Kinetics of Cystine Catalysis of the iodine-Azide Reac ti o n W. E. DAHLl and H. L. PARDUE Department of Chemistry, Purdue University, lafayette, Ind.
b An instrumental technique is applied to a kinetic study of the cystine catalysis of the iodine-azide reaction. This reaction is essentially first-order in cystine, but minor pH-dependent side reactions appear to cause a higher-order effect a t high cystine concentrations. It is first-order in iodine and has an iodide dependency and - 1.0 order. between -0.5 The azide dependency ranges from first-order at high azide (1.OM) and low iodide ( 1 X 10-3M) concentrations to almost second-order at low azide (0.1M) and high iodide concentrations ( 1 X 10-W). The reaction shows a maximum rate at p H 5.2 to 5.4 and a minimum rate a t pH 7.5. A reaction pathway is proposed which is in agreement with the kinetic data and reaction characteristics observed. Conclusions are drawn concerning certain features of disulfide compounds which influence their activities as catalysts for the iodine-azide reaction.
C
by sulfur compounds of the oxidation of azide by iodine has attracted significant interest (7, IS, 14). This reaction has been investigated, taking advantage of a new instrumental method for reading numerical values of reaction rates automatically. I n this manner, high precision kinetic data have been obtained near zero reaction time with the result that a more complete understanding of these reactions is obtained. ATALYSIS
EXPERIMENTAL
The concentration cell and measurement system have been described (9, IO). For purposes of determination of reaction order dependence on initial concentration of reagents, a combination of the isolation and differential methods ( I ) was applied. Isolation was achieved by adjusting the reagent concentrations to obtain a reaction which was pseudo-first-order in iodine. Dependencies were then observed by varying individual reagent Concentrations and observing the different rates of iodine consumption. The standard conditions upon which the relative rates of the reaction were Present address, Central Research Dept., Monsanto Co., St. Louis, Mo. 1382
ANALYTICAL CHEMISTRY
based were as follows: 4.92 X 10-4X Iz, 5.0 X 10-3M I-, 0.9iM NaN3, 6.65 X 10-5Akf catalyst, p H of 6.0, and temperature of 25' + 0.05' C. Under these arbitrary conditions with cystine as the catalyst, the initial rate of iodine consumption was determined by calibrating the potentiometric response. This initial rate, -dIz/dt = 1.1 X moles 1 2 per liter-second, was assigned a relative value of 1.00. Rates for cystine catalysis measured under other conditions were assigned relative values to this. A number of other catalytic species were investigated under the standard conditions and assigned catalytic activity relative to that of cystine. RESULTS
The relative rates reported in this study are averages of a t least three runs per test. Relative standard deviations less than 1% were achieved in these measurements. All rate data for cystine have precision and accuracy comparable to those reported previously (9). The electrodes of the concentration cell exhibited Nernstian behavior. dlthough the cell potential became more positive with decreasing azide concentration and pH, the change in this potential for a constant change in iodine concentration did not vary significantly. Interpretations of reaction orders are made from log-log plots of the data in the linear plots reported here. Thus, any effect of azide or p H or the electrodes in the range of this work did not affect the response-curve slope. Effect of Iodine Concentration. The slope of the potentiometric response from t h e iodine-azide reaction is independent of iodine concentrations from 5 x 10-4M to 1 x 10-4.11. This result, combined with the observed Nernstian response of the indicator electrode, confirms the assumption that the reaction is pseudo-first-order in iodine. All measurements made in these studies were completed well within this range. Effect of RH. The profile for the reaction is displayed in Figure 1. The pH range of primary interest is 5.2 t o 8.8. A t values greater t h a n 8.8, t h e added NaOH apparently partially destroys the activity of t h e catalyst. At low p H levels, loss of N3- in the form of the volatile HN3 is an important factor.
