I
Figure 5. The quantity, (E” - E& as a function of dielectric constant (D)for acetic acid-chloroform The base-base perchlorate mixtures (at 0.0013M) are half-neutralized lithium acetate ( I ) and tri-nbutylamine (2) (12). A gradual increase in AmV with decreasing dielectric
constant ( D ) can be noted in Table V. The quantitative verification of the electrostatic effect of the solvent upon the base perchlorate dissociation constant is shown in Figure 5 and conforms to theoretical expectations in part (13). The linear portion of the function, (EHN- EB)us. 1/D, indicates (12) 0. Kolling, ANAL.CHEM., 40,956 (1968). (13) L. Pettit and S. Bruckenstein, J. Amer. Chem. Soc., 88, 4783 (1966).
that this effect is a dominant one over the mole fraction range 0.0 to 0.52 in chloroform. The sharp rise in each curve is found for those solvent mixtures in the region of the minimum in the dielectric constant-mole fraction plot in Figure 1 . The mixed solvent of 1 :1 (v/v) proportion used as the titration medium for antihistamines (2) has a dielectric constant of about 4.7 at 25 O C . Since this solvent composition is close to the end of the linear portion of Figure 5 , the effect of increasing EXN - EB by a decrease in D is near the limit attainable in acetic acid-chloroform mixtures. On the other hand, the differential titration of group IA acetates with perchloric acid (3, 14) has been made in chloroform-rich mixtures beyond the minimum in Figure 1 and within the solvent composition range where changing dielectric constant has a very minor effect upon ERN. ACKNOWLEDGMENT
We are indebted to Wilton L. Cooper for the development of gas chromatographic procedures for the analysis of chloroform. RECEIVEDFebruary 1, 1971. Accepted March 23, 1971. Financial support for this investigation came from the National Science Foundation Grant No. GP 10015. (14) C. Pifer, E. Wollish, and M. Schmall, ANAL.CHEM., 26, 215 ( 1954).
Automation of Nitrogen Analysis of Grain and Grain Products D. E. Uhl, E. B. Lancaster, and Charles Vojnovich Northern Regional Research Laboratory, Peoria, Ill. 61604
A procedure was developed to determine the nitrogen content of some agricultural commodities in an automatic analyzer with accuracy and precision comparable to the Kjeldahl. Standards of material similar to the samples being analyzed contributed to better agreement between the two methods. The overall precision of both methods is estimated to be 2 or 3% of the measured mean value.
THETECHNICON NITROGENAUTOANALYZER is a continuous flow device for automatically digesting nitrogen-containing samples and mixing the digest with phenol and hypochlorite in alkaline solution to transform the ammonia into a n indophenol blue proportional to the nitrogen present. This reaction was first reported by Berthelot (1) and since has been discussed at length by others (2-4). The developed color is continuously measured by a recording spectrophotometer. The AutoAnalyzer was designed for the routine analysis of large numbers of samples of the same type of material. However, it can also be used for mixed samples. (1) M. P. E. Berthelot, Repert de Chim.Appl., 1859,282. (2) L. T. Mann, ANAL.CHEM., 35,2179 (1963). (3) W. T. Bolleter, C. J. Bushman, and P. W. Tidwell, ibid.,33, 592 (1961). (4) C . W. Gehrke, F. E. Kaiser, and J. P. Ussary, J . Ass. Ofic. Anal. Chem.,51, 200 (1968). 990
ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
Of particular interest in our laboratory are the following materials:
zN f Corn fractions Millet fractions Wheat fractions Sorghum fractions Formulated cereal foods Crambe, raw and defatted Soya, raw and defatted Fermentation media from grain products
0.25-2,50 0.50-4.00 1. W 4 . 0 0 0.863.00 1.00-3.50 4.00-9.00 6.00-8.50 0.0025-0.25
We have used the AutoAnalyzer to determine nitrogen of these diverse materials and concentrations. Good results were obtained when the samples were properly treated and when a suitable standard was selected. We will discuss here our modifications of the Technicon equipment and procedures and will compare statistically analytical results from the AutoAnalyzer with those obtained by the Kjeldahl method or the micro-Kjeldahlmethod (5). A number of changes made to improve the sensitivity and linearity of the standard curve met with varying degrees of (5) “Methods of Analysis” of Ass. Offic. Anal. Chem., 10th ed., Washington, D. C., 1965, p 14.
