Automated Kjeldahl Nitrogen DeterminationA Comprehensive Method for Aqueous Dispersible Samples Donald G . Kramme,' Richard H. Griffen, Clark G. Hartford, and Joseph A. Corrado2 Miles Laboratories, Inc., Marschall Dicision, Elkhart, Ind. 46514 IN 1960, ANDRESFERRARI (I) succeeded in automating the Kjeldahl nitrogen procedure. It was a general method which required adaptation for each class of sample assayed. Digestive conditions were adjusted in accordance with sample concentration and the severity of digestion required (2). Different standards were employed for each class of sample analyzed. Ideally, ammonium sulfate is the standard choice since all organic nitrogen is converted to this form. However, since some compounds are more readily digested than others, a standard having the same refractory properties as the unknowns is employed. Standards which have been employed include beta alanine for meat samples, and 2benzyl-2-pseudo-thiourea for milk samples (3). Jacobs ( 4 ) used a mixture of nicotinamide and urine as the standard in the analysis of urine, feces, and food. In other work, glycine was the standard of choice for urine and feces samples (5). In many of these adaptations, the digestive system of the procedure was optimized for the specific sample class of interest, However, difficulty arises when samples of unknown composition or mixtures of sample classes must be analyzed. In these instances, the digestive conditions and standards must be consistent. Little work has been published on the development of comprehensive systems which could be used for all classes of samples. Therefore, a standardized procedure was developed which could be employed without modification regardless of the composition of the sample received for analysis by our research services laboratory. EXPERIMENTAL
Apparatus. A basic Technicon AutoAnalyzer System for nitrogen analysis is used. The modules used include sampler 11, two pump I modules, continuous digestor, colorimeter, heating bath, range expander, recorder, and vacuum pump. A special custom made gas aspiration pipet is employed in the continuous digestor (see Figure 1). This pipet is made from 8-mm 0.d. glass tubing and has a ground glass fitting on one end. The other end which is placed in the helix has a drip ring 8 inches from the tip. Three 1/a2-in.-1/16-in.diameter holes are evenly spaced on the bottom side of the pipet between the drip ring and the tip. The tip has a 1/16-in.diameter opening. Reagents. The digest acid was prepared by dissolving 3.0 grams of selenium dioxide in 100 ml of distilled water, adding 100 ml of perchloric acid (70%) and mixing. To this, 1800 ml of sulfuric acid were added and the mixture was diluted to 2 liters with distilled water. The distilled Present address, Worthington Foods, Inc., Worthington, Ohio 43085-Subsidiary of Miles Laboratories, Inc. * Deceased May 13, J 969. (1) A. Ferrari, Ant?. N.Y. Acad. Sci., 87, 792 (1960). (2) J. F. Marten and G. Gatanzaro, Arzalysf (Lor~don:,91, 42 ( 1 966).
(3) Technicon Corporation, Tarrytown, N. Y.,Industria! Method 31-69A (1969). (4) S. C. Jacobs, 1. Clir7. Pathol., 21, 218 (1968). ( 5 ) J. L. Cox and B. G. Harmon, Automat. Arid. Chem. Technicon S,ymp., 1, 149 (1967).
12"
I
i-- 8"
i
1
8rnrn O D GLASS TUBING
E!