Effect of Azide Concentration. The results of the azide dependence study are presented in Figure 2. The reaction dependency on azide approaches first-order at high azide concentrations for iodide concentrations less than 5 x 1 0 - 3 ~ . It approaches second-order at low azide concentration for iodide concentration higher than 1 x lO-*X. When 4J1 NaN03 replaced water as the diluting agent for the stock 4M N a y 3 , a 4% greater decrease in rate was observed for a reduction in ?it- concentration from 0.9iOJI to 0.325JI. This indicates a possible slight enhancement in rate as ionic strength is decreased. However, this effect is small compared to changes observed with varying azide concentrations. Effect of Iodide Concentration. The iodide ion dependence d a t a are given in Figure 3. A shift in order from about -0.5 t o -1.0 exists as iodide concentration is increased. Effect of Catalyst Concentration. The reaction has essentially firstorder dependence on cystine concentration as indicated in Figure 4. However, this dependency tends toward slightly higher order a t high catalyst concentrations. This tendency is p H dependent (greater a t higher pH) and may be due to a side reaction. Comparison of Cysteine with Cystine as a Catalyst. When the cysteinecatalyzed reaction is allowed to proceed until most of t h e iodine is consumed, t h e slope of t h e potentiometric response curve is very large at first, but decreases steadily. The amount of iodine consumed is very much in excess over t h a t required t o oxidize cysteine to cystine. However, upon adding more 13- solution, the response is linear and indicates a rate such as is obtained for an equivalent amount of cystine under the same conditions. This indicates that the sulfhydryl compound is a very active catalyst for the iodine-azide reaction as it is being oxidized to the disulfide. Effect of Azide or Iodine on Cystine. The abilities of azide or iodine to individually cleave t h e disulfide bond of cystine were tested under t h e standard conditions. Cystine was stirred vigorously for over 30
AZIDE
Figure 1.
Figure 2. Relative reaction rate vs. azide concentration as a function of iodide concentration
Effect of p H on relative reaction rate
x x
A. D.
IzN3- 2 IN3
N3-
e Iz?J3-, K,,
= [I2N3-] [I21
ihT1-1
(3)
The Br3- complex was reported as being considerably less stable. Because the relative stabilities of 12x3- and IN3 have not yet been satisfactorily established, it is possible that either or both may occur in the reaction mixture. I n this treatment, I & - will be dealt with because it is at least a required intermediate for the possible formation of IN3. Under the standard conditions of this study the NaN3 at 1M is 200 times the I- concentration. I n this case, if an 12N3- equilibrium constant of 8.4 is correct, most of the IZ is in the 12N3- complex.
Before discussing possible reaction pathways, consideration should be given to the tendency for i,he reactants to form competing complexes. An 12hT3- complex has been repor1;ed ( 8 )
+
+ 1-
However, BrN3 is formed from the BrzN3- complex (6). I n this case, BrN3 was indicated as favored over BrzN3- at bromide concentrations less than 0.5M as shown in Equation 4.
DISCUSSION
I2
I- = 1 10-3~ I- = 5 10-3~ I- = 1 X 1 0 % i I- = 5 X 10%I
B. C.
minutes in t h e reaction compartment with azide in one case and with iodine i n t h e other. I n each case, upon addition of t h e final reagent, t h e rate was identical t o t h a t observed when cystine was added t o t h e iodine-azide mixture. If either azide or iodine had cleaved a significant amount of cystine, the presence of the resulting cysteine would have caused a much faster initial rate. Other Catalytic Species. T h e relative rates of reaction achieved using various catalytic species are listed in Table I.
(2)
with an estimated value of the equilibrium constant of 8.4 relative to 800 for the 1 3 - complex. I n that study, no consideration was g,iven to the possible formation of IN,.
