenylmercury compounds are reduced in two steps. both steps being irreversible. ElIC, polarographed in 1 0 - ~ ~solutions 1 in various buffers a t pH 2 to 12 in the absence of maximum suppressors, also gives two waws. The log
i
-
os. E plot of the first wave
7. Zd 2
VOL. 33, NO. 2, FEBRUARY 1961
0
195
i
i / A
PH Figure 2. Half wave potential of A, C2H5HgCI, and B, C2HsHgSR, vs. pH 1. 2. 3.
HCI-KCI Acetate Phosphate
4. Borate 5. "3, KCI 6.
NaOH
gives a straight line of slope 0.058, and thus could correspond to a reversible 1electron reduction. The diffusion current of the first wave is proportional to the concentration of EhIC in the entire pH range investigated. In the presence of sulfite a t alkaline p H the first wave of EMC is shifted to a more negative potential. Thus, in a boras buffer a t p H 9, the half wave potential of a 10-4M solution in the absence and presence of 0.1M sulfite is -0.36 and -0.50 volt, respectively. Apparently EMC forms a complex nith sulfite. Since the second reduction step of EMC is not used in sulfhydryl titrations, a discussion of this wave which is drawn out and irreversible is not given in this paper. An extensive study of the polarography of alkylmercury compounds a t the R.D.M.E. and a t the conventional D.M.E. is under way in this laboratory. Titrations with E M C and Phenylmercury Hydroxide. Air-free solutions of cysteine and reduced glutathione were titrated in acetate, phosphate, and borate buffers, and in hydrochloric acid-potassium chloride as well as in dilute sodium hydroxide solutions. Cystine was titrated in the presence of sulfite in borate and phosphate buffers and in dilute sodium hydroxide (0.021M). Complete polarograms were taken after the addition of various amounts of titrant. A few polarograms of mixtures of cysteine and E M C in a borax buffer are illustrated in Figure 1. At a mole ratio of cysteine to EMC of 2 to 1, the diffusion current of cysteine is about half of its original value, and at a potential of -0.8 volt, a cathodic wave is observed which corresponds to the reduction of ethylmercury cysteinate, C2H6HgSR. If the mole ratio of cysteine to E M C is 1 to 1, no anodic cysteine wave is observed and the polarogram consists of the two reduction steps of ethylmercury cysteinate. Upon further addition of EMC, the wave of the excess EMC precedes the ethylmercury cysteinate wave, the cysteinate being a stronger complex than the chloride. Cysteinate waves
196
ANALYTICAL CHEMISTRY
were obtained a t vaiious pH values ranging from 2 to 12. At p H 2 the log i -.us. E plot is a straight linc of slope id - z 0.058 while solutions a t higher pH give irreversible m-aves. The shift of the half wave potentials with pH of both the EMC and cysteinate wave is illustrated in Figure 2. The difference between the half wave potentials of the two waves decreases markedly a t pH values lower than 5 and the two waves merge into each other below p H 2. The potential a t which a titration of cysteine with E I I C can be performed thus varies nith the p H and extends over the diffusion current region of the first excess EMC wave. For practical purposes a pH lower than 4 is not recomniended for a titration of cysteine with EMC, since the diffusion plateau of the first excess C l I C wave becomes very short and the end point is not sharp at this IOK pH region. The most suitable potentials for titrations of cvsteine us. EMC are -0.7, -0.6, -0.5, and -0.44 volt for p H ranges of 12 to 10, 10 to 7 , 7 t o 6. and 6 to 4.6, respectively. A titration curve obtained by plotting the current measured a t -0.6 volt us. the volume of EMC solution added is illustrated in Figure 3 4 . The intersection of the excess reagent line with the line representing the residual current corresponds to the mole ratio of EMC to RSH of 1 to 1. Use of these observations is made in the amperometric titration of sulfhydryl. At a suitable pH, disulfide reacts with sulfite according to the equation ( I S ) RSSR
+ s03-' e RS- + RSSOS-
(1)
On the addition of increasing amounts of EMC, RS- reacts to form slightly dissociated ethylmercury cysteinate and the reaction of Equation 1 goes to completion, the final products being the mercaptide and the sulfonate. Current-voltage curves obtained with titrations of cystine in the presence of sulfite are similar to those illustrated in Figure 1, indicating that the sulfonate has no effect on the EMC and ethylmercury cysteinate wave. Of course, the excess EMC wave is shifted to more negative potentials in the presence of sulfite. Mixtures of reduced glutathione and EMC, a t various molar concentration ratios, were also polarographed and gave current-voltage curves which were practically identical with those obtained with cysteine. Experiments with undenatured albumin mere carried out in borate and phosphate buffers. E M C reacts rapidly with sulfhydryl of albumin to form a complex CeHbHg-albumin, which is not reduced at the mercury electrode. Titrations of albumin can therefore be performed on the entire diffusion plateau of the first excess E M C wave a t potentials which are about 0.1 volt more negative than the half wave potential of this wave. Considering that aromatic mercury compounds have been more widely used than aliphatic compounds a$ reagents
1
I
/
2
x
1
63M
TITRANT ADDED, ml.
