1670
ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
-=La -
L. B. Marshall, G. D. Christian, and A. Kuma, Analyst(London),102, 424
I
(1977). M. G. Hardinge, J. B. Swarner, and H. Crooks, J . Am. Diet. Assoc., 46,
197 (1965).
YI
,
-."
- . 222
-i
'=CCd=TJEf.~,'
^J (L.
..-
_-
ZCL
2;
N. W. Tietz, Ed.. "Fundamentals of Clinical Chemistry", W. 8 . Saunders, Philadelphia, Pa., 1970,pp 409, 809. M. W. Kanter, "Clinical Chemistry", The Bobbs-Merrill Co., Indianapolis, Ind., 1975,p 169. R. Lees, "Food Analysis: Analytical and Quality Control Methods for the Food Manufacturer and Buyer", 3rd ed., CRC Press, Cleveland, Ohio, 1975,pp 68 and 168. G. G. Birch and 0. M. Mwangelwa, J . Sci. Food Agric., 25, 1355 (1974). Sigma Chemical Co., Form No. 322, St. Louis, Mo. "Enzymatic Assay of a-Amyhse (E.C. 3.2.1lr',December 1971,Revised August 1977,Sigma Chemical Co., St. Louis, Mo. January 1978,Sigma Chemical Co., St. "aGlucosidase (E.C. 3.2.1.20)", Louis, Mo. "The Enzymatic Assay of Invertase", November 1977,Sigma Chemical Co., St. Louis, Mo. "Lactase", Sigma Chemical Co., St. Louis, Mo. G. G. Guilbault, "Handbook of Enzymatic Methods of Analysis", Marcel Dekker, New York, N.Y., 1977. E. H. Hansen, A. K. Ghose, and J. Ruzicka, Ana&st(London), 102, 714 (1977),and references therein. F. S. Cheng and G. D. Christian, Ana/yst(London), 102, 124 (1977). J. Cerning-Beroard, Cereal Chem., 52, 431 (1975). G G.Guilbauk, P. J. Brignac, Jr., and M . Juneau, Anal. Chem., 40, 1256
Figure 4. Comparison of results obtained by sample injection into a closed-flow-through system and by the saccharogenic method. Results represent determinations in 32 samples of human blood serum with amylase content mostly in the normal range. (Pearson's correlation coefficient: 0.99; slope obtained by linear regression: 0.973,intercept:
11968).
3.14 U/100 mL)
(1967).
ACKNOWLEDGMENT The authors are indebted to David Jackson (Oklahoma State University Student Hospital and Clinic) for samples of human blood serum. LITERATURE CITED (1) Ch-Michel Wolff and H. A. Mottola, Anal. Chem.. 50, 94 (1978). (2) Ch-Michel Wolff and H. A. Motfola, Anal. Chem., 49, 2118 (1977).
G. G. Guilbauk, M. H. Sadar, and K. Peres, Anal. Biochem., 31,91 (1969). G.G. Guilbault and E.B. Rietz. Clin. Chem. ( Winston-Salem, N.C.),22,
1702 (1976). H. A. Mottoia and A. Hanna, Anal. Chim. Acta, in press. G. C. Toralballa and M. Eitingon, Arch. Biochem. Biophys., 119, 519 I.Asfaha, Oklahoma State University, unpublished observations, 1978. "WorthingtonEnzyme Manual", L. A. Decker, Ed., Wwthington Biochemical Corp., Freehold, N.J., 1977, p 195. G. Reed, "Enzymes in Food Processing", 2nd ed., Academic Press, New York, N.Y., 1970, p 90.
RECEIVED for review April 10, 19'78. Accepted July 20, 1978. This work was supported by the National Science Foundation. One of the authors (D.P.N.) gratefully acknowledges a leave of absence and support from the University of Athens, Greece.
Unexpectedly Rapid Esterification of Nitrite Applied to the Determination of Nitrite in Water Marcus Keh-Chung Chao, Takeru Higuchi, and Larry A. Sternson" Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66045
Nitrite is converted to an alkyl nitrite by passing an acidified solution through a bed of packed beads (XAD-2) coated with 1-decanoi, the latter acting both as a reactant and as an extractant. The reaction proceeds by rate limited diffusion of nitrous acid into the aikanoi phase where it is rapidly esterified. Esterification by this pathway is qulte rapid (obsd f,,2 = 11 s) and efficient. The product, decyl nitrite, is retained in the aikanoi phase [partition coefficient (C,,,H,,OH:H,O) = 2.5 X lo'] where it is resistant to hydrolysis because of the limlted solubility of water in the alkanoi phase. Under optimized conditions, 6 8 % of the nitrite present in the original water soiutlon was retained on the column as decyi nitrite. Decyi nitrite was eluted from the column with acetone and then converted to a highly colored azo dye by sequential reactlon with sulfanilamide and N-( 1-naphthyl)ethyienediamine. The chromophore was monitored spectrophotometrically. The detection limit for nitrite quantitatlon by this method was 5 X 10-e M (200 pg/mL).