( M O L E S I LITER1
Table I. Relative Catalytic Activity of a Variety of Divalent Sulfur Compounds at 6.65 X 1 O - W
Catalyst h!i ethionine L-Cystine D-Cystine (Cysteine)z Ni (Reduced glutathione)2 Ni DL-Cvstine Oxidized glutathione Cystamine r-Carboxypropyl disulfide Dithiodiglycollic acid 0-Hydroxyethyl disulfide 2,2 -Dithiodibenzoic acid DL-a-Lipoic acid Cysteine Reduced glutathione Alercaptoethanol hlercaptoacetic acid
Relative activity 0.00 1.00 1.00 1.08" 1.12 1 15 1.19 2.05 2.47 3.20 3.61 4.23 >20
Very Very Yery Very
reactive reactive reactive reactive
At 3.12 X 10-5M. * A t 2.08 x 10-51M.
a
Y
c W
z 3 0.0 -
I-
Y
a 0.4
0
-
4
6
CYSTINE
L '.05 0 AI -00 IODIDE
Figure 3.
e
.04
.os
I
fYOLESILITER1
Relative reaction rate vs. iodide concentration
8
IO
(MOLES I LITER)(IO'l
Figure 4. Relative reaction rate vs. cystine concentration as a function of pH A. B.
C.
pH = 5.2 pH = 5.8 pH = 6.3
VOL. 37, NO. 11, OCTOBER 1965
1383
-4reasonable reaction pathway must account for the observation that although the over-all reaction requires two moles of KaN3 to react with one mole of Iz,the reaction order in azide varies from first-order to second-order depending on the relative amounts of azide and iodide present. Three more observations must be considered. First, RSH activity is much greater than that of RSSR,but RSH is subject to osidation to the disulfide. Second, both N3- and Izhave been reported to have some capability toward cleaving a disulfide bond, but neither is observed to effect this significantly with cystine in the absence of the other. Third, methionine, RSCHS, shows no catalytic activity even though it contains divalent sulfur. Proposed Reaction Pathway. With the preceding results and observations in mind, a reaction pathway is suggested. It is based on the premise t h a t the activity of the disulfide as a catalyst is due t o a cleavage of the disulfide bond to produce the active species. This cleavage is assumed to be the principal rate-determining step. Reaction step RSSR
Ns-
1.
13-
e Iz S I&;3- $
(12Na-RSSR)
e RSI
+ RSN3
+ N3- s RSN3 + I3. RSS, + 3Nz + RS4. RS- + RSI + I- f 5. RSI + RS- e RSSR + I6. RSN3 + RS- RSSR + N3-
+ I-
2. RSI
K3- +
12x3-
~
N3-
~
This pathway is for a constant pH, and the equilibrium of H + with the negative species above has been omitted. The effect of pH on the reaction will be discussed separately. I n step 1, 12N3- is indicated as the cleaving agent for the disulfide. If IN3 were considered, the pathway would be similar except the I- on the far right in steps 1 and 3 would be omitted. I n the case of cystine, if either azide or iodine alone is able to cleave the disulfide bond, the equilibrium is so unfavorable that the result is negligible. I n the presence of both Nt- and Iz,the cleavage could continue because of the displaced equilibrium resulting from the catalyzed reduction of azide involving concomitant electrophilic (I+) and nucleophilic (K3-) attack on the disulfide bond (6). Steps 5 and 6 represent likely paths wherein two reaction intermediates (RSN3 and RSI) may combine with RS- to regenerate the catalyst. These steps, along with step 1, represent a catalyst cycle. Steps 2, 3, and 4 are an inner cycle 1384
ANALYTICAL CHEMISTRY
involving the reactive intermediate species and the initial reagents. For sulfhydryl compounds, step 4 would be the starting point in the reaction. It is important to consider that although step 1 of the reaction pathway is the principal rate-determining step, changes which affect the rates of the other steps will also affect the over-all rate because the pathway is cyclic. To appreciate the relative likelihood of various steps of this proposed pathway, consider the order of sulfur(I1) nucleophilicity of several species, including the paticipants in the reaction (11, 1 2 ) .
