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-
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Equation
spectrometer. The ratios were significantly different for all the mono-, di-, and tetrachlorodibenzo-p -dioxin isomers (see Tables 11-IV), thus indicating energetically dissimilar decomposing molecular ions. It should be noted that although the differences between the ratios obtained for the 2,8- and 2,7-dichloro isomers were small, the values obtained were the averages of 4-5 scans in which the standard deviation for both ratios was less than 0.005. The results could be interpreted to indicate that either the decomposing molecular ions retained their substituent identity (Le., chlorine) or that these decomposing ions possessed differences in internal energy content. The data also indicated that IKE spectrometry yields distinctive fingerprint spectra for all the isomeric chlorinated dibenzo-p -dioxins and thus provides an additional spectrometric technique for the identification and analysis of structurally similar organic molecules. The technique is not as sensitive as ET mass spectrometry or gas chromatography but it is conceivable that with additional technical improvements and the use of mass-analyzed IKE spec-
4
trometers this techniaue coulc be used in the future or examination of environmental samples by focusing on certain diagnostic peaks.
LITERATURE CITED (1) B. Pring and N. E. St. Jernstrom, Acta Chem. Scand., 22, 549 (1968). (2) E. N. Givens, L. Alexakos. and P. B. Venuto, Tetrahedron. 25, 2407 (1969). (3) N. P. Buu-Hoi, G. Saint-RuF. and M. Mangane, J . Heterocycl. Chem., 9, 691 (1972). (4) J. H. Beynon. R. G. Cooks, J. W. Amy, W. E. Baitinger, and T. Y. Ridley, Anal. Chem., 45, 1023A (1973), and references cited. (5) S. Safe. 0. Hutzinger, W. D. Jamieson, and M. Cook, Org. Mass Spectrom., 7 , 217 (1973). (6) S.Safe and 0. Hutzinger. Chem. Commun., 260 (1972). (7) S. Safe, 0. Hutzinger, and W. D. Jamieson, Org. Mass Spectrom., 7 , 169 (1973). ( 8 ) W. D. Jamieson and F. G. Mason, Rev. Sci. Instrum., 41, 778 (1970) (9) A. E. Pohland and G. C. Yang, J. Agr. FoodChem., 20, 1093 (1972).
RECEIVEDfor review December 10, 1973. Accepted July 1, 1974.
Application of an Improved Spectrophotometric Azide Determination to Azides Used for Enzymes Immobilization Oreste Brenna, Mario Pace, and P. G. Pietta lstitufo di Chimica Organica ed Analitica 2, Via Celoria-20
733 Milano, /fa@
Enzymes have been covalently bound to insoluble polymeric matrixes by several methods ( 1 - 5 ) . One of these methods is based on the reaction of the free amino groups of enzymes with the azido groups introduced by chemical modification of natural and synthetic polymers (6, 7 ) . An easy and rapid method to quantitatively determine the azido groups present on a polymeric matrix would be useful for its characterization, e.g., for the estimation of the active groups following the modification of the polymer under varying conditions and for a stability test of the activated polymer during storage.
The elemental analysis is not the method of choice because the low nitrogen content of the derivative cannot be measured with sufficient accuracy. The methods of assay of organic azides already described (8-10) have as a common feature the release of the azido groups as inorganic azide ions. These are then assayed by gravimetric, volumetric, or spectrophotometric methods. First we improved the spectrophotometric method of Anton et al. (11) because of its simplicity as compared with other methods. It is based on the spectrophotometric determination of the complex Fe3+-N3-. We studied the dependence of the absorbance of the complex from pH, fer-
A N A L Y T I C A L CHEMISTRY, VOL. 47, NO. 2 , FEBRUARY 1975
*
329
01
c m n
10.
. -
L
0 yi
n
4
-
.
05 -
05
~
10
15
20 PH
25
75
50
100
Figure 3. Absorbance of the complex Fe3+-N3-. dependence on pH
Fe3'(rnM/I )
Figure 1. Absorbance of the complex Fe3+-N3-: ferric ion concentration
0.1
0.25
05 ionic
dependence on
0 75 strength
1.0
Flgure 2. Absorbance of the complex Fe3+-N3-: dependence on ionic strength
(0)Fe3+ = 10 rnM/I. (x) Fe3+ = 22.2 rnMII.
ric ion concentration, and ionic strength. Then we applied this method to determine the azide content of azido derivatives obtained from carboxymethylcellulose, polyacrylamide, and other polymers.