Recently, a great deal of interest has been generated concerning potential health hazards of nitrites. Nitrites are 0003-2700/78/0350-1670$01 .OO/O
frequently used as preservatives in food products and their precursors are widely distributed in nature because of the use of nitrogen fertilizers. Nitrites have been regarded as potentially hazardous compounds ( I ) . They oxidize hemoglobin to methemoglobin which is unable to transport oxygen (2-4) and they react with amines and amides to form nitrosamines, which are potent carcinogens (5-9). In view of the ubiquitous presence of nitrites in the environment (IO),a sensitive method for trace level determination of nitrites is desirable. Although nitrites can be determined polarographically ( I I ) , the classical methods are colorimetric procedures. The nitrite is reacted with a primary aromatic amine (e.g., sulfanilamide) in acid solution to yield a diazonium salt which is coupled with an electron rich aromatic molecule [e.g., N-(1-naphthyl)ethylenediamine (NEDA)J to produce a highly colored azo compound which is measured spectrophotometrically (12,13). The detection limit for nitrite ion by this method using sulfanilamide and NEDA as reagents is lo4 M (13). Sensitivity has been improved (by a factor of 30) by employing a concentration step after formation of the azo dye. The azo compound has been concentrated by a series of extractions into increasingly smaller volumes of solvent ( 2 4 , 15); or by retention on a strong cation-exchange resin and G 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
subsequent elution into a small volume of acetic acid (16). In this report, the feasibility of concentrating the aqueous nitrite solution prior t o diazotization is described. The approach which was investigated involves the conversion of nitrite t o an alkyl nitrite by passing the solution through a bed of packed beads coated with a hydrophobic alkanol, the latter acting both as a reactant and as an extractant, retaining the nitrite as alkyl nitrite in the alkanol phase on the inert support. The alkyl nitrite would subsequently be eluted from the support with a small volume of organic solvent and detected spectrophotometrically after conversion to an azo dye. The feasibility of this approach is dependent on (1)favorable reaction kinetics for on-column alkyl nitrite formation, (2) retention of the alkanol and ester on the column, (3) minimal hydrolysis of the alkyl nitrite on column, (4) quantitative removal of alkyl nitrite from t h e column in a small volume of eluent, and (5) effective measurement of trace amounts of alkyl nitrite. Allen (17)previously studied the reversible reaction between n-propanol and nitrous acid in 72% (w/w) dioxane:water (Equation 1).
CBH7ONO
khydr
HC
+ HzO 2 C3H70H + HONO hem H+
(1)
T h e reaction is described by the rate equation presented as Equation 2 and shows a first-order dependency on p H (over t h e range p H 2.5-3.5).
= k”hyd[C3H70NO] - k”,,,[HONO]
(2)
Based on the pseudo-first-order rate constants (k’? calculated for these reactions (k”hyd= 9.08 X lo4 s-l; k’kst = 5.22 X s-l at 0 “C and p H 2.53) and the equilibrium constant determined for the process ( K = (k”,,,/k”hyJ = 0.57), nitrite esterification in aqueous solution is a slow and inefficient process. Therefore, the postulated analytical approach would be ineffective if esterification proceeded via the mechanism described by Allen, i.e., reaction of the alcohol with nitrous acid in the bulk aqueous solution to produce the corresponding alkyl nitrite which then diffuses into the alcohol phase where it is retained. It was hypothesized that the esterification pathway could be modified by using a hydrophobic, water insoluble, alkanol as the substrate and carrying out the reaction in a flowing stream. It was postulated that under these conditions, nitrous acid, formed in the aqueous phase, would partition into the alcohol phase and subsequently react with the alkanol to yield the alkyl nitrite. If it is assumed that formation of the nitrite ester in the corresponding alcohol phase is rapid (Equation 31,
ROH
+ HONO
ki
RON0 k-1
+ H20
(3)
then diffusion of nitrous acid into the alcohol phase is rate determining and the process can be expressed by Equation 4
-d [ H O N O ] dt
=
kl [HONO] - k-l[RONO]
(4)
where k, and k_l are complex constants which include the diffusion rate constant, the total surface area, and thickness of the diffusion layer. Under these conditions, nitrite esterification would occur rapidly and the ester should be re-
Inlection System Spectrophotometer equipped w i t h a micro f l o w cell
Figure 1.