+
+
RS - SR X - e RSX RS- (5) X-: R - > CzHsS- > CgIsS- > CY-
> s03-’> OH- > 2,4-(Noz)zc&&s> N3- > SCN-, ISteps 2, 5, and 6 are favored to the right in accordance with this information. Step 1 can be expected to be slow in the forward direction. Viewing the experimental data in the light of this pathway, one may see that when Ns- concentration is high enough that essentially all the Iz present is tied up as IzN3-, the reaction rate with regard to iY3- will approach first-order as principally dictated by step 3. Further addition of K3- will not increase IzN3- concentration. Hence, the rate of step 1 will be unaffected. At lower N3- concentration or higher I- concentration, further azide addition will result in an increase in the forward rate of step 1 as more 12N3- is formed. The expected over-all result is that as azide concentration is increased, the reaction order dependence on azide diminishes from higher-order to firstorder. At low azide and high I- concentrations, a second-order azide dependence would be predicted. These effects were observed esperimentally. The iodide dependence according to the pathway is essentially negative and is the result of several equilibrium effects. Xote in addition to 1 3 - formation, the rate inhibiting position of Iin steps 2 and 4. The iodine concentration shows up only as its equilibrium contribution toward IzS3-. As a result, the reaction dependence in iodine is first-order. The reaction should have a simple first-order dependence upon catalyst concentration in following the indicated pathway. This was observed experimentally a t low catalyst concentrations. Effect of pH. The pathway shows no dependence on pH. However, H + concentration may influence the rate of reaction in a number of ways. As previously mentioned, it can remove N3- from the reaction as volatile HN3 (pK,’ = 4.72). Steps such as 5 and 6 are known to be slowed a t lower values of p H as the RS- is further converted to
RSH (4). Reduction in rates of these steps should result in a faster over-all rate. This would occur as the rapid inner cycle (steps 2, 3, and 4) would proceed to a greater extent because of the longer lifetimes of the reactive intermediates, RSN3 and RSI. Furthermore, it has been suggested that acid cleavage of the disulfide bond accelerates halogen (electrophilic) cleavage of similar bonds (11).
+ + +
+
RSSR H + e RSH RS+ RS+ 1 2 z RSI If (6) I + RSSR + RSI RSf
+
+
I n the presence of N3- this would give rise to a concomitant electrophilic and nucleophilic cleavage. To explain the anomalously high optical rotation in its neutral solution, it has been suggested that cystine exists in the chelated endocyclic ring structure. 0.* * + H HOC
/
1
\
”
I
HCCH~S-SCH~CH
I
HN
I
(7)
COH
.4 structure such as this will cause steric interference with disulfide cleaving agents. I n addition, the two hydrogen bonds mill impede separation of the cleaved halves. These factors may contribute to the decrease in rate from pH 5.2 to 7.5. At higher pH, the OH- can act as a nucleophilic cleaving agent more active than S3-. Thus, alkaline cleavage of the disulfide bond must be considered (3)* RSSR
+ OH- s [RSOH] + RS-
(8)
This added cleavage may enhance the over-all rate of the reaction. The alkaline cleavage of the type shown in Equation 9 results in gradual destruction of the disulfide catalyst. 3[RSOH] + RSSR
+ RSOiH + 2H+
(9)
An effectof this type was observed a t pH 8.8. When the reaction catalyzed by cystine was allowed to proceed to completion, the cystine was destroyed and no reaction took place upon addition of more iodine. Following a similar procedure at a pH of 6.0, it appeared that virtually no cystine was destroyed. A side reaction such as this alkaline cleavage may explain the slight nonlinearity of the plot of rate us. cystine concentration. This nonlinearity does not exist at pH 5.2 but becomes more noticeable with increasing p H (Figure 4). Activity of Other Catalytic Species. The final features investigated with
respect t o the reaction of iodine by azide were the relative initial reaction rates obtained by using a number of other divalent sulfur compounds as catalysts. -111 of t8hesulfhyclryls or mercaptans tested exhibited general behavior similar to cystine. These rates decreased regularly until the reactions were proceeding as though catalyzed by disulfides. I n the cases of cysteine and reduced glutat.hione, complexation by Si(I1) converted their catalytic act,ivity to the level found for equivalent concent'rations of the respect,ive disulfides. This result may be understood when it is realized that (RS)?Ni is formed (6), greatly reducing the initial concentration of the RS-reactive intermediates. The Ni(I1) forms coordinate bonds with the nitrogen atoms of the amino groups and the sulfur atoms of the sulfhydryl groups. This observation emphasizes t,he importance of t,he RS-- species in this reaction. Complexation with Ni(I1) was not effective for mercaptans which did not, contain both amino and