Figure 3. Amperometric titration of cysteine in 40 ml. of 0.04M borax a t pH 9.2 A. B.
4 ml. of 1.002 X 1 O-3M RSH titrated with 2.003 X 10-'M CgHEHgCI at -0.6 volt 3 ml. of 1.992 X 10-3M RSH titrated with 2.1 X 10-aM CsHsHgOH at -0.5 volt
for sulfhydryl titrations, an attempt was made to use phenylmercury hydroside as a titrant also. Current-voltage curves taken with PMOH and cysteine are similar in appearance to those obtained with mixtures containing EMC. PMOH waves are generally drawn out and PMOH as well as phenylmercury cysteinate are reduced at somewhat less cathodic potentials than the corresponding ethyl compounds. The potentials for titrations with PMOH are therefore, by about 0.1 volt, more positive than those applied for titrations with EMC at the same pH. At pH lower than 10 this can result in an overlapping with the anodic cysteine wave, and in titration curves like the one illustrated in Figure 3,B. The reaction line is not straight. The excess reagent line is used for the detection of the end point in the same way as indicated for titrations with ERIC. Generally, the reaction between cysteine and PMOH is slower than that with EMC. I n titrations of cysteine with PMOH the current increases abruptly upon each addition of the titrant and then decreases until a constant value is obtained. The reason is that the initial excess of PMOH is slowly used up by the cysteine. The reaction is still slower in titrations of cystine in which cysteine is formed by the interaction of cystine and sulfite. The rate of this reaction with PMOH decreases markedly with increasing pH as observed in a p H range between 7.7 and 12.2. For analytical purposes EMC is therefore recommended as the better titrating agent.
PROCEDURES
Cysteine and Glutathione. Introduce 40 nil. of a solution which is 0.0SM in disodium phosphate (NazH P O J a n d 0.01M in monosodium phosphate (h-aH2P0J into a 100-ml. beaker. Immerse the R.D.M.E. and rovcr with a rubber stopper with holes for electrode, salt bridge, buret, and inlet tube for nitrogen. Remove air with pure nitrogen and pass nitrogen through the solution during the entire titration. Immerse thc salt bridge in the solution and determine the residual current a t -0.G volt. To the air-free solution add mough sample so that the cysteine or glutathione concentration in the mixture is between arid 10-4M. P u t the tip of the buret into the beaker and titrate with EMC of suitable conceiitration ( 5 x lop4 to 2 x 10-311f) a t a n applied potential of -0.6 volt 08. S.C.E. The intersection of the excess reagent line with the residual current is the end point. One inilliliter of 10-3;C1 EMC corresponds to 0.121 mg. of cysteine, 0.307 mg. of glutathionc~,or 0.033 mg. of sulfhydryl. The titration can also be performed in 0.023f sodium hydroxide (pH 12) a t a potential of -0.7 volt us S.C.E., in 0.0451 borax (DH 9.2) at -0.6 volt. in a phosphite mixture 0.0251 in Sa?HPOdand NaH9POI (DH 6.7) at -0.5 volt,^ and in an acet'ate mixture 0.02.11 in CH3COOH and 0.025f in CH,COONa a t -0 44 volt us S.C.E. The borax buffer can be substituted by an ammonia buffer, 0.04:M in ammonia and 0.04V in ammonium chloride. Ammonia buffers a t p H 8 (0.04M NH3, 0,431 NH4CI)arid a t p H 10 (0.4A1 NH8, 0.04M NH4C1) can be used a t titration potentials of -0.G and -0.7 volt, respectively. If an ammonia buffei is used the cysteine (glutathione) voncentration of the titration mixture should not be less than 10-45f and the nitrogen must first be passed through a solution of the same compoFitioii as that of the buffer in the titration mixture. Cystine. I n t o a 100-ml. beaker place 15 ml. of 0.1-I4 borax solution, 20 ml. of water. and 8 ml. of 0.5M sulfite solution. Introduce salt bridge and electrode into the solution; pass nitrogen through the solution, determine the rwidual current, and add enough saniple to make the solution 10-5 to 10-4;Tf in cystine. Titrate with EMC solution of the proper molarity at a potential of -0.7 volt. One milliliter of 10-3X EAIC corresponds to 0.240 ing. of cystine. The boras buffer can be substituted by a phosphate buffer (O.OSA4 Sa2HP0+ 0.01M NaH2P04,p H 8.0) or by 0.02M sodium hydroxide (pH 12.0). In each case the titration potential is -0.7 volt us.