1671
Syrir.qe P UmP
I
Block diagram of the micro flow cell apparatus
sistant to hydrolysis (k, > k1)due t o the low solubility of water in the alkanol phase and the presumed large partition coefficient of the long hydrocarbon chain alkyl nitrite between alkanol and water phases. Thus, Equation 4 simplifies t o Equation 5 -d[HONO] = kl[HONO] (5)
dt
The feasibility of concentrating nitrite in aqueous solution by a n extractive alkylation technique is described in this report.
EXPERIMENTAL Reagents. Sodium nitrite was obtained from J. T. Baker Chemical Co. and was dried for 24 h over calcium chloride prior to use. Sulfanilamide, N-(1-naphthy1)ethylenediamineand 1decanol were purchased from Eastman Kodak Co. Amberlite XAD-2, manufactured by Rohm & Haas Co., was in the form of white beads (20-50 mesh). Micro Flow Cell Apparatus. The apparatus was assembled using a Sage (Model 341) single direction infusion pump, an Altex Septum Injector, a Varian Techtron 635 spectrophotometer equipped with an Aerograph micro flow cell (8-pL capacity; 1-cm path length), and a Varian Techtron Model 135 strip chart recorder. Components in the flow system were interconnected with Altex tubing (0.8-mm id., 1.5-mm 0.d.). A block diagram of the detection apparatus is shown in Figure 1. Mobile phase [methanol:water (5050) v/v] was pumped through the system a t a flow rate of 6.5 pL/min. Column Packing. XAD-2 beads were washed thoroughly with water and the fine particles removed by decantation. The mixture was filtered and rinsed with acetone. Forty-five milliliters of beads were slurried in acetone containing 3 mL of 1-decanol. The acetone was removed at reduced pressure on a rotary evaporator and the coated beads were then packed into a disposable capillary pipet (5.75 in. length X 0.3 in. o.d.), and held in place by a glass wool plug. On-Column Formation and Extraction of Alkyl Nitrite. The aqueous sample solution containing sodium nitrite was acidified with concentrated sulfuric acid (0.1 mL) and pumped through the column with a reciprocating pump (Milton Roy Instrument Co.) at a flow rate of 300 mL/h. The column was then washed with water (5 mL) to remove any acid residue; the water was subsequently removed by pumping air through the system for 2 min. Acetone was then pumped through the column a t a rate of 0.3 mL/min to elute the 1-decanoland decyl nitrite, until 1.2 mL of eluate was collected (in a graduated centrifuge tube). Colorimetric Determination of Decyl Nitrite, Sulfanilamide (50 pL of a 5% solution) was added to the acetone eluate and the solution mixed thoroughly and allowed to stand for 1min. N-(1-naphthy1)ethylenediamine(50 pL of a 0.25% solution) was then added and the absorbance of the find solution (1.3 mL) was measured at 550 nm in a micro cuvette (1-mLcapacity; 1-cm path length) after allowing 8 min for complete color development. Micro Flow Cell Measurements. The final acetone solution (1.3 mL) containing the azo compound was evaporated under reduced pressure to remove the acetone. The residue was centrifuged to yield a biphasic system. The upper layer contained 1-decanol and was removed; the lower layer (200 pL) contained the azo dye. The syringe pump controlling the micro flow cell assembly was stopped and an aliquot (-20 pL) of the aqueous solution containing the azo compound was injected into the system and the pump was started again. Absorbance was recorded (at 550 nm) as a function of time on the recorder, and the area under the resulting curve integrated.
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
Table I. Effect of Amount of Decyl Alcohol Coated onto XAD-2 Beads on the Observed Yield of Decyl Nitrite Formed on the Column decyl alcohol (mL) yield, %a per 45 mL of beads 2 3 4 5
a ACETONE A THF
1.3 .
0
DIOXANE ACETONITRILE
36
48 51 50
a The yield was determined from the ratio of the amount of decyl nitrite in the eluate and the total amount of nitrite ions in the sample solution. The value obtained is the average of three separate runs in which 100 mL of 5 X IO-' M NaNO, solution at pH 1 . 7 was pumped through the column a t 300 mL/h.