sulfhydryl groups in arrangements similar to cysteine. Akseen in Table I, the cat'alytic activity of the disulfide compounds st'udied varied over about a fourfold range from cystine (1.OO) to 2,2'-dithiodibenzoic acid (4.23). It is evident that more than one effect ia required in order to correlate t,his catalytic act,ivity wit'h structure. Two of the disulfides studied showed behavior sufficiently different from cystine to be discussed separately. The 2,2'-dithiodibenzoic acid was insoluble in water, but went into a fine suspension. This suspension dissolved in NaK3 solution, apparently as a result of N3- cleavage of the disulfide bond. If the suspension and azide solution were added first to the reaction cell followed by iodine, the observed rate had the characterist.ics of sulfhydiyl catalysis. However, when the sample was added last, the rate was of the disulfide type. This was the only disulfide studied which showed this degree of susceptibility to Sa-nucleophilic sttack. This was probably made possible because of stabilization by the phenyl group of the RS- intermediate. DL-a-Lipoic acid, which has a disulfide bond as part of a strained five-membered ring (11), exhibited high catalytic activity. The response curve was similar in shape to that for sulfhydryl compounds, but the relative rate was significantly slower than obtained by RSH catalysis. The rate appeared to be proportional to t.he square of this catalyst concentration when t'his concent'ration was low. This indicates that cleavage of the disulfide bond was no longer the principal ratedetermining step. Upon examining ea,ch of the remaining cata1yst.s in terms of features which will
affect cleavage of the disulfide bond, the variations of activity among them may Two be more readily understood. effects seem to be import'ant. The higher the possibility of intramolecular hydrogen bonding bet\+-een constituents on opposite sides of the disulfide bond, the greater the resistance t o cleavage of that bond and the lower the catalytic activity. The greater the electronwithdrawing ability of functional groups of the catalyst, the more susceptible the disulfide bond to nucleophilic attack and cleavage. As a test of these conclusions, consider the relative activities of several disulfides wit,h structural features differ.ent from cystine or oxidized glutathione. Cystamine differs from cystine only by itmslack of a carboxyl group on each side of the disulfide bond. The increase in activit,y of this catalyst may be explained by its inability to form intramolecular hydrogen bonds across t,he disulfide bond. This effect' appears to be more important than the loss of the electron-withdrawing carboxyl groups. Dithiodiglycollic acid differs from cystine by its lack of an amino group on each side of the disulfide bond. I n addition, t,he carboxyl groups are on the a-carbons (carboxyl groups of cystine are on the p-carbons). The greater increase in activity with this compound may be esplained by its loss of hydrogenbonding ability as well as the closer prosirnitmyof the carboxyl groups t,o the disulfide bond. I n the case of ?-carbosypropyl disulfide, the activity would not be expected to be as high as with the cat,alyst, just discussed because the carboxyl groups are on the ?-carbons. The increme in activit,yof P-hydroxyethyl disulfide over dithiodiglycollic acid is less readily explained. There is a reduction in the electron-withdrawing effect of the funct,ional groups, so anot,her effect must be important. One possibility is a hydrogen-bond formation betiyeen the hydroxyl hydrogen and the sulfur atom on the same side of t'he disulfide bond. This might tend to st'abilize any mercaptide resulting from cleavage while increasing the susceptibility of the disulfide bond to nucleophilic attack. COMPARISON WITH MECHANISMS SUGGESTED BY OTHER INVESTIGATORS
I n comparing the results of this study with those of the earlier works, the manner in which the data were obtained should be considered. I n the earlier works, the measurements were taken after relatively long reaction times and at best represented equilibrium states. The measurements reported here were obtained within 20 seconds reaction time and related to initial reaction rates. L@vtrupreported no investigation of the effect of iodide on the reaction and the data are not complete enough for
adequate comparison. He proposed the mechanism given in Equation 10. RSSR
+ N3-
1
I2
2 RSSRN3-1
+
2
h-a-
RSSRXJ +I-
+ RSSR + 3x2 --c
I-
3
(10)
This mechanism provides no means for interpreting the effect iodide concentration has on the azide dependency of the reaction. According to this mechanism, the only effect of changing p H from 6 to 5 would be to reduce azide concentration, with a resultant decrease in reaction rate. The actual increase in rate is explained in the present study as caused by effects not indicated in L6vtrup's mechanism. Strickland, JIack, and Childs gave a more detailed mechanism in the report of their study (14).