S.C.E.
Protein Sulfhydryl. I n t o a 100ml. beaker introduce 40 ml. of a solution which is 0.01M in monosodium phosphate (SaH2P04), 0.0864 in disodium phosphate (Na?HP04), and 0.2U in potassium chloride. Add 0.05 to 0.08 nil. of octyl alcohol. To the airfree solution add enough of the protein solution to make the mixture 1 t o 7 X 10-5-Tf in sulfhydryl. While passing
nitrogen through the solution titrate with a n EMC solution a t a potent,ial of -0.5 or -0.6 volt US. S.C.E. RESULTS
Table I gives the results of amperometric titrations of cysteine and glutathione in acid and alkaline media with EMC as titrating agent. An inspection of this table shows that cystcine as 1 ~ 1 1
Table
I.
Concn. of EMC Eoln., hl 2.003 x 10-3
as glutathione can be titrated accurately in these media. I n ammoniacal media the residual current changes with fluctuations in the ainnionia concentration. This can result in large errors if the cysteine concentration in the titration mixture is markedly smaller than 5 x lO-5M. Thus with a cysteine concentration of 1.3 X 10-5M the titration gave a n error of -6% whereas no error was found n i t h the same cystcine con-
Titrations of Cysteine and of Reduced Glutathione with EMC
Init. RSH (GW Concn. of Soln. Titrated
Titration Potential. Supporting Electrolyte Buffer, M PK
CYSTEIS E cH3c0OHI 0.02 CH~COO?ra.0.02 2 . 5 x 10-5 CHsCOOH,O 02 CH3COOKa, 0.02 Na2HP04,0.02 10-4 xaH1P04, 0.02 2.5 X Sa2HP04,0.02 SaH,P04. 0.02 10-4
4 67
-0 44 0 485 0 481
-0 8
4 67
-0 44 0 121 0 122
tl 0
6.7
-0
6 7
-0 5
0 121 0 123 $1 0
-0 6
0 486 0 486
0.0
-0 6
0 061
Ob1
0.0
-0.6
0.122 0.122
0.0
-0.6
0.485 0.488 4-0.6
-0 6
0 . 1 2 1 0.123
-1.6
9.2 9.2 10.8
-0 6 -0 6 -0.7
0 486 0 486 0 121 0 121 0 485 0 484
0.0 0.0 -0 2
10 8
-0 7
0 121 0.124 f 2 . 5
10.8
-0.7
0 061 0 057
12.0 12.0
-0.7 -0.7
0.486 0.486 0.121 0.121
0.0 0.0
1.229 1.229
0.0
10-4
5.008 x 10-4
1 . 3 x 10-5 Na2HP04, 0.08 NaH,PO, 0.01 2 . 5 x 10-5 Na2HP0,, 0.08 SaH2PO4, 0.01
7. i
-
1 . 1
2.003 X 5.008 x 10-4
2.5
x 10-5
2.003 x 10-3
Borax, 0.04 10-4 2 . 5 x 10-5 Borax, 0.04 KH3, 0.08 10-4 KC1, 0.04 5.008 X 2 . 5 X low6 KH?, 0 08 KCI, 0 04 "1, 0 08 1.3 X KCl, 0 04 NaOH, 0 02 2.003 x 10-3 10-4 NaOH, 0 , 0 2 2.5 x
5
0 485 0 486
0
$0 2
-6.5
GLUTATHIONE 2.00 x 10-3
10-4 2.6
x lo-'
2.003 x 10-3 5.008 X lo-'
10-4
2.5 X
2.003 X 5.008 X 2.003 x 10-3 2.00 x 10-3
2.5 X 10-4
10-4 10-4 10-4 2.6 X
CH3COOH,0.02 4 . 6 -0.44 CHICOONa, 0.02 KCl, 0 . 4 CH&OOH,O.02 4.6 -0.44 CH,COONa, 0.02 Na2HP04,0.02 6 . 5 -0.50 NaH?P04,0.02 KCl, 1 . 0 -0.50 NaZHPO4, 0 02 6 8 YaHgP04, 0.02 Na2HP04,0.02 6 . 8 -0.50 XaH2P04, 0.02 Na2HP0,, 0.08 7 . 7 -0.60 KaH2P04, 0.01 Na2HP04,0.08 7 . 7 -0.60 NaH2P0,, 0.01 Borax, 0.04 9 . 2 -0.60 Borax, 0 . 0 4 9.15 -0.60 KC1, 0 . 4 NaOH, 0.02 12.05 -0.70 SaOH, 0.02 12.05 -0.70 KC1, 0 . 4 NaOH, 0 , 0 2 12.05 -0.70
0,282 0 280 1.234 1.234
-0.7
0.0
1.231 1.225 - 0 . 5 0.308 0.310 + 0 . 6 1.243 1.243
0.0
0,311 0.312 1 0 . 3 0.305 0 306 $0 3 1 2 2 9 1.228 - 0 . 1 1.229 1.218 -0.9 1.128 1.128 0 0 0.282 0.280 - 0 . 7
VO1. 33, NO. 2, FEBRUARY 1961
197
Table II.