R E S U L T S A N D DISCUSSION Preliminary experiments indicated that water samples could be analyzed for nitrite by extractive esterification, by passing acidified aqueous solutions containing sodium nitrite (5 x M) through a glass column (5.75 in. X 0.3 in. 0.d.) containing Amberlite XAD-2 beads (20-50 mesh) coated with 1-decanol. The column was washed with water to remove residual nitrous acid and then an organic solvent was used to elute the alcohol and alkyl nitrite from the column. The amount of nitrite ester in the eluent was determined colorimetrically by diazotization. Parameters such as the amount of decanol coated on XAD-2 beads, eluent, pH, flow rate, temperature. and sample volume were investigated to optimize analysis conditions. Amount of Decanol on XAD-2 Beads. The effect of the amount of decyl alcohol used to coat XAD-2 beads was determined empirically to select optimum reaction conditions. The alcohol is assumed to be partially incorporated within the beads because of their high porosity. However, the depth and extent of penetration of alkanol or alkyl nitrite are unknown. Using a constant volume of beads (45 mL), maximum yields of alkyl nitrite were obtained when 3 mL of decanol was used (Table I). Lesser amounts of coating gave poorer yields of decyl nitrite; increasing the amount of decanol beyond 3 mL failed to increase esterification efficiency. Therefore, a 3-mL loading of 1-decanol per 45 mL of XAD-2 beads was used for all subsequent studies. E l u e n t Selection. The eluent must remove decyl nitrite from the column in a minimum volume and must be somewhat miscible with residual water in the column. Low molecular weight alcohols are not suitable because they undergo rapid transesterification producing volatile nitrous esters (18). Dioxane, acetone, tetrahydrofuran (THF), and acetonitrile were investigated as potential eluents. The distribution curves for these eluents (Figure 2) show that the absorbances measured in T H F and dioxane are less than absorbances of solutions eluted with acetone or acetonitrile. Acetone is superior to acetonitrile because it elutes the decyl nitrite more rapidly and in a narrower band from the column and therefore was chosen as the eluent. Maximum elution of decyl nitrite was achieved when 1.2 mL of eluent was collected. Effect of pH on On-Column Esterification. The yield of decyl nitrite obtained by passing aqueous nitrite solutions (5 X M) of varying pH's (pH 0.9-6.0) through a decanol-loaded column was independent of pH below pH 2 (Figure 3). Reaction yield declined with increasing pH and fell to zero above pH 6. The inflection point of this curve occurred a t pH 3.43, coinciding with the pK, of nitrous acid (pK, = 3.35) (19). The extent of on-column formation of decyl nitrite was dependent only on the concentration of nitrous acid. The pH dependence of the on-column reaction appeared to reflect the effect of pH on the distribution of nitrite species between the alcohol and aqueous phase as shown by the close agreement between the experimentally-generated curve and
I
1
I
I
I
I
I
3
5
7
9
NUMBER OF FRACTIONS ( 0 , 3 m I / F R A C T I O N )
Figure 2. Elution curves of decyl nitrite from the column by various eluents. Absorbances were measured at 550 nm and arose from the azo compound produced by the successive addition of sulfanilamide and NEDA to the acetone eluate containing decyl nitrite collected in
0.3-mL fractions from the column
50
t-%
I
40t
"1
i
20
g
lot
1
I
1
1
2
3
4
I
5
6
0 w I I-
7
PH
Flgure 3. pH profile (-) of the extent of on-column formation of decyl nitrite when 100 mL of a 5 X lo-' M NaNO, solution was pumped through a column packed with decyl alcohol-coated XAD-2 beads at 300 mL/h. Yield was determined from the ratio of the amount of decyl nitrite in the eluate and the total amount of nitrite ions in the sample solution. Theoretical curve (- - -) constructed based on the distribution coefficient for nitrous acid (pK, 3.35) between decanol and water
a theoretical partition curve (Figure 3). The effect of pH on on-column esterification is different from that predicted from Allen's study (17), where the rate of formation of alkyl nitrite in aqueous solution showed a first order dependency on hydrogen ion concentration. In reinvestigating the kinetics of n-propyl nitrite formation from n-propanol and nitrous acid (over the pH range 1-21, the rate constants, obtained from enthalpimetric measurements, were in agreement with those calculated by Allen and the reaction was found to be first order with respect to hydrogen ion concentration. Thus, on-column nitrite esterification appears to proceed via a different mechanism than that described by Allen for esterification in aqueous solution. The kinetics of on-column formation of alkyl nitrite are, however, consistent with the hypothesis that esterification occurs after rate limiting diffusion of nitrous acid into the decanol phase (Le., rate depends on nitrous acid concentration; but is not first order with respect to hydrogen ion concentration).
ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
Table 11. Observed Yield of Decyl Nitrite Formed on Column with Variations in Flow Rate and Residence Time flow rate, mL/h
residence time," s
3OOc 225= 15oC 300d
9.6 12.8 19.2 19.2
yield,
%b
47 56 67 68
" Volume of aqueous phase in column/flow rate; in this study t, = 0.8/flow rate. The yield obtained is the average of two separate runs in which 100 mL of 5 x lo-' M NaNO, solution at pH 1.7 was pumped through the column. One pass through the column. Two passes through the column. Effect of Flow R a t e on On-Column Esterification. Water samples containing nitrite (5 x M) were acidified to pH 1.7 with H 8 O 4 and then passed through decanol-loaded XAD-2 columns at various flow rates (15Ck300 mL/hr). The residence time and corresponding yield of decyl nitrite for several flow rates is shown in Table 11. Yield was inversely related to flow rate and when the line describing this relationship was extrapolated to zero flow rate, an intercept approaching 100% yield was observed. Thus, at zero flow rate, the nitrite would be quantitatively captured on the column as decyl nitrite. The effective rate constants ( k , ) (Equation 3) for on-column formation of the alkyl nitrite were calculated based on the definition that the half-life of the reaction is the residence time, t,, ( t , = volume of aqueous phase in the column/flow rate; in this study the volume of aqueous phase was 0.8 mL) when a 50% yield of decyl nitrite is obtained. If diffusion of nitrous acid into the alkanol phase is rate determining the kinetics of (Equation 4) and it is assumed that k , >> k1, the extractive alkylation process are described by Equation 5 and the rate constant for the process can be readily calculated
A 50% yield of decyl nitrite was obtained at a flow rate of 275 m L / h which corresponds to a half-life for the reaction of 11 s ( k , = 0.07 s-'). Extractive esterification of nitrite from aqueous solution into decyl alcohol is therefore a very rapid process. Yield of decyl nitrite is a function of sample residence time. The same yield of decyl nitrite was obtained by passing the solution through a column once at 150 mL/h or twice at 300 mL/h (Table 11); in both cases, sample residence time was 19.2 s. The 68% yield obtained from two consecutive passes of solution at 300 mL/h approaches the theoretical value for two passes calculated on the basis of the yield from a single pass (47%) at 300 mL/min. If the second pass recovers 47% of the nitrite remaining in solution after one pass through the column (53%), a theoretical yield of 72% decyl nitrite is obtained from the two passes. The discrepancy between the experimental (68%) and theoretical (72%) yields is due, a t least in part, to the fact that the theoretical yield was calculated based on the assumption that a fresh alcohol phase was used for the second pass. These studies indicate that on-column nitrite esterification is a rapid reaction, in contradiction to Allen's work, further suggesting that the reaction does not occur in the aqueous phase, but rather takes place in the alcohol phase and is controlled by the diffusion of nitrous acid into decanol-coated beads. T e m p e r a t u r e Effects. On-column nitrite esterification of decanol was also studied as a function of temperature by
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Table 111. Variation of the Yield of the On-Column Formation of Decyl Nitrite with Temperature temp, yield, "C %a ; b Ink, 22.5 47 67 85
45.0 56.4 61.4 66.6
0.0622 0.0864 0.0991 0.114
2.777 - 2.448 -2.311 -2.170 --
a Yield obtained when 100 mL of 5 X 10.' M NaNO, solution at pH 1.7 was pumped through the column at 300 mL/h. k , = -[ln (1- Y ) ] / t , ,where (1- Y ) is the fraction of nitrite ions remaining unreacted in the aqueous solution, and t, is the residence time (Equation 8).