Input Manifold
[Inches]
0
I
01 E
0Acidflex
Pulse Chamber
/ 0.110 j
Dig. Mixli 0.110
; 0.110 ;
I
Sampler and Mixer
50% NaOH
m
tn Waste
Air Scrubber 5% HtSOl
HpSO4 Solution
Dig. Sample II
-
Dip. Sample
I
Output Manifold
2x
2x
Mixer
Mixer
II I I
I
1
0
; 0.045 I
0Water
I o.lti 1
i t O~Sampi!j0,06Ooj NaOH IO.081'1
0
0 Jacketed Mixer
-
Waste
II I I
1
Air
I
I
I0.065
j
I
I
OAlk. Phen.10.051'1 I
Time Delay Coil
NaOCl
I
Pull Thru 15 mm Tubular flowtell 630 m m pp Filters Filters 630
Colorimeter
Recorder lO"/hr
I
1 0.035 I I
10.073
\
Waste
Figure 1. The AutoAnalyzer flow system used incorporates several changes from the Technicon Manual. Changes are marked with an asterisk. The HgSO4 solution contains 5 Z Hg(OOCCH&, 10 % HzS04,and 10 % K S 0 4 . A7 is a jacketed mixing chamber success. Russell (6) reported that the presence of manganous ion improves the sensitivity of the color reaction. We were unable to substantiate Russell's work with the reagents recommended in the Technicon bulletin (7),probably because of the complexing salts in the sodium hydroxide solution. EXPERIMENTAL
Reagents and Equipment. The reagents used are those recommended by Technicon (7) with the following exceptions: Both Catanzaro (8) and Garza (9) suggested mercuric sulfate be added to aid digestion. Mercury or mercury salts have been used as catalysts for years in Kjeldahl digestions. They (6) J. A. Russell, J . Biol. Chem., 156,457 (1944). (7) Technicon Methodology Bulletin N-3b, Technicon Corp., Ardsley, N. Y.,1965, 5 pp. (8) E. W. Catanzaro, Technicon Symposium on Analytical Chemistry, New York, N . Y . , 1964. (9) A. C. Garza, Technicon Symposium, Automation in Analytical Chemistry, New York, N . Y . , 1965.
also aid in AuioAnalyzer digestions. We add mercuric ion in the form of mercuric acetate. Since mercuric salts are insoluble in the concentrated digestion mixture (7), we use a solution of 5 grams of mercuric acetate, 100 ml of concd %Sod, 100 grams of K&O4 made up to 1 liter with distilled water and introduce it into the continuous flow system just before the digestor at the rate of 1 ml/minute. We add 5 grams of ethylenediaminetetraacetate (EDTA) t o a liter of the standard 35 % sodium hydroxide, 5 potassium sodium tartrate solution. This amount of EDTA gave the best improvement in linearity of the standard curve with the least depression of sensitivity. A continuous supply of distilled dilution water (filtered if not absolutely clear) is mandatory, since a cessation of water flow can cause clogging in the manifold by sodium sulfate and consequent loss of operating time. It is also recommended that all reagents used after the digestor be filtered t o ensure a smooth recorder line. The flowsheet (Figure 1) includes our changes to the system ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
991
by soaking in 10 ml of water containing a wetting agent (Brij-35 supplied by Technicon). This step prevents a hard carbonized shell forming around the particles when sulfuric acid is added. Generally, the samples were wetted by overnight soaking although occasionally a sonic bath was used to expedite the wetting and solution process. After the wetting step, 10 ml of concentrated sulfuric acid was added to the samples. Heat of solution helps to &solve the sample. A loss of volume of 1.5 ml due to shrinkage when the sulfuric acid was added and to evaporation during soaking was compensated for by adding 1.5 ml of water at this point. The concentrations of samples to be run were kept below 30 mg of nitrogen per 100 ml SO that the final color remained in the most linear region of the relationship between absorbance and concentration. The low concentrations were usually achieved by weighing a suitable amount of the sample, based on estimated nitrogen content, into a small beaker and adding 20 ml of total liquid as previously mentioned. Keeping the concentration low also makes it possible to expand the concentration base line of the standard curve and so read the concentration of the unknown more accurately. Figure 2 shows a typical plot of a corn standard. The manufacturer suggests ammonium sulfate, urea solutions, or organic materials such as soy flour or crystalline albumin as standards (7). These materials, made up as solutions and standardized by Kjeldahl analysis, were all tried along with several others. Although the suggested standards were convenient, they were not generally satisfactory for our purposes. The best results have been ob-. tained by using as standard a solid material that has the same refractory properties as the unknown, and that has been analyzed by the Kjeldahl method. Wherever possible a grain of the same kind as the unknown was chosen for the standard. In this procedure both standard and unknown can be assumed to undergo the same reactions all the way through the analyzer. This assumption is not necessarily correct when a liquid standard is used. Corn, wheat, and sorghum grains yield similar standard curves. However, not enough data are available to say what materials are safely interchangeable as standards. Differences in particle size between samples or between sample and standard do not appear to be important in the range of 40 to 80 mesh. Several samples of the same material have been run in succession, each of different particle size with no significant difference in results. This uniformity is probably due to the use of the electric mixer on the samples. The solid standard, of known nitrogen content, was weighed out in the amounts necessary to give solutions of several different concentrations of nitrogen and then solubilized in exactly the same manner as the unknowns. The added accuracy resulting from this procedure is considered to be worth the extra time involved. In Table I, a combarison is made between the results when an ordinary standard (liquid standard) was used and when solid grain materials were the standards.
