On the other hand, the hardware controlled titrator, while less versatile and less accurate than the computerized titrator, is much simpler and less expensive. It is particularly well suited to routine applications where relative standard deviations of the order of 0.4% are adequate. The titrant delivery device could also be modified to produce smaller droplets of titrant ( 3 ) .This would enable titration of very small samples, such as single cells, provided that suitable micromonitors are available.
LITERATURE CITED (1) (2) (3) (4)
(5) (6) (7) (8) (9)
Hieftje and B. M. Mandarano, Anal. Chem., 44, 1616 (1972). D. G. Mitchell and K. M. Aldous, Analyst, 98, 580 (1973). G. M. Hieftje and H. V. Malrnstadt, Anal. Chem., 40, 1860 (1968). H. V. Malrnstadt, C. G. Enke and S. R. Crouch, "Electronic Measurements for Scientists,"W. A. Benjamin, New York, N.Y., 1974. A. Savitzky and M. J. E. Golay. Anal. Chem., 36, 1627 (1964). J. Steiner, Y. Terrnonia, and J. Deltour. Anal. Chem., 44, 1906 (1972). G. Gran, Analyst, 7 7 , 661 (1952). J. Frazer, personal communication, 1974. H. V. Malrnstadt and E. H. Piepmeier, Anal. Chem., 37, 34 (1 '365). G. M.
ACKNOWLEDGMENT
RECEIVEDfor review August 8, 1974. Accepted. November
The authors express their appreciation to Maurice Williams and Larry Sexton for their help in construction of some of the apparatus used in this study. Thanks are also due to B. M. Mandarano, who performed some of the first fixed-level titrations with the reported titrator.
27, 1974. Support of this work by grant GM17904-03 from the National Institutes of Health is gratefully acknowledged. Presented in part at the 25th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1974.
Indirect Polarographic Microdetermination of Fluorine in FluoroOrganic Compounds after Oxygen-Flask Combustion Y. A.
Gawargious, Amir Besada, and B. N. Faltaoos
Microanalytical Research Laboratory, National Research Centre, Dokki, Cairo, Egypt
Two indirect polarographic methods are described for the microdetermination of fluorine In fluoro-organlc compounds after oxygen-flask combustion. In one, the liberated F- is precipitated as PbCIF, In the presence of CI- and 70% ethanol, wlth excess Pb(N03)2 and is measured indirectly by recording the polarographic cathodic wave of the unconsumed Pb2+. In the other, the F-, in about 62% acetone medium, is Precipitated as CaF2 with excess calcium iodate and is measured indirectly by polarographically recording the cathodic reduclion wave of the free iodate released. Nine fluoro- and perfluoro-organic compounds were analyzed by the two methods. For each method, the error is about f0.4%.