enclosing the column in a water jacket attached to a constant temperature circulating pump. Yield of decyl nitrite increased with an increase in temperature (Table 111). The apparent rate constants for on-column formation of decyl nitrite (k,) was calculated at various temperatures from the corresponding product yields, as follows. Integration of Equation 5 gave Equation 6. [HONO] In = -k,t, (6) [HONOI, where [HONO], is the original concentration of nitrite ions applied to the column, [HONO] is the concentration of nitrite ions exiting the column and t , is the residence time. If Y is defined as the fraction of nitrite ions in the aqueous solution that react with the alcohol to produce decyl nitrite, ( I - Y) is the fraction of nitrite ions remaining unreacted in aqueous solution and
(7) Combining Equations 6 and 7 gives Equation 8, (8) k l = -[ln (1 - Y)]/t, which allows calculation of k l from the yield of decyl nitrite (Table 111). The apparent activation energy for on-column nitrite esterification was determined from an Arrhenius plot to be -2 kcal mol-'. This value is considerably less than anticipated for most chemical reactions (15-100 kcal mol-') but is close to that for liquid phase diffusion processes (2-5 kcal mol-') (20). This suggests that the rate determining step for the on-column reaction is a diffusion process rather than a chemical reaction, again supporting the rate-limiting diffusion mechanism. Effect of S a m p l e Volume on Detection Limits. Since extractive esterification concentrates the nitrite into a small volume of decyl alcohol, the sensitivity of the method should increase as the sample volume applied to the column (i.e., the absolute amount of nitrite) increases. When varying volumes (100-1500 mL) of aqueous solution having an initial conM in sodium nitrite were passed through centration of 1 X the column at a flow rate of 300 mL/h, the amount of decyl alcohol formed increased linearly but approached a limiting value (Figure 4) at larger sample volumes (>500 mL), indicating that an equilibrium is reached between alkyl nitrite in the alcohol phase and nitrous acid in the aqueous phase. The equilibrium constant ( K = [RONO],,/[HONO],, = k l / k l , where kl and k-l are the effective rate constants for formation and hydrolysis for decyl nitrite, respectively, on the column) represents the distribution constant of nitrite between the decanol and aqueous phase (D = [RONO]d,/[HONO],,). This distribution was determined by pH 1.7 by an extractive method. Decyl alcohol was shaken in a sealed tube with 25 mL of an aqueous 1.5 x M NaN02 solution (adjusted to pH 1.7) for 1 h at 25 "C. The final concentration of decyl nitrite in the alcohol phase was 6.74 X M and the con-
1674
2 I
ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
IC
I
/
'Id 100
I
I
300
500
I
I
I
700
900
1100
VOLUME
I
1300
I
1500
(mi)
Figure 4. Amount of decyl nitrite formed on-column as a function of the volume of sample (aqueous solution at an initial concentration of 1 X lo-' M NaNO,). The solution was acidified to pH 1.7 and pumped through the column at 300 mL/h. Decyl nitrite concentration was determined spectrophotometrically as described in the Experimental section of the text
centration of nitrous acid in the aqueous phase was 2.7 X M; consequently, D = 2.5 X lo3. This large distribution coefficient ( D ) indicates the high affinity of decyl nitrite for the alkanol phase and, since D = K , supports the original assumption used in deriving Equation 5 that k , > k1,i.e., decyl nitrite is relatively stable toward hydrolysis (Equation 3) in the alcohol phase. Spectrophotometric Measurement of Decyl Nitrite. The decyl nitrite formed on-column was eluted with acetone and converted t o an azo dye by reaction with sulfanilamide (50 MLof 5% solution) followed by addition of NEDA (50 r L of 0.25% solution). The resulting solution was monitored spectrophotometrically a t 550 nm. The concentration of nitrite in unknown water samples was determined by comparing the absorbance produced in the acetone eluate with a standard curve generated by carrying known amounts of nitrite through the analysis sequence. The measurement was then verified by the standard addition method. The calibration curve was linear in the range from 1 x 10-7-1 x M sodium nitrite. Samples were analyzed with an accuracy of *7% and a precision of &5%. The sensitivity of the method has been improved by concentrating the acetone solution containing the azo dye from 1.3 mL to 0.2 mL and monitoring the resulting sample spectrophotometrically in a micro flow cell. The sample (20 gL) was injected into a capillary leading to the flow cell (Figure I) and the absorbance recorded as a function of time. A Gaussian-shaped peak was produced (instead of a square wave) as a result of the laminar flow in the capillary. Peak area was directly proportional to the concentration of the absorbing species (in the range 5 x 104-1 X lo4 M sodium nitrite) and inversely proportional to flow rate (1.5-15 pL/min), as is expected since the peak area is the integration of the absorbance of the azo dye over the sample residence time (Q. By measuring the absorbance-time curve produced by a 20-kL sample containing a specific amount of azo species pumped through the system a t a flow rate of 6.5 kL/min, the sensitivity of the method was increased by one order of magnitude compared with the direct absorbance reading. Under these conditions, the minimum detectable concentration of nitrite measurable by the micro flow technique was 100 pg/mL of sodium nitrite obtained originally from a 100-mL water sample, with precision of &5%. Interferences. The possible interferences with nitrite determination caused by trace organic and inorganic compounds commonly present in ordinary water were investigated. Chloroform was reported as the most abundant (133 ppb)