Titration of Cystine with Ethylmercury Chloride at -0.7 Volt vs. S.C.E.
(Titrating agent, 2 003 X lo-3.U CZHbHgC1) Init. RSSR Concn. of Soln. Titrated, M 10-4
x 10-6
2 5
10-4 2 5 x 10-5 10-4 2 5 x 10-5
Supporting Electrolyte Buffer, JIG PH Xa?HPOa,0.08 XaH9POd.0.01 Na2HPOI;O 08 NaHzPO,,O 01 Borate, 0 04 Borate, 0 04 SaOH. 0 02 S a O H , 0 02
RSSR,N g .
Error,
Taken
Found
c /O
8.0
0 (371
0 969
-0.2
8 0
0 243
0 241
-0 8
9 9 12 12
2 2
0 0
0 0 0 0
971
243 971
24.7
0 0 0 0
973 241 970 241
$0 -0 -0 -0
2 8 1 8
All solutions 0 1Jf in SalSO,.
5
Table 111.
Titration of 40-MI. Albumin Solutions in Various Buffers
In presence of 0 08 nil. of octvl alcohol a t -0.5 volt vs. S.C.E. with 2 X 10-3Jf E l I C as titrating agent Mole Ratio of Concn. of Albumin, Supporting Electrolyte Hg to Albumin Buffer, Jf PH at End Pt. M 5
x 10-6 10-4
1 . 2 5 x 10-5 5
x 10-5 10-4
1 . 2 5 x 10-6
5
x
10-5
10-4 1 , 2 5x 10-6 5
x
30-5
10-4
5
x 10-6
2 5 x 10-5 5
x 10-5 10-4
1 25 x 10-6 10-4
HC1, 0 005 KC1, 0 2 CH,COOH, 0 1 KC1, 0 2
CH,COOH, 0 02 CH3COOSa,0 O l b CHYCOOH, 0 02 CHICOOla, 0 01 CH,COOH, 0 02 CH~COONEI, 0 Olb Sa2HPOI,0 02
TaH?P04,0 02b Sa2HPO4, 0.02 NaHZPOI, 0 02* SarHPO4, 0 02 IYE~H~PO:, 0 ,0zb Sa2HPOd,0 08 Ir'aHIPOd, 0 0 l b Xa?HPOI, 0 08 SaHlPO,, 0 O l b Sa?HPO?,0 08 KaH?PO;, 0 O l b Boras. 0 04 IiC1, 0 . 2
3.0 3.4 1.5
4.5 4.5 G. 7 6.7
G.7
7 8
-( . I
7.8 9 2
Borau, 0 04
9.2
Bomu. 0 04
9 2
Boras, 0 04
9 ,2
Boras, 0 04
9.2
Boras, 0 04 IiC1. 0 2
$1 , 2
IiCI. 0 2
IiCI, 0 2 IiC1, 0 2
IiC1, 0 . 2
Titrant was 5 X 10-43f E N C . Plus 0.2M KCl. Titration mixture contained 0 05 ml. of octyl nlcohol Titration prrformed at -0 6 volt us. S.C.E. e Titrant was 5 X lO-;Jf HgCl?. I Titrant was 2 X 10-3AlfHgCI?. 0 Solution did not contain octj-1alcohol. a
198
ANALYTICAL CHEMISTRY
centration in a phosphate buffer. The average error is around 0.5% with misto 10-521 tures containing 2 x cysteine or glutathione in acetate, phosphate, and boras buffers and in dilute sodium hydroxide, At sulfhydryl concentrations smaller than IO-'J/ a sharp end point is not obtained. The results of cystine titrations are summarized in Table 11. Good results are obtained in solutions a t p H 9 to 12 wit'h cyst,ine concentrations not lower than 10-$31. The sulfite concentration should not be lower than 0.05JI; othern-ise the reaction is s l o (6) ~ and the end point, not sharp. The average error is around 0.57,. Table I11 gives the results of titrations of crystallized albumin a t various concrntrations and in various buffers. Taking a molecular wight of albumin of 70,000, the end point in these titrations corresponds to a molr ratio of 0.66 i 0.01, which is in good agreement with results obtained in titrations a t the rotatcd platinum electrode with niercuric