Several methods exist for the determination of fluorine in organic compounds; the subject has already been reviewed by Macdonald ( I ) . After the sample is decomposed by one of various techniques (2, 3 ) , the F- can be determined gravimetrically (4-6), titrimetrically (7, 8 ) , potentiometrically (9-1 1) using fluoride ion-specific electrode, or spectrophotometrically (12-14). Titration of the F- with thorium nitrate (7), the procedure most commonly used, has limitations; the reaction is reported ( 1 5 ) to be nonstoichiometric over the F- range 1to 50 mg. As already known (15), many difficulties are associated with the methods available for the determination of fluorine in organic samples, particularly for the microdetermination of perfluoro-organic compounds. Moreover, no polarographic methods, neither direct nor indirect, exist for such a determination, primarily because F- does not exhibit polarographic characteristics (16). Although previous work in this laboratory has already established polarographic methods for the microdetermination of other halogens in organic compounds (17, 181, such methods for fluorine determination are still lacking. 502
*
In the present work, two new indirect polarographic methods were developed for the microdetermination of fluorine in fluoro-organic compou.nds after oxygen-flask combustion. One depends on precipitation of the released F- as lead chlorofluoride (PbClF) with Pb(N0& followed by polarographic recording of the cathodic wave of the excess Pb2+. The equations involved in the precipitation and redox reactions may be represented as follows: F-
+
Pb(NO,),
+
Cl-
-+
PbClF1
Pb2+ 1- 2e ---
+
2NO,-
Pb
(1) (2)
In the other method, F- is reacted with calcium iodate, Ca(IO&, and the cathodic reduction wave of the free 103liberated is measured polarographically. The precipitation and redox reactions involved may be expressed as follows: 2F-
+
IO,-
Ca',IO,),
+
6H'
+
-
6e
CaF,
-
I-
1 + 2103-
(3)
+
(4)
3H,O
EXPERIMENTAL Apparatus. An Orion-KTS 510 polarograph (Hungarian make) with accessories was used. The combustion was carried out in a 500-ml oxygen flae k. The electrolytic vessel was an ordinary Kalousek cell with a i:athode compartment that allowed sample solutions as small as 4 ml to be polarographed. The dropping mercury electrode (DME) had a drop time of 3 to 4 sec under an open head of 75 cm of mercxry. A saturated calomel electrode (SCE) was the anode. Reagents. A l l reagents were AR or MAR grade, and doubly distilled water wiis always used. Lead nitrate, about 0.01M solution, prepared by c'iissolving about 3.3123 grams of Pb(N03)z in doubly distilled water and made up to one liter with water. Potassium iodate, about 0.01M solution, prepared by dissolving about 2.1401 grams of K'J.03 in doubly distilled water and made up to one liter with water Calcium iodate was measured as a 10% suspension in water. Proced ures. The sampling, weighing, and combusting of the or-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, M A R C H 1975
A p p i i r d p o t e n t i a l , Ed.. m I
S C E
Volts
Figure 1. ( A ) Cathodic waves of Pb2+ [ 10 ml of 0.01M Pb(N03)2 solution]. (a) Blank (no F-). (a') Sample (1.520 milligram F-). Start, -0.2 V; sensitivity 10 pA full scale deflection. (6) Cathodic wave of IO3- liberated. (6) Blank (no F-). (b')Sample (1.330 mg F- content. Start, -0.9 V; sensitivity 30 p A full scale deflection
ganic compounds by the oxygen-flask method were similar to those described by Macdonald (19). The fluoro-organic samples analyzed included liquid and solid aliphatic and aromatic compounds. The Lead Chlorofluoride Method. For Solid Fluoro-Organic Compounds. Place about 10 ml of doubly distilled water in a 500ml oxygen flask. Weigh accurately 3 to 6 mg of the fluoro-organic compound onto a piece of filter paper and add to the paper about 15 mg of NaN03. Wrap the solids and combust the sample. Shake the combustion flask for 5 min; then allow it to stand for 2 to 3 min. Remove the stopper; rinse down the walls of the flask and the platinum gauze with 10 ml of absolute ethanol. Quantitatively transfer the contents of the flask