organic compound in the New Orleans water supply (21). At 200 ppb, chloroform did not interfere with nitrite determination (nitrite concentration = 5 X lo-' M). Methanol, ethanol, and dimethylamine (100 ppb) also did not interfere with the nitrite determination. Low molecular weight alcohols were considered as possible interferents because they may react with nitrite ion in acidic aqueous solution to form volatile nitrite esters prior to the on-column extraction. However, a t low alcohol concentration, the rate of alkyl nitrite formation is slow (17), thus accounting for the lack of difficulty encountered from these compounds. I t was thought that nitrite might be similarly consumed by reactions with amines to form nitrosamines; however, no interference was observed. In addition neither nitrate ion (the precursor of nitrite) nor chloride ion (at concentrations of 1 ppm) interfered with the nitrite determination. Nitrite ion is, however, rapidly consumed in the presence of oxidizing agents, including chlorinating mixtures and therefore nitrite cannot be detected in their presence, e.g., nitrite could not be detected in drinking water which had been chlorinated. Applications to Real Samples. Samples of Kansas River water (100 mL) were carried through this analysis sequence and were shown to contain between 1.02 and 16.7 X lo-@M nitrite. CONCLUSION The mechanism of on-column esterification of nitrous acid with 1-decanol is different from that occurring in aqueous solution ( I 7); an alternate pathway is suggested. Rather than assuming that esterification occurs in the aqueous phase followed by diffusion of the alkyl nitrite into the alcohol phase, we have proposed that, in an acid environment, nitrite, in the form of nitrous acid diffuses into the alkanol phase when it then reacts to form alkyl nitrite. Diffusion of nitrous acid into the decanol phase was thought to be the rate-limiting step in the on-column reaction. This proposed mechanism is supported by the experimental data generated in this study. Allen showed that nitrite ester formation in aqueous solution has a first-order dependency on hydronium ion activity. On-column formation of decyl nitrite, however, depends only on the concentration of nitrous acid in solution (Equation 5) and therefore is in contradiction to Allen's mechanism, but is consistent with the proposed mechanism. The rapidity of on-column formation of decyl nitrite is again inconsistent with Allen's mechanism, but can be explained if the reaction proceeds by rate-limited nitrous acid diffusion into the alkanol phase followed by rapid esterification. The proposed mechanism is further supported by kinetic studies of on-column esterification carried out as a function of temperature. The calculated activation energy for the process (2 kcal molt1) was considerably less than that observed for most chemical reactions, but was close to that determined for liquid phase diffusion processes, suggesting that the rate determining step for the on-column reaction is a diffusion process rather than a chemical reaction. All the data support a mechanism for on-column esterification based on rate limiting diffusion of nitrous acid into the alkanol phase followed by rapid esterification. LITERATURE CITED (1) W. Lijinsky and S. Epstein, Nature (London). 225, 21 (1970). (2) A. Sinios and W. Wodsak, Msch. Med. Wochenschr.. 90, 1856 (1965). (3) 2. Knotek and P. Schmidt, Pediatrics, 34, 7 8 (1964). (4) B. Ronceche, "Eleven Blue Men and Other Narratives of Medical DetectaY', Little, Brown, Boston, Mass., 1954. (5) P. Magee and J. Barnes, Br. J. Cancer, 10, 114 (1956). (6) I. Wolff and A. Wasserman, Science, 177, 4043 (1972). (7) J. Sander, Hoppe-Seyler's 2. Physiol. Chem., 349, 429 (1968) (8) N. Sen and D. Smith, J. Assoc. Off. Anal. Chem., 52, 47 (1967). (9) J. Sander, Hoppe-Seyier's Z . Physiol. Chem., 52, 1691 (1968). (IO) Y . Lam and D. Nicholas, Biochem. Biophys. Acta, 178, 225 (1969). (11) Princeton Applied Research, Princeton, N.J., Application Brief N-1 (1974). (12) E. Sawicki and T. Stanley, Talanta, 10, 641 (1963).
ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978 (13) M. Shinn, Ind. Eng. Chem., Anal. Ed., 13, 33 (1941). (14) G. Macchi and B. Cescon, Anal. Chem., 42, 1809 (1970). (15) M. Nishimura, K. Matsunaga, and K. Matsuda, BunsekiKagaku, 19, 1096 (1970). (17) A. Allen, J . Chem. Soc., 1968 (1954). (18) W. Fischer, 2.Phys. Chem., 65, 61 (1908). (19) T. Turney and G. Wright, Chem. Rev., 59, 497 (1959). (20) F. Helfferich, "Ion Exchange", McGraw-Hill, New York, N.Y., 1962.
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(21) "New Orleans Area Water Supply Study", US. Environmental Protection Agency, Dallas, Texas, 1974.
RECEIVED for review June 16, 1978. Accepted July 25, 1978. Work supported in part by NIH Grant CA-09242 awarded by the National Cancer Institute (PHS, DHEW).