chloride as titrant ( 7 ) . Good results are obtained in solutions a t p H 9.2 to 3.0 with albumin concentrat'ions betn-een 5 X t o 10-~31. Nost of the albumin titrations ~ w r e performed with mixburw containing sniall amounts of octyl alcohol n-hich was addcd to prevent excessive foaniing. Results obtainrd in the presence and absence of octyl alcohol are ident,ical (Table 111). This was confirnied in titrations xi-ith the rotatcd platiiiuni electrode. Octyl alcohol can thrrefore be addcd to the albumin solution n.itliout' any d f c c t on the titrata)-)lesulfli!-tlryl content. Titrations n-crc also carricd out nitli albumin n-hich had bern s u b j c r t d to denaturation in 431 guanidine hydroc~liloritle (GHCI) or 831 urea in the prescnccl of 0.05JI sodium sulfitc. Thus in one instance 40 nil. of a phosphatc. buffer (0.02331 Sa2HPO4.0.02331 S a H 2 POa. JA1fGHCl. 0.0531 Sa2S03, p H 6.5) was made 3.5 X 1OP631in albumin hy addition of 1 nil. of 1.3 x 10-4Alf albumin stock solution and allon-ed to stand for 30 minut'es. 11-hcrrupon tlie solution was titrated n-ith 2 X 10-331 EJIC' at' -0.7 volt 2's. S.C.I-7. The point' of intersection of til(, r w g m t linr n-ith thc, liiic corresponding t o t,lie residual of the mixture befo1~3adding thc titrant girts thc rntl point. Table IV s h o w that the, number of disulfitle groups pcr mole of albumin is 17.5 +0.2. rr-hich is in good agrecmcwt with rcsults obtained by Koltlioff et nl. (4)Iyith tlie mercxry pool electrode and with niercuric chloride as reagent. Sonic 40 titrations of ( cystine a t p H ranges of 4.4 to 12 and of 8.2 to 9.2. rrspectively, n-ere pwfornied with PJIOH as titrant. For the sake of brevity the results are not tabulated. Tablrs can be obt'ained from the authors.
Cysteine titrations can give results with a n error of about 1%, the results being better at p H 9 to 12. Because of the slowness of the reaction with PAIOH, the reproducibility of PRIOH titrations is generally poorer than t h a t of titrations n i t h EhIC. This is particularly pronounced in titrations of cj.stine in the presence of sulfite. Rcxliable results cannot be obtained a t cystine concentrations loner than 10-a-lI. An advantage of PRIOH is that pure commercial products are obtainable, n hile commercial ERIC must hc purified and is inore eapensive than PAIOH.
Titration of 40 MI. of 3.56 X 10-6M Albumin Solution in Buffers a t Various pH Values In presence of 4114 GHCl or 8.M urea and of 0.05M sodium sulfite nith 2 x l O - + J f EJIC a t titrating agent. All solutions allon-ed to stand 30 min. before titration Table IV.
Supporting Electrolyte Buffer 4 B
1
-4. B.
This inwstigation n as supported by r r ~ ~ ~ rgrant c h from the Satioiial Sctirnce Foundation.
D.