RSSR + 1 3 -
RSN3
+ IN3
S2
+
+ I-
(11)
+ 3x2
(14)
RSI
RSI
I n the case of cysteine (RSH), Equation 15 would replace Equation 11. RSH
+
1 3 - --c
RSI
+ H + + 21-
(15)
Several of the observations used by them in arriving a t the mechanism could not be reproduced in the present study. This is probably a direct result of the time which the reaction had been allowed to proceed when the respective measurements were made. They reported the catalytic effect of cysteine to be half that of cystine. In t,he study report'ed here, cysteine had a much greater catalytic effect than cystine. Cystine mas not observed to bleach iodine by action independent of its catalytic effect. I n addition, a titration of cystine by 1 3 - yielding RSI as indicated in Equation 11 seems unlikely since RSI is very unstable and is reported to be readily converted to RSSR (4). If the reactions in Equations 11 and 15 both proceed to coni'd \ I erable extents, as would be indicated by the titrations these authors describe, the initial rates of reactions catalyzed by RSH and RSSR should be similar. To the contrary, this study shows that the initial rate as catalyzed by R 3 H is much faster than that for RSSR. Finally, their observations that no reaction occurred when either NaN3 or 1x3 was added to the titration mixture do not agree with their mechanism. Since Equations 11 and 13 are reversible, addition of Na- to the titration mixture would result in the formation of the RSN3, via Equation 12, and the IN3, VOL. 37, NO. 1 1 , OCTOBER 1965
1385
via Equations 11 and 13, required for the reaction to proceed as written. A similar condition applies to IN3 addition. Since Strickland, Alack, and Childs suggested no rate-determining step for their mechanism, its analysis in terms of the present kinetic results is difficult. However, it is interesting to note the similarities between their mechanism and the one proposed here. Combining Equations 11 and 12 yields step 1 of the reaction pathway. Step 2 is similar to Equation 12. The possible existence of IN3 as appears in Equation 14 was discussed earlier. d parallel may be drawn between Equation 14 and a combination of steps 3 and 4. Finally, the recombination steps (5 and 6) are approximated by the reverse direction of the equilibrium in Equation 11. The basic difference betneen the mechanism proposed by Strickland,
Mack, and Childs and the pathway suggested in the present study is the principal rate-determining step reported here. Indications of the relatively slow cleavage of the disulfide bond were made possible by the new technique which enabled measurements approaching the initial reaction rates. A more definitive statement of the relative magnitudes of the various steps proposed must await a detailed study of the catalysis by sulfhydryls.
meister, H. L., Chem. Rev. 39, 269 (1946). ( 5 ) Kice, J. L., Venier, C. G., Tetrahedron Letters 48. 3629 (1964). (6) Lenz, G: R., Marten: A . E., Biochemistry 3, 745 (1964). (7) Lgjvtrup, S., Compt. Rend. Trav. Lab. Carlsberg 27, 72 (1949). ( 8 ) Neyerstein, D., Treinin, 8., Trans. Faraday SOC.59, 1114 (1963). W. E . -.T.. (9) Pardue. H. L.., Dahl. . . . -. ~-~~~ Electroanal. Chem. 8 , 268’( 1964). (10) Pardue, H. L., Shepherd, S. A,, ANAL.CHEM.35, 21 (1963). (11) Parker, A. J., Kharasch. N.. Chem. Rev. 59, 583 (1959). (12) Parker, A. J., Kharasch, N., J . A m . Chem. So?. 82, 3071 (1960). (13) Raschig, F., Chemiker Ztg. 32, 1203 (1908): (14) Strickland, R. D., Mack, P. A., Childs, W. A., ANAL. CHEM. 32, 430 (1960). I
~
’
I
LITERATURE CITED
(1) Benson, S. W., “The Foundations of
Chemical Kinetics,” McGraw-Hill, New York. 1960. (2) Griffith, R. O., Irving, R., Trans. Faraday SOC.45, 563 (1949).