to a 100-ml volumetric flask using 60 ml of ethanol. Add 8 ml of 0.01M NaCl solution followed by 10 ml of Pb(N03)Z solution (0.01M). Shake the flask gently; a dropwise until the white precipitate is formed. Add 0.2M "03 solution is just acid to methyl red indicator (0.2 ml of a 0.15% ethanolic solution of methyl red). Dilute the solution to the mark with water and mix it completely. Record the polarogram of an aliquot of this solution; use a starting potential of -0.2 V us. SCE and a sensitivity of 10 FA full-scale deflection under otherwise appropriate polarographic conditions. Repeat the same procedure without sample combustion using 10 ml of the P b ( N 0 3 ) ~solution (0.01M) and record the Pb2+ reduction wave under identical conditions. The difference in the height (in mm) of the wave for the blank [Figure IA, ( a ) ] and that for the sample [Figure l A , ( a ' ) ] corresponds to the amount of Pb2+ consumed in the precipitation of the PbClF. Calculate the fluorine content from a previously constructed calibration curve, Figure 2A. This curve was obtained by running increasing amounts of NaF, (0.380 to 1.520 mg F-) as a standard, through the same procedure and recording the cathodic waves of Pb2+ remaining in solution after the precipitation of PbClF. Deduction of the height of the wave of the unconsumed Pb2+ from that of the blank gives the respective wave height of the consumed Pb2+ (shown as the ordinate in Figure 2A) which is equivalent to the amount of F- present in the sample solution. For Liquid Fluoro-Organic Compounds. Weigh a cellotape (glued cellophane roll, English make, commercially available) bag ( 2 0 ) ,prepared from a strip (3 X 4 cm) lined with a small L-shaped filter paper (2 X 1 cm) on the glued side, which contains about 20 mg of cotton wool, a linen thread about 3 cm long, and about 5 mg of NaN03. Absorb 3 to 6 mg of the liquid sample on the thread by holding one end of the thread with platinum-tipped forceps and immersing the other end in the sample. Introduce the thread into the bag; then close the bag tightly and reweigh it. Carry out the
/
0
I
I
1
I
0.6 0.9 F-concentration, mg 1 5 0 m l
I
I
I
1 .?
Figure 2. Calibration curves for the indirect polarographic determination of F- in fluoro-organic compounds. ( A ) PbClF method. ( B ) Ca(103)* method
combustion and absorption and complete the procedure as described above for solid fluoro-organic compounds. The Calcium Iodate Method. Weigh accurately 3 to 6 mg of the solid or liquid sample as already described and add about 20 mg of benzoic acid instead of NaN03, which is used in the PbClF procedure. Combust the sample according to the oxygen-flask procedure using 5 ml of doubly distilled water for absorption; shake the flask thoroughly. Rinse down the platinum gauze and the walls of the flask with 5 ml of water. After 2 min, add 2 ml of the suspension of a 10% aqueous solution of Ca(I03)2, heat the mixture for 1 min, and set the flask aside for 2 min to cool. Add 20 ml of acetone and allow the solution to stand for 10 min to permit complete precipitation of the CaF2 produced and of the excess Ca(I03)2present. Filter the contents by suction through a sintered-glass funnel (G5) into a conical flask; for the washing, use 5 ml of a 3:5 wateracetone mixture. Quantitatively transfer the filtrate to a 50-ml volumetric flask, add 2 ml of 5M NaOH solution, and dilute the resulting solution to the mark with doubly distilled water. Polarograph an aliquot of the solution and record the cathodic reduction wave of IO3-; use a starting potential of -0.9 V us. SCE as anode and a sensitivity of 30-pA full-scale deflection under otherwise suitable polarographic conditions. Measure the height of the wave due to the liberated 103- and calculate the fluorine content from a calibration curve (Figure 2B) constructed previously between increasing amounts of sodium fluoride (0.380 to 1.330 mg F-), as standard carried through the same procedure, and the height (in mm) of the cathodic reduction wave of the sodium iodate liberated. A typical polarogram representing the cathodic wave of 103released is shown in Figure 1B.