Stability Constants for the Complexation of Copper(I1) Ions with Water and Soil Fulvic Acids Measured by an Ion Selective Electrode William T. Bresnahan,' Clarence L. Grant, and James H. Weber* Department of Chemistry, Parsons Hall, University of New Hampshire, Durham, New Hampshire 03824
We discuss the coordination of Cu2+ to welltharacterized soil fulvic acid (SFA) and water fulvic acid (WFA) samples as studied by electron paramagnetic resonance (EPR) spectroscopy, differential pulse polarography (DPP), and a Cu2+ ion selective electrode (ISE). Identical titrations of Cu2+with SFA using DPP and ISE techniques showed that the DPP peak current was not proportional to the hydrated Cu2+ concentration. Cu2+ ISE experiments were done at pH 4.0, 5.0, and 6.0 for both SFA and WFA and also at pH 4.7 for WFA, and conditional stability constants were calculated by two methods. The more useful method-Scatchard plots constructed for each experiment-indicated the presence of two classes of binding sites (confirmed by EPR) with stability constants in all cases of about 1 X 10' and 8 X lo3. From pH 4.0 to 6.0, the total number of binding sites per molecule increased from 0.8 to 4.2 and 0.6 to 2.6 for the soil and water fulvic acids, respectively; most of the increase occurred between pH 5.0 and 6.0. We speculate that the difference in the two classes of sites is not due to the type of donor atoms, but to the geometry of the site.
The speciation of trace metals in natural waters together with their overall concentration has an important effect on the quality of our water resources. The physiochemical state of trace metals in water systems affects their availability to biota, their transport, and their accumulation in sediments. Particularly important is the extent of complexation of trace metals by humic and fulvic acids found in natural waters. The stabilities of metal-organic complexes in natural waters are greater than those of the corresponding inorganic metal complexes. Unfortunately most thermodynamic models have been developed to include the behavior of only inorganic ligands (I),or of inorganic and synthetic organic ligands ( 2 , 3 ) . I t is often assumed that because of the low concentration of the organic matter or the relatively high concentration of major cationic species, the chelation of trace metals is insignificant. In reality, the most important reason humic matter has been omitted from thermodynamic models is that these molecules are chemically very complex and not yet well understood ( 4 ) . 'Present address,Department of Chemistry,University of Michigan, Ann Arbor, Mich. 48104. 0003-2700/78/0350-1675$01,00/0
To measure metal-humic matter interactions in natural waters, it is necessary to: (1) know and define the concentration of humic matter in the system; (2) isolate and characterize this mixture of compounds; (3) propose model compounds based upon the chemical information obtained in characterization; and (4) evaluate and compare the complexation-chemistry of the natural organic matter and the model compounds. It is the aim of this research to focus upon the fourth point, specifically to measure conditional stability constants between Cu2+and well-characterized water fulvic acid (WFA) and soil fulvic acid (SFA). Reuter and Perdue ( 4 ) recently reviewed the importance of heavy metal-organic matter interactions in natural waters. The review includes a discussion of the advantages and limitations of several analytical techniques that have been used for stability constant determinations. References not in the review include potentiometric titration (5),equilibrium dialysis (6), and ion selective electrode (ISE) techniques (7). Differential pulse polarography (DPP) and differential pulse anodic stripping voltammetry (DPASV) have also been used to determine humic matter-metal ion stability constants (8-10). Several groups (8-12) have emphasized the difficulty of doing D P P and DPASV measurements of free metal ion concentration in the presence of humic matter and other surfactants. For this reason we compared the results of the titration of Cu2+by fulvic acid (FA) using both the DPP and ISE techniques. Because we found the former method to be unreliable in our system, all the useful conditional stability constants originated from the ISE experiments. EXPERIMENTAL Apparatus. The differential pulse polarograms were obtained with a Princeton Applied Research (PAR) model 174 Polarographic Analyzer, PAR model 315 Automated Electroanalysis Controller, PAR model 172A Mercury Drop Timer and recorded with a Houston Omnigraph 2000 XY recorder. A platinum wire counter electrode and PAR 9331 saturated calomel reference electrode with salt bridge were used in a PAR 9301 cell fitted with a glass water jacket. Dissolved oxygen was removed from solutions by bubbling nitrogen through the cell for at least 10 min and passing it over the solution during electrolysis. All experiments were performed at 25.0 OC. An Orion model 94-29A Cu2+ISE, the reference electrode of a Corning model 476050 combination electrode, and an Orion model 701 digital pH meter were used for all ISE experiments. The PAR 9301 cell was used to hold the test solutions; again all C 1978 American Chemical Society