(3) Hiskey,
R. G., Thomas, B. D., Kepler, J. A., J. Org. Chem. 29, 3671
(1964). (4) Kharasch, N., Potempa, S. J., Wehr-
,
RECEIVEDfor review May 17, 1965. Accepted July 29, 1965. Investigation supported by a David Ross XR Grant from the Purdue Research Foundation.
Continuous Chloride-Ion Cornbustion Method Applied to Determination of Organochlorine Insecticide Residues F. A.
GUNTHER, T. A. MILLER, and T. E. JENKINS
Department of Entomology, University of California, Riverside, Calif.
A new continuous chloride-ion system has been developed for use with a completely automatic combustion apparatus to determine organochlorine insecticide and other residues. This dynamic, continuous flow detector system uses a silver-silver chloride vs. slow-leak calomel electrode combination in routine analysis of samples of as much as 2 grams of plant extractives, Because the present equipment accommodates samples containing from 1000 to approximately 0.03 pg. of insecticide, containing from about 70 to about 8% organically bound chlorine, the useful range of detectability is from 500 p.p.m. to approximately 0.01 p.p.m.
0 (4,
chloride method 6) consists of producing hydrogen chloride gas by combustion, trapping this gas in a dilute solution of sodium carbonate-containing chloridefree nitric acid to suppress the ultimately interfering (4, 6) carbonate ion, and measuring the chloride ion concentration by simple direct potentiometry with a silver-silver chloride us. slow leak calomel electrode system. Amounts of original insecticides are then calculated from their percentage NE TOTAL ORGANIC
1386
ANALYTICAL CHEMISTRY
chloride compositions. This combustion chloride (1, 4 , 6 , 9 , I f ) direct potentiometric method (4, 6 ) is more advantageous (1, 4 , 6 , 9 ) than the usual wet oxidation and other combustion (8) techniques for handling large numbers of samples routinely. The only two operating disadvantages of the so-called “manual” method ( 1 , 4 , 6 , 9 ) involve the two components of the union of combustion ( I , 4, 9) with direct potentiometry (4, 6 ) : manually controlled combustions, which require up to 23 minutes each, can result in lost samples from incomplete burning or from too-rapid burning, and the transfers and other operations in “batchwise” direct potentiometry readily lead to inadvertent contamination by extraneous chloride ion in the laboratory atmosphere (inadequately cleaned glassware, etc.) in this extremely sensitive detection method. Complete automation of the combustion operation and continuous chloride-ion measurement would minimize both disadvantages. A completely automatic system has been developed which burns up to 2 grams of sample extractives in a burning cycle of 7 minutes, continuously monitors and measures any chloride ion in the combustion products, and graphically displays the resulting
chloride measurement. This system is more sensitive than the earlier (1, 4 , 6 , 9 ) versions, reduces the number of process steps, and requires less time per sample. DESCRIPTION OF APPARATUS
The automatic combustion furnace has been described by White et al. (11); slight modifications for the present purpose are discussed below. A block diagram of the new detection system is shown in Figure 1. Combustion products leave the furnace A under a slightly positive pressure. Any watersoluble gases present are dissolved in a dilute nitric acid carrier solution flowing into the system a t constant rate. After temperature equilibration in bath B , the concentration of chloride ion in this flowing carrier solution is continuously measured by the electrode system E with voltage amplification through a p H meter connected to a multirange recorder. Details are presented in later sections. Furnace. The automatic furnace (11) used for combustion was built from plans kindly supplied by the Shell Development Co., with a few minor modifications. For example, a switch was added to the 2-minute program controller allowing extension of the final 2-minute burn-out period, and the bubbler was not used because our vacuum line opened to the atmosphere.