EXPERIMENTAL Materials. All materials used were reagent grade. Polyacrylamide P-300 was obtained from Bio-Rad, carboxymethylcellulose CM-11 from Whatman, and carboxymethyl CM-Sephadex C-50 from Pharmacia. PMA-cellulose (cellulose polymethylacrylate) (12) with add-ons of 10, 35, and 100% were kindly given us by B. Focher of the Stazione Sperimentale Cellulosa Carte Fibre Tessili Vegetali ed Artificiali, Milan, Italy. Methods. I m p r o v e m e n t i n A z i d e Determination. The absorbance of the N3--Fe3+ complex depends chiefly on three parameters, namely, ferric ion concentration, ionic strength, and pH. To understand the relative importance of these three parameters, a constant amount of sodium azide was assayed while keeping constant two of the parameters and varying the third. Dependence on Ferric Ion Concentration. A constant quantity of NaN3 was assayed a t varying molar amounts of ferric ion. The ionic strength was kept equal to 1 by addition of solid NaC104 and the p H equal to 2 by addition of concentrated HC104 or NaOH. The absorbance changed with the increasing ferric ion concentration and attained a constant value a t concentrations greater than 25 mmoles/l. (Figure 1). Dependence on Ionic Strength. A constant quantity of NaN3 was assayed, keeping both the molarity of the ferric ion and the
330
pH constant (equal 2 , as above), but varyirig the ionic strength by addition of solid NaC104. The absorbance changed with the increasing ionic strength and attained a plateau at ionic strength greater than 0.80 (Figure 2). Dependence on pH. A constant quantity of NaN3 was assayed, keeping the molarity of the ferric ion a t 0.1M and varying the pH by addition of concentrated HC104 or NaOH. The absorbance increased with pH, reaching a maximum value near pH 2.2 (Figure 3).However, ferric hydroxide begins to precipitate a t this pH. According to these experimental findings, a 0.1M solution of NH4Fe(S04)2*12H2O in distilled water was chosen for the assay. P r e p a r a t i o n of Azide Derivatives. Azido derivatives of P-300 were obtained as described by Inman and Dintzis (6);CM-11 derivatives were obtained in accord with Wharton et al. ( 7 ) . Carboxymethyl Sephadex (2-50 azido derivatives were prepared as follows. A suspension of the polymer ( 5 g) in methanol (50 ml) and concentrated HC1 ( 2 ml) was refluxed for 2 hr. The product was filtered, washed with water to remove the acid, then with 60% methanol, absolute methanol and dried overnight. The hydrazide derivative was obtained by reaction with 250 ml on 2M hydrazine a t 40 "C for 12 hr. The polymer was washed with water until the washings gave a negative test with trinitrobenzensulfonic acid ( 6 )and was suspended in 200 ml of 0.5N HC1. The stirred suspension was cooled a t 0 "C and reacted with 50 ml of 0.5M NaN02 added dropwise over a 10-minute interval. The polymer was allowed to react for a further 20 minutes and then transferred to a Buchner funnel and quickly washed with icecold water. Azide derivatives from PMA-cellulose were obtained similarly by reacting the polymer with 75% hydrazine for 2 hr and then with 0.5N HC1 and 0.5M NaN02.
PROCEDURE FOR AZIDE ASSAY The freshly prepared azido derivative was washed 2-3 times with ice-cold water to remove the acid. Five to 10 ml of the packed suspension were stirred with 20 ml of 0.1N NaOH in a covered 50-ml polyethylene beaker for 2 hr at room temperature. The suspension was allowed to settle and the clear supernatant was used for the assay. One-tenth ml supernatant was added to 4.9 ml of 0.1M NH4Fe(S04)2.12 H20. The absorbance was measured us. a blank (4.9 ml of 0.1M NH4Fe(S04)2. 1 2 H20 + 0.1 ml of 0.1N NaOH) using a Beckman Acta I11 spectrophotometer. It was necessary to read the adsorbance within five minutes because hydrazoic acid is slowly released in acid medium. Beer's law is followed from 0.01 to l.5mM azide concentration (c458,1 cm = 1.2 x lo6 cm2 A 4 - I ) . The azide concentration can be calculated from the following formula:
N,-(mequiv/l.) =
~
A 1.2
5
X __
(1)
0.1
where the azide concentration is referred to a liter of supernatant. The azide concentration in mequiv per gram of dry polymer can be obtained from the following relation
ANALYTICAL CHEMISTRY, VOL. 47, NO. 2, FEBRUARY 1975
N,-(mequiv/g) = N,-(mequiv/l.) x b x
1 C
(2)
where b is the total water content of the suspension in liters and c is the dry weight in grams of the assayed polymer. b can be obtained by the following relation:
b = P - (T + c )
(3)
where 7' is the weight of the polyethylene beaker; P is the sum of the weight of the beaker, of the azido polymer suspension and of the base used for the hydrolysis. c is obtained by precipitating the hydrolyzed polymer with 60% methanol, washing the precipitate with absolute methanol, and drying to constant weight in a vacuum desiccator over CaC12 a t room temperature. The density of the polymers and of the hydrolysing solutions were assumed equal to 1.