1’. D., J . -1iu. (’hem. Soc. 7 6 , 4331 (1954). ( 2 ) Edelhoch, H., Katchalski, E., LIajburg, R. H., Hughes, J. T., E d d l , J., 1 0 1 d . 75, ~ 5058 (1953). ( 3 ) H:ita. T.. J i e i r i . Research I n s t . Food
6 5
GHC‘1
(1958). 1.5) \ -
(1) ,!o>.er,
6 5
-0
6 -0 7 -0 6 -0 6 -0 8 -0.8
S o . of -S--SGroups per Mole
of .Ilbumin 18.3 18.5 17.7 17.4
17.2 17.5 17.0
B. H., J . .Inz. C h e m . Soc. 80, 3235
ii
LITERATURE CITED
pH 6.5
F’olt 1’s. S.C.E. -0 6
6 5 Urea 6 5 c GHCl 8 3 Urea 9.7 D 0 023M SarHPO,, 0 0235i SaHzPOa. Same ab 9 plus 0 06 nil. octj 1 alcohol. 0 6M borau, 0 06 nil. octyl alcohol. Same as C, e\cq)t 0 01.2/ borax and 0 1Jf siilfite. B B
P, ACKNOWLEDGMENT
Denat. lgent GHCl GHCl GHCl
Titration Potential,
Kolthoff. I. 11..Stricks. IT.. ~~-
~ H E X23,’763 ( I 951).
>
I
-1s.4~.
(6) Icolthoff, I. JI., Strick., IV., J . A m . Chem. Sac. 72, 1932 (1950). ( 7 ) Kolthoff. I. 11..Stricks. IT., Morren, ’L., ASAL.’CHEM. 26, 366 ’( 195k). (8) IlacDonnell, I,. R.,dilva, R. B., FeeneJ-, R. E., Arch. Biochem. Biophus. 32, 288 (1951). (9) Matousek, L., Lancikova, O., Chem. list!/ 47, 1062 (1953).
f
10) Pihar, O., Ibitl., 47, 1617 (1053).
(12) ‘Sidg&ck; N. V., ments and Their Comoounds.” D. 298. Clarendon Press, Oxfoid, 1950: (13) Stricks, IT., Kolthoff, I. M., J . Am. Chenz. SOC.73, 4569 (1951). (141 I b i d . . 75. 5673 (19531. (15) Ibzd., 78; 2085 (1956). RECEIVED for revieTT- .lugust 15, 1960. Accepted Soveiiiber 9, 1960. Division of ilnalytical Chemistry, 138th Xeeting, AC8, S e n 1-ork, S . Y., September 1960.
Use of Four-Electrode Conductometry for the Automatic Determination of Carbon Dioxide and Ammonia in Concentrated Scrubbing Water of Coke Oven Gas EMBRECHT BARENDRECHT and NORBERT G. L. M. JANSSEN Centrad Laboraforium der Staafsmijnen in Limburg, Geleen, Holland
b Four-elect rod e cond uctometry has proved to be a very useful tool in analyzing solutions with good conductivity and solutions contaminated with oil, tar, and the like. Two electrodes carry a 50- or 60-count-persecond alternating current through Ihe solution electrolyte to be analyzed, and the potential difference produced i s measured with the two other electrodes in such a way that the conductivity can be recorded directly. The use of a cell of special construction renders the measurement remarkably independent of the flow rate of the scrubbing water through the cell. Moreover, the grounding problem could b e resolved. Application of this method for the automatic determina-
tion of carbon dioxide and ammonia in concentrated scrubbing water of coke oven gas i s described.
u
non-, most industrial conductonietric ana1yst.s (batchn iscl or continuous) have bcrn ( arried out with the aid of audio-frequency alternating current antl t n o platinizrd electrodes. I n caws, honevcr. nhere rather concentrated solutions (specific conductance larger than lo-* o h n - l cni.-l) are to be measured. the main difficulties for setting up a n automatic analyzer are causrd by the rcquired geometric dimensions of the rrll, because the cell constant, expressed in cni.-’, must be large. Moreover, in industry, such concentrated solutions STIL
are oftc’n coiitaniinatd n ith orgaiiic suhstanws such as oil, tar, and the like, so that the platinized clectrodes nil1 soon be poisoned and the conducting liquid path betwcen these electrodes be blocked. There are two interesting methods to eliminate these difficulties. The first method was originally proposed a hundred years ago by Reetz (9) n here induction currents grnerated in a closed loop of the solution to be measured are practically proportional to thc conductivity of the solution. This electrodeless method has been rediscovered lately and seems to bf a proniisiiig tool (2, 4, 6, 10, 1 1 ) . Another nwthod that can be adapted is also rather old (Q), antl makes use of four electrodes as described herr. SwVOL. 33, NO. 2, FEBRUARY 1961
199