RESULTS AND DISCUSSION The Lead Chlorofluoride Method. Preliminary work has shown that quantitative recoveries of fluorine can be obtained only after mixing the sample, prior to combustion, with an auxiliary oxidizing agent. Sodium nitrate was selected for this purpose since, besides functioning as a powerful additional oxidant, its combustion products do not interfere in the subsequent polarographic recording of the Pb2+ wave. The use of NaN03 proved efficient for the complete decomposition of both partially and fully fluorinated organic compounds. ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
503
-
Table I. Results of the Indirect Polarographic Microdetermination of Fluorine i n Fluoro-Organic Compounds by Use of the Pb2+ PbO Reduction Wave F, 56 Compound
Sample weight, mg
Calcd
Found
Enor, %
o -Fluorobenzoic acid
4.500-7.500 4.03 0- 6.430 4.620-6.605 2.955-3.345 4.905- 5.450 3.910-4.500 3.005-4.050 2.950-3.606 2.960-3.585
13.56 11.58 11.58 17.09 17.25 19.78 30.14 40.11 55.85
13.19-13.15 11.29-11.14 11.26- 11.91 16.79-17.70 17.01- 16.74 19.34- 19.30 29.80-29.70 39.79-40.65 55.4 1- 55.25
- 0.37--0.41 - 0.29--0.44
6 -Fluorocumarone 8 -Fluorocuniarone p -Fluoroaniline p -Fluorotoluene Fluor o benzene Trifluoroacetanilide Ethyltrifluoroacetate Perfluoronaphthalene
-0.32-+O. 33 - 0.30- to.61
-0.24--0.51 -0.44--0.48 -0.34--0.44 - 0.36-+O. 54 -0.44-0.60
Std dev, 0
10.38 i-0.39 10.37 k0.46 i.o.01 k0.01 k0.05 k0.40 10.12
-
Table 11. Results of the Indirect Polarographic Microdetermination of Fluorine in Fluoro-Organic Compounds by Use of the 1 0 3 I- Reduction Wave F, % Compound
Sample weight, mg
Calcd
Found
Error, %
o -Fluorobenzoic acid
3.950-4.650 4.330-6.230 3.920-6.415 4.150-5.050 3.500-4.765 3.225-4.900 3.175-4.470 2.855-3.605 2.755-3.050
13.56 11.58 11.58 17.09 17.25 19.78 30.14 40.11 55.85
13.10- 14.00 11.30- 11.25 11.90- 11.23 16.90- 17.61 17.65-16.80 19.30- 19.40 29.61-29.81 39.67-39.51 55.20- 55.30
-0.46-+0.44 -0.28--0.33 +0.32--0.35 -0.19-~0.52 +0.40--0.45 -0.48--0.38 -0.53--0.33 -0.44--0.60 -0.65--0.50
6 -Fluorocumarone 8 -Fluorocumarone p -Fluoroaniline p -Fluorotoluene Fluorobenzene T rifluoroacetanilide Ethyltrifluoroacetate Perf luor onapht halene
The appreciable solubility of the PbClF precipitate is significantly minimized in 70% ethanolic medium, which contributes greatly to the success of the method. The consumption of the Pb2+ increases proportionally with the increase of the fluorine content, as can be deduced from the calibration curve (Figure 2 A ) , constructed between milligram amounts of NaF and the wave height (in mm) corresponding to the Pb2+ consumed. This is obtained by subtracting the height of the wave due to the unconsumed Pb2+ (remaining in the sample solution after precipitation of the PbClF) from the height of the wave of the blank experiment. The fact that the straight line relation obtained passes by the origin on extrapolation indicates that no solubility of the PbClF precipitate occurred in the medium at least along the F- range tested under the conditions selected. On applying the established procedure, reasonably satisfactory results (Table I) were obtained for nine representative fluoro-organic compounds that included partially and fully fluorinated solid and liquid aliphatic and aromatic compounds. The absolute error is f0.60% and the total average recovery is 98.89%. T h e Calcium Iodate Method. The success of the polarographic methods developed previously (17, 18) for the microdetermination of chlorine, bromine, and iodine in organic compounds encouraged the search for a parallel method for fluorine. A suitable approach was found in reacting F- with Ca(I03)2.This reaction was studied (21) for the iodometric estimation of some alkali fluorides; a correction factor was reported to be essential to compensate for the partial precipitation of the liberated alkali iodate in the 65% isopropanol medium used. Moreover, an504
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