RESULTS AND DISCUSSION Since only one standard solution is required for azide determination, the improved spectrophotometric method described here is very simple and rapid. No pH adjustments are required. In fact the 0.1M solution of NH4Fe(SO4)2. 12 H20 gives pH 1.98 f 0.01 and the addition of 0.1 ml of 0.1N NaOH to 4.9 ml of the above solution does not cause pH changes greater than the experimental error in the pH determination. This pH is just below the maximum colorimetric yield obtainable without precipitation of iron hydroxide in the assay medium. Furthermore, the ferric ion concentration and the ionic strength of the 0.1M solution of ferric ammonium sulfate are in the range in which the N3--Fe3+ complex gives the maximum colorimetric yield (Figures 1-3). From the examination of the azido derivatives of the matrixes, we obtained the following results. The capacity was measured as mequiv of azido groups per dry gram of polymer. The capacity of the CM Sephadex C-50 azido derivatives prepared as described was 2.4 mequiv Ns- per dry gram. A greater capacity was obtained by increasing the time of the esterification. However, the degradation of the polymer became appreciable because of the very acidic medium. Azido derivatives of PMA-cellulose showed a capacity of 0.7, 1.5, and 3 mequiv N,-/dry gram of cellulose PMA with add-ons of 10,35, and 100%respectively. Add-on is defined as copolymer weight (after treatment with acetone to remove the homopolymer PMA) less the initial weight of cellulose to be treated, divided by this last weight, %. Azido derivatives for CM-cellulose were found to reach the maximum capacity when the reaction time of CM-11 methylester derivative with hydrazine hydrate in methanol a t 40 "C to yield the hydrazido derivative was about 12 hr (Figure 4a ). The capacity of P-300 derivatives was also dependent on the reaction time of the polyamido polymer with 6M hydrazine at 47 "C to yield the hydrazido derivatives (Figure 4b). Inman and Dintzis (6) reported a linear relationship be-
L
- 05
1
>' u
E 2
I
02
1
b 2
4
6
8
reaction
1 2 2 4
time ( h )
&.e
O1
72
Figure 4. Capacity of polyacrylamide P-300 (a ) and carboxymethylcellulose CM-11 derivatives ( b ) : dependence on the reaction time with hydrazine during hydrazido derivatives preparation
tween the reaction time of activation and capacity in the hydrazido derivatives. We also found this linear relationship, at least for the first 8 hr. Further reaction times had to be avoided because the successive conversion of the hydrazido polymers so obtained to the azide form yielded gummy polymers not suitable for enzyme immobilization.
PRECISION AND ACCURACY OF THE METHOD The capacity values shown in Figure 4,a and b, were the averaged values of three analyses of the same samples. The precision reported as relative deviation from the mean was 1.4 and 3.1% for the CM-11 and P-300 azide derivatives, respectively. The accuracy of the method, defined as % error, was 0.8 and 1.7% for the above mentioned polymers. These data were obtained by comparing the averaged value of five analyses with the value obtained using a known amount of NaN3 added to the untreated polymer. LITERATURE CITED Y. Levin. M. Pecht. L. Goldstein, E. Katchalsky. Biochemistry, 3, 1905 (1964). H. H. Weetall and N. Weliky, Nature (London),204, 896 (1964). D. H. Campbell, E. Luescher, L . S. Lerman, Roc. Natl. Acad. Sci. US., 37, 575 (195 1). R. Axen, J. Porath, and S. Ernback. Nature (London), 214, 1302 (1967). M. A. Mitz and E. Summaria, Nature (London),189, 576 (1961). J. K. lnman and H. M. Dintzis, Biochemistry, 8, 4074 (1969). C. W. Wharton, E. M. Crook, and K. Brocklehurst, Eur. J. Biochem., 6, 565 (1968). E. L. Grove, R. S. Braman, H. F. Combs, and S. B. Nicholson, Anal. Chem., 34, 682 (1962). A. Messmer and S. Mlinko, Acta Chem. Sci. Hung., 29, 119 (1961). P. J. Staples. Chem. Ind. (London), 1210 (1960). A. Anton, J. G. Dodd, and A . E. Harvey, Jr., Anal. Chem., 32, 1209 (1960). P. Cremonesi, Cellul. Chem. Techno/., 2, 145 (1968)
RECEIVEDfor review June 28, 1974. Accepted September 18, 1974. This work was supported by Grant No. 73.00232.11.115.7151 of Consiglio Nazionale delle Ricerche, Rome.
ANALYTICAL C H E M I S T R Y , VOL. 4 7 , NO. 2, F E B R U A R Y 1975
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