GROUND GLASS FITTING
l / 3 2 " - I 16' DIAMETER HOLES EVENLY SPACED
Figure 1. Custom made gas aspiration pipet water is safely added, using a 3-mm 0.d. glass tube immersed well below the surface of the mixture. The sodium hydroxide solution contained 350 grams of NaOH per liter. The alkaline phenol was prepared by dissolving 150 grams of sodium hydroxide in 750 ml of distilled water, adding 250 ml of 90% (w/v) phenol solution and mixing. Undiluted Clorox (Procter and Gamble) is used for the sodium hypochlorite reagent. Standards. A stock solution of 1 (10,000 ppm) nitrogen from ammonium sulfate was prepared. Dilutions were made to give 10, 25, 50, 100, and 150 ppm N standards. No other standards are necessary. Modified Procedure. The modified flow diagram of the automated system is shown in Figure 2. Sample cups containing the appropriately diluted aqueous unknowns and standards between 10 and 150 ppm nitrogen content were loaded into the sampler module. The digestor module was set with Stage 1 at 400-420 OC drawing 4.4 A and Stages 2 and 3 at 330 "C drawing 6.8 A. The sampler was set at the rate of 20 per hour. An aliquot of the digest is alkalized and reacted with alkaline phenol in the presence of sodium hypochlorite to produce indophenol. The blue color was measured at 630 nm, using a 15-rnm flow-cell. The range expander for the recorder was set at 2 X . A calibration curve was prepared and the ppm nitrogen of the unknowns was read directly from the strip chart. Digestion and Color Development Modifications. The Technicon Procedure ( 6 ) was modified to produce a tenfold increase in sensitivity. First, the reagent proportions of the output manifold were changed to give increased sensitivity (Figure 2). The ratio of digestor effluent to water and sodium hydroxide was increased as much as possible, still retaining sufficient alkalinity for properly controlled color development. The sodium hypochlorite and alkaline phenol concentrations were then adjusted to give optimum color development. The digestor input manifold (Figure 2) was changed to increase the sample size by 50%. The digestion mixture was changed and the temperature increased to completely digest the sample. At the higher temperature, the original short fume aspiration pipet allowed condensation of vapors in the cooling stage. A straight tube inserted past the cooling stage was inadequate in fume removal and fumes condensing on the outside of the tube corroded the mounting bracket. Therefore, a more efficient gas aspiration pipet (Figure 1) was (6) Technicon Corporation, Tarrytown, N.Y., Method N-3b(1965).
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Metal ion Li+ K+ Mg2+
Ca2+ Ba2+ ~ 1 3 +
Table I. Metal Ion Interference and Effect of Chelating Agents (yoresponse, no ion equals 100%) 5.0% sodium No chelating agent 4 . 0 2 Naz EDTAa potassium tartrate 400ppm 50ppm 400ppm 50 ppm 400 pm 50 PPm 89 85 76 100 96 100 130
100 98 93 95 96 96 111 117 89 135 91 105 107 100 93 93 100
102 90 98 102 102 98 112
96 87 93 102 102 91 107 189 87 91 102 140 95 102 95 82 100
98 100 98 94 100 100 106 143 104 113 98 98 104 100 98 98 100
Cr 3+ b b Mn2+ 96 112 Fe 3+ b 168 cot+ Ni z+ 76 105 CUZ' 139 112 130 115 Zn2+ 117 102 Cd2+ Hg2+ 87 107 85 83 Pb2+ 100 100 No ion a Accuracy was impaired by an unstable base line with 4 2 Nar EDTA. Precipitate deposited in the flow-cell of the colorimeter.
designed to alleviate these problems. It is much longer, extending well into the helix. It has three holes placed along the bottom of the pipet to effectively remove the digestant fumes. The drip ring collects the remaining condensate which drops into the waste ring of the helix. In the original high temperature stages (2 and 3), some excess fumes still persisted. These hot fumes, when aspirated, caused melting of the rubber tubing connected to the water aspirator, as well as severely corroding the water aspirator. To solve this problem, the heating stages were reversed. The first stage became the hottest (420 " C ) and the second and third stages cooler (330 "C). With the second and third stages as the cooler stages, the Se02catalyst was found to precipitate in these stages. By reducing the SeO, concentration from 0.3 to 0.15%, the precipitation problem was eliminated and good digestion still maintained. RESULTS AND DISCUSSION
Nitrogen Recovery. Various amino acids and other compounds including glucosamine, casein, urea, and nicotinamide were analyzed. These compounds represent the spectrum of refractability that may be encountered. Using the same conditions for all compounds (temperature, digestant, etc.); recoveries near 100% of theory are obtained. These recoveries are obtained by Ferrari ( I ) with autolyzed yeast samples. Gehrke (7) has achieved 99-1002 recoveries in the analysis of commercial fertilizer samples. Complete recovery of less refractory materials, such as glycine and urea has been reported by Kammerer (8); however, only 65-80z recovery of the more refractive compound nicotinamide was obtained. In contrast, complete recovery of all of these sample classes was achieved. To determine the carbohydrate digestive capacity of the system, ammonium sulfate standards were added to high carbohydrate solutions. Solutions containing 50, 100, and 150 ppm N were added to 0-25 2 w/v sucrose solutions. The solutions were then assayed for nitrogen. Complete diges( 7 ) C . W. Gehrke, F. E. Kaiser, and J. P. Ussary, Automat. Anal. Chem. Technicon Symp., 1,239 (1968). (8) P. A. Kammerer, M. E. Rodel, R . A. Hughes, and G. F. Lee, Eucirori. Sci. Techriol., 1, 340 (1967).