Std dev, 0
i0.47 k0.35 *O. 38 k0.36 *O. 50 k0.46 +O. 04 &O. 08 +O. 08
other source of error is associated with the end-point detection. Since starch cannot be used in such highly alcoholic medium, visual detection by the disappearance of the yellow color of iodine is taken as the end point, which is known not to be sharp enough on the micro scale. In the present work, these two sources of error were eliminated. The first was overcome by use of about 62% acetone medium, which not only is efficient in precipitating the excess Ca(IO3I2 but also does not cause any precipitation of the liberated sodium iodate when NaF was used as the standard for the construction of the calibration curve (Figure 1B).The lack of error was verified experimentally since a linear relation extrapolatable to the origin was obtained on plotting the increasing mg amounts of NaF taken us. the corresponding measured wave heights (in mm) of the sodium iodate released; proportionality is obvious within the F- range tested. The cathodic IO3- wave vanished completely in all media that contained even the least amount of isopropyl alcohol, which is, of course, unsatisfactory both chemically and polarographically in the present method. After the conditions necessary for reproducible results for inorganic fluoride were established, preliminary work on fluoro-organic compounds showed that NaN03 cannot be used as an auxiliary oxidant, since NaOH, most probably produced after combustion and absorption, interferes seriously with the reaction between F- and Ca(I03)2.This interference is evident from the excessively high fluorine recovery, which apparently is due to the reaction between the NaOH formed and the Ca(I03h added, the result being the liberation of additional sodium iodate. However, the use of benzoic acid (about 20 mg) as an alternative decomposition (10) aid proved successful with both partially and
(9) J. Pavel, R. Kuebler, and ti. Wagner, Microchem. J., 15, 192 (1970).
fully fluorinated fluoro-organic compounds. Furthermore, benzoic acid was found to be as efficient as NaN03 when used in the PbClF method. The procedure developed gave satisfactory results (Table 11) for the whole series of fluoro-organic compounds analyzed. The absolute error is f0.60%, and the total average recovery is 99.30%. Although these results are generally less accurate than those expected (11)for the fluoride ion-sensitive electrode methods, yet the present work offers two new indirect polarographic methods that can also serve valuably in other cases.
(IO) D.A. Shearer and G. F. Morris, Microchem. J., 15, 199 (1970). (11) W. Selig, Fresenius'Z. Anal. Chem., 249, 30 (1970). (12) N. I. Larina and N. E. Gel'man, Zh. Vses. Khim. Obshchest., 15, 231 (1970). (13) E. C. Olson and S. R. Shaw, Microchem. J., 5, 101 (1961). (14) M. E. Fernandopulle and A. M. G. Macdonald, Microchem. J., 11, 41 (1966). (15) G. Ingram, "Methods of Organic Elemental Microanalysis," Reinhold, New York, N.Y., 1962, p 209. (16) I. M. Kolthoff and J. J. Lingane. "Polarography," 2nd ed., lnterscience Publishers, New York and London, 1952. (17) Y. A. Gawargious, G. M. Habashy, and E. N. Faltaoos, lndian J. Chem., 7, 610 (1969). (18) G. M. Habashy, Y. A. Gawargious, and E. N. Faltaoos, Talanta, 15, 403 (1968). (19) A. M. G. Macdonald. Analyst (London), 86,3 (1961). (20) W. I. Awad, Y. A. Gawargious, S. S. M. Hassan, and N. E. Milad, Anal. Chim. Acta, 36, 339 (1966). (21) W. I. Awad. S.S. M. Hassan, and M. E. Elsayes, Mikrochim. Acta, 1969, 688.
LITERATURE CITED A. M. G. Macdonald. Advan. Anal. Chem. instrum., 4, 100-103 (1965). T. S.Ma, Anal. Chem., 30, 1557 (1958). T. S.Ma and M. Gutterson, Anal. Chem., 42, 105R (1970). R. Belcher and A. M. G. Macdonald, Mikrochim. Acta, 1957, 510. T. S.Ma, Microchem. J., 2, 91 (1958). W. Selig. Fresenius'Z. Anal. Chem., 234, 261 (1968). R. Belcher, E. F. Caldas, S. J. Clark, and A. M. G. Macdonald, Mikrochim. Acta, 1953, 283. W. Schoniger, Mikrochim. Acta, 1956, 869.