9 0.01
0.02
0.03
Nitrogen, %
Figure 2. A typical standard curve showing the relationship between transmittance and increasing concentrations of nitrogen contained in severaI solutions of a standard corn sample in sulfuric acid. The transmission is on a logarithmic scale Table I. Comparison of Per Cent Nitrogen Obtained by Use of Liquid and Solid Standards Liquid (NH&SOn Solid Titrimetric Material standard standard Kjeldahl Corn 1.20 1.27 1.27 Crambe 8.75 8.35 8.32 Sorghum 1.98 2.50 2.50 SOY 7.36 8.64 8.65 Amino ethyl flour 1.49 1.98 1.95 Corn germ 3.20 3.62 3.56 shown in the Technicon Methodology Bulletin N-3b (7). The changes in recommended equipment are marked by an asterisk. The changes in tube diameter expressed in inches were: Input manifold: air segmentation to sampler reduced from 0.045 to 0.040; output manifold: diluted sample reduced from 0.065 to 0.060, NaOH reduced from 0.090 to 0.081, alkaline phenol increased from 0.045 to 0.051. These conditions have given us good results, but other combinations might also be satisfactory. The other changes shown on the flowsheet include addition of HgSOa solution and the air scrubber. The air scrubber, which contains dilute sulfuric acid, was added so that the possible contamination of the reaction mixture by ammonia vapors from the ambient air could be prevented. An electric mixer was added to the sampler (Model No. 11) of the standard Technicon AutoAnalyzer equipment to keep sample suspensions well mixed while the sample was being drawn into the system. The digestor heating units were set at 4.5 A for the first heater and 3.5 A for the second heater; the helix was set to rotate at 6.5 rpm. Procedure. All solid samples were ground and then presolubilized. The finer any sample is ground, the more easily and quickly it can be digested. Samples should be fine enough to pass a 60 mesh or finer U. S. Standard screen. The proper amount of ground sample was thoroughly wetted 992
ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
APPLICATIONS AND STATISTICAL EVALUATION
In preliminary analyses, the arithmetic standard deviation was greater for samples having a high nitrogen content, such as soy flour, than it was for corn flour, for example, but the relative standard deviations were about the same. The standard deviation (arithmetic) was as much as three orders of magnitude lower for the biopolymer solutions (fermentation media) than for the usual cereal grain product submitted for analysis, but the relative standard deviation (coefficient of variation) was about the same as for samples of high nitrogen content. For data in which the standard deviation is highly correlated with the mean, a data conversion is recommended (10). For analytical data such as these nitrogen data, where (10) G. W. Snedecor and W. G. Cochran, “Statistical Methods,” 6th ed., Iowa State Univ. Press, 1967, pp 325-330.