102 103 103 100 102 98 103 121 98 103 107 105 98 98 102 97 100
5.0z sodium potassium tartrate-0.5 Nas EDTA 400ppm 50ppm
z
95 88 93 100 102 91 109 181 88 163 98 123 95 98 100 77 100
106 102 104 100 104 102 108 119 100 123 100 106 100 102 100 98 100
tion is accomplished at 5 and 10% sucrose levels. At higher sucrose concentrations, digestion is incomplete and results in caramelization. Caramelization causes an increase in optical density of the solution and higher peak heights are observed on the strip chart, resulting in an apparent increase in nitrogen content. A similar experiment was performed, using dextrose. Again, complete digestion occurs with 5 and 10% dextrose and caramelization occurs at the 15, 20, and 25 levels. Therefore, carbohydrate concentrations up to 10 2 may be successfully assayed without adding a comparable level of pure carbohydrate to the standards. The linearity of the method was established by assaying various concentrations of casein. A stock solution containing 1.895 mg/ml (dry basis) casein was diluted to give solutions containing 303, 379, 455, 530, and 605 mg protein/ml. These solutions were assayed in duplicate. When the ppm found was then plotted us. the grams protein added, a straight line was obtained which passed through the origin. The automated method was compared with the manual Kjeldahl method by analyzing four unknown samples by both procedures. Good agreement of the two methods was achieved. Metal Ion Interferences. The effect of certain metal ions on the color development reaction was investigated. Sixteen metal ions were tested (Table I). Solutions containing 400 and 50 ppm metal ions were added to known ammonium sulfate standards. These mixtures were introduced into the color development system and the relative peak heights observed. Significant interference was observed with Cr 3+, Mn2+,Fe3+,and Co2+at the 50-ppm level. At the 400-ppm level, interference was also observed with Li+, K+, Mg2+, Ni2+, Cu2+, Zn*+, Cd2+,Hg2+, and Pb2+. Chelating agents such as EDTA or sodium potassium tartrate are sometimes employed to minimize these effects. The ability of these agents to minimize the effect of interfering metal ions was investigated. Three different chelating systems were evaluated; 4.0 2 Na2 EDTA, 5.0% sodium potassium tartrate, and a 0 . 5 2 Na2 EDTA-5.0 % sodium potassium tartrate solution (9). The
z
(9) D. E. Uhl, E. B. Lancaster, and C. Vojnovich, ANAL.CHEM., 43,990 (1971).
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interferences of Cra+,Fea+,and C12+ are diminished to some extent by all three systems. The enhancement effect of Mn2+ is not appreciably diminished by any of the systems. At the 400-ppm level, most interferences are decreased by the chelating agents ; however, the inteferences of certain metal ions are increased. The interference of Pb2+ is significantly increased by the 0.5 % Nas EDTA-5.Oz sodium potassium tartrate system. These findings indicate an inherent weakness of the color development system, that of the metal ion interference. The use of EDTA or sodium potassium tartrate results in a significant reduction in interference with some metal ions; no change in the interference of others; and with certain other ions, enhances the interference. In this comprehensive method, chelating agents were not included because sample composition is often unknown or widely varied. This is consistent with the conclusions of others-(7, 8, IO)-which have elected not to include chelating agents in the color development system, although 0.5 % EDTA and 5.0 % sodium (10) J. R . Todd and J. H. Byars, Auromat. Anal. Chem. Technicon Symp., 1, 140 (1967).
potassium tartrate system in the 35 sodium hydroxide solution is now recommended by Uhl(9). CONCLUSIONS
Sensitivity has been substantially increased as a result of optimizing reagent ratios and increasing the proportion of digested sample being analyzed. This eliminates the need for a specially built longer flow-cell and its related problems. The digestion procedure has been improved to eliminate the need for “similar matrix” standards and enables one to use the same digestion procedure for a variety of nitrogen compounds. Liquid samples containing up to 10% carbohydrate can be digested without interference or further dilution. In more than five years of routine laboratory use, we have found ammonium sulfate standards directly correlate with all types of proteins analyzed. This enables one to confidently analyze proteins of unknown types as well as mixtures of several proteins in one sample.
RECEIVED for review May 16, 1972. Accepted September 5, 1972.
On the Drawing of the Base Line for Differential Scanning Calorimetric Calculation of Heats of Transition Charles M. Guttman and