RECEIVEDfor review April 22, 1974. Accepted September 11, 1974.
Automated Determination of Nitrogen in Milk Products H. W. Schafer and N. F. Olson Department of Food Science, University of Wisconsin-Madison, Madison, Wis. 53706
Procedures were developed to determine concentrations of nitrogen in milk and cheese and fractions of these products by a modlflcatlon of the Kjeldahl method using the Technicon AutoAnalyzer. Mean percentage differences between concentrations of nitrogen determined by the automated and AOAC methods ranged between 0.7 and 2.9% of the nitrogen concentratlon measured in the various types of samples. Much better precision between replicate measurements was obtained with the automated method. No statistically significant differences were observed between methods in measurement of total nitrogen in milk and cheese and of noncasein and nonprotein nitrogen in milk. Statistically significant differences were found in measurement of noncasein nitrogen of cheese and total nitrogen of cheese whey. Differences between the methods were equal to 2.9% of noncasein nitrogen concentration in cheese and 1.1 % of nitrogen concentration In whey.
Measurement of recovery of milk proteins during cheese manufacture and monitoring subsequent proteolysis of cheese are important observations in quality control and research functions of the cheese industry. The time and expenditures required for manual nitrogen analysis have led to the development of automated Kjeldahl nitrogen analyses (1-7) and alternatives to the Kjeldahl method (8-11). Automated Kjeldahl analyses of organic materials using the Technicon AutoAnalyzer generally required adjustment of digestion conditions and choice of a suitable standard for the type of sample being analyzed. The standards included @-alanineand freeze-dried meat for meat (12-14 ), 2-benzyl-2-pseudo-thiourea for milk (12-14 ), mixtures of nicotinamide and urine for urine, feces, and homogenates of hospital patients' diets ( 1 5 ) , and glycine for urine and feces samples ( 1 6 ) . Various ammonium salts have been
used as standards for soil and feed samples which were predigested in a block heater and analyzed with the Technicon AutoAnalyzer ( 7 ) . In the present study, total nitrogen content of milk, whey, and cheese and the amounts of noncasein nitrogen in cheese, and milk, and nonprotein nitrogen in milk were determined using the Technicon AutoAnalyzer I1 (17). Subsequent to initiation of this study, Kramme et al. ( 1 8 ) described a modified Technicon AutoAnalyzer system which was used for analysis of amino acid mixtures, casein, urea, and nicotinamide. The modifications included higher digestion temperatures, longer time for color development after adding phenol and hypochlorite, and expansion of the range of the recorder. This procedure allowed use of ammonium sulfate as a standard for all types of samples tested. Ammonium sulfate was used in another study, for analysis of a variety of foods, by increasing residence time of the sample in the digestor helix and adjusting the perchloric acid concentration and digestor temperature (19). However, agreement between this procedure and AOAC Kjeldah1 method was not very satisfactory for samples of condensed milk. The Kjedahl nitrogen analysis system of the Technicon AutoAnalyzer I1 was used without modification in our investigations. A stock standard of acid-precipitated, whole casein in distilled-ionized water or 0.1M sodium citrate was diluted with various solutions to make working standards for analysis of various milk-based products. We did not choose 2-benzyl-2-pseudo-thiourea as a standard as suggested by Brisson (13), because of nonlinearity of standard curves obtained during preliminary trials.
EXPERIMENTAL Apparatus. Analyses were made with the basic Technicon AutoAnalyzer I1 system consisting of sampler IV, proportioning pumps I and 111, continuous digestor, single-channel colorimeter, ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, M A R C H 1975
505