Table II. Analysis of Duplicate Samples (Precision for Four Commodities Calculated from Duplicate Samples of Each) No. of Precision5 Commodity samples Autoanalyzer Kjeldahl 9 2.5 0.8 Corn germ Corn 14 1.1 1.6 Millet 9 3.1 2.5 Average of above 2.2 2.2 8 6.3 2.5 Crambe a Log analysis yields s (standard deviation). Precision is [antilog($)- 11 X 100,and is close to the relative standard deviation.
the relative standard deviation is more or less constant, a logarithmic conversion is indicated. All statistical tests reported here are therefore carried out on the logarithms of the nitrogen determinations. The results have been converted back from their logarithmic form to the usual units for use in the tables. Four series of comparisons with the Kjeldahl method were made. In the first, several samples each of four commodities were analyzed in duplicate by each method (Kjeldahl and AutoAnalyzer). The duplicate analyses were done on different days. A three-way analysis of variance was performed on each commodity. None of the first-order interactions were significant with the exception of a sample X method interaction for corn germ, which was significant at the 5 % level. The interaction term was assumed to be a good estimate of analytical error for subsequent two-way analysis of the data. For the three-way data, the standard deviation for each method was calculated. The results (Table 11) show that, with the exception of crambe meal, the standard deviations of the two methods are the same and that the relative standard deviation is about 2.2 of the numerical value of the nitrogen content of the sample. A word of explanation for the precision may be in order. F, the antilogarithm of the standard deviation, s, computed from the logarithms of the observations, was 1.025 for crambe meal. The precision, P,in the tables is thus (1.025 - 1) X 100 = 2.5%. To obtain F from the tabulated values, the calculation is reversed: F = PjlOO 1. For crambe, F =
+
Sample type Soybean Sorghum Corn Corn germ
+
2.5/100 1 = 1.025. For a sample of nitrogen content N, the 66 % limits are N X F and N/F. The 95 limits are approximately N X F4 and N/F2.As a rough guide, however, the 66% limits are N f NP/lOO. Another finding from the three-way experiment was that for two commodities, corn germ and millet, the AutoAnalyzer results were significantly higher than the Kjeldahl values by a small amount. The importance of this finding must be viewed in connection with the findings discussed next. The second comparison with the Kjeldahl method provided data on a large number of samples of several commodities and some broths and solids from production of biopolymer experiments. A single value for nitrogen content was obtained by each method: AutoAnalyzer and Kjeldahl. The standard deviation was taken to be the sample X method interaction. Table 111 shows that the average (pooled) standard deviation for all the commodities is only slightly higher than the average standard deviation in Table 11, with the exception of the biopolymer data. The precision for the biopolymer data is about the same as the average precision of crambe analyses in Table 11, but there is no way of showing what caused the decrease in precision. It is suspected that this lower precision was due to sample nonuniformity, since both of these materials are somewhat more heterogeneous than are the other materials tested. Earlier data (not shown) on biopolymers and corn germ indicated the same sort of difficulty, the trouble seeming to occur with about one fourth of the samples. Also, some of these earlier data were based on different standards than the ones now in use. The difference between the nitrogen values was significant for three materials, all in the direction of lower results by AutoAnalyzer. For all the data examined, including those of Table 111, eight examples of significant difference between methods occurred. Of these, exactly half showed the AutoAnalyzer data higher than Kjeldahl, and half showed the reverse. Since there is no way of knowing the true value of the nitrogen, it is concluded from the data that the bias (mean error) is random and that the true value from analysis lies within limits slightly larger than the values of the standard deviation shown. In a third series of analyses of compounds with known nitrogen content, both the AutoAnalyzer and Kjeldahl results were generally lower than the theoretical nitrogen content (Table IV). The results from the AutoAnalyzer were
Table 111. Analysis of Single Samples Run by AutoAnalyzer and Kjeldahl No. of Mean errore samples Precisiona Meanb 9 3 12 8 4 8 8 16 12
2.31 1.28 2.69 3.12 3.80 1.78 2.19 3.55 2.21
8.12 2.49 1.21 2.93 1.37 2.56 3.24 2.00 2.99
-0.008 $0.021 -0.047 -0.144 -0,051 -0.033 $0.007 f O . 028 +O. 042
Lecithin Dry food blend Macaroni Millet Wheat Average of above 2.75 Biopolymer Low concn 7 5.1 0.00172 -0.ooo22 Medium concn 31 5.4 0.0173 $O.OOO26 High concn 23 0.183 -0.0023 6.0 Log analysis yields s. Precision is [antilog (s) - 11 x 100,and is close to the relative standard deviation. Geometric mean, N. Difference between mean by AutoAnalyzer and mean by Kjeldahl.
Significance
** **
**
ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
993
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~
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Table IV. Comparison of Methods on Pure Chemical Compounds Nitrogen, % TheoAutoChemicals retical Analyzern Kjeldahl Ammonium persulfate 12.0 12.0 11.2 Tris hydroxy methyl amino methane 11.5 11.1 10.4 Ammonium thiocyanate 36.5 36.6 36.2 Ethylenediaminetetraacetate
7.46
7.10
7.29
Urea 46.6 46.9 46.1 Lysine monohydrochloride 15.3 14.9 14.2 Amino-naphthol sulfonic acid 5.86 5.38 5.49 Acetamide 23.7 22.9 23.3 a AutoAnalyzer values were obtained using ammonium sulfate in 10% sulfuric acid as standard.
method X samples interaction as error, gave relative standard deviations from 1.9 to 2.7, with the AutoAnalyzer results being significantly higher than those from the other two methods. These data indicate that all three methods have about the same precision, since the precisions are in satisfactory agreement with the results already shown. Apparently any grain can be accurately analyzed for nitrogen on the AutoAnalyzer if the sample of grain is presolubilized and if the standards consist of the same grain. By keeping the sample concentration in the 10 to 30 mg per 100 ml range, determinations are as accurate as those made by the Kjeldahl method. Relative standard deviations for both methods are usually around 2 %, but if heterogeneous materials and long-term variability are included, an average relative standard deviation of 3 would be a better estimate of precision. ACKNOWLEDGMENT
higher than those determined by Kjeldahl in five of the eight instances. These data indicate that the AutoAnalyzer was perhaps a little more accurate than the Kjeldahl method on these samples. In the fourth comparison, nitrogen analyses were made on chemically modified corn flour, where the nitrogen content varied from 1.2 to 2.275 Single determinations were available by each of three methods: AutoAnalyzer, Kjeldahl, and micro-Kjeldahl. A two-way analysis of variance, using the
We thank Dr. W. F. Kwolek for advice and assistance with the statistical interpretation of the data and C. H. VanEtten for invaluable criticism and advice.
RECEIVED for review January 25, 1971. Accepted March 17, 1971. Mention of trade or company names is for identification only and does not imply endorsement by the Department of Agriculture.
Conducting Glass Electrode in a Thin-Layer Electrochemical Cell with Application to the Analysis of Neptunium R. C . Propst Savannah River Laboratory, E. I . du Pont de Nemours and Co., Aiken, S. C . 29801
A cylindrical cell for coulometry in thin layers of solution at a conducting glass electrode is described. Volumes were reproducible to &0.3%. Techniques are recommended to reduce edge diffusion and iR gradient efiects. Application of the method to determine neptunium in process samples is described.
RECENTLY, A CONDUCTING GLASS (antimony-doped tin oxide) electrode was found to be well suited for the coulometric determination of actinides (1-3). However, coulometry in stirred solutions is slow, and rapid methods are desirable for routine control analyses. By combining the advantages of the wide workipg range of the conducting glass electrode (CGE) with the fast response of thin-layer cells (4), the analysis time for actinides is reduced to about one minute. For radioactive solutions, thin-layer cells should be compatible with containment equipment, rugged, and easily (1) R. C. Propst, ANAL.CHEM., 41,910 (1969). (2) R. C. Propst and M. L. Hyder, Nature, 221, 1141 (1969). (3) R. C. Propst and M. L. Hyder, J. Inorg. Nucl. Chem., 32, 2205 (1970). (4) C. R. Christensen and F. C. Anson, ANAL. CHEM., 35, 205 (1963).
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ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
cleaned and refilled with fresh sample solution. Also, they should provide results reproducible to &0.5z. Several of the designs described in the literature offer excellent volume reproducibility; however, none were completely satisfactory for this purpose. The elegant micrometer-based designs (5-7) are difficult to decontaminate and clean, and the variable solution thickness feature is not required for routine applications. The sandwich-cell design (8) with parallel glass plates appeared suitable. When equipped with a side arm, this cell would provide flow-through containment (total enclosure from solution reservoir to waste container), and the incorporation of reactant-getter electrodes would eliminate edge effects (5). However, the spacer material could not be permanently bonded to the glass plates. Different spacer materials including glass solder and Teflon (Du Pont)-FEP were tried, but the seals were either initially defective or else failed after a short time. The rate of failure was aggravated ( 5 ) D. M. Oglesby, S. H. Omang, and C. N. Reilley, ANAL.CHEM., 37, 1312 (1965). (6) A. T. Hubbard and F. C. Anson, ibid., 40,615 (1968). (7) J. E. McClure and D. L. Maricle, ibid., 39,236 (1967). (8) A. Yildiz, P. T. Kissinger, and C. N. Reilley, ibid., 1018 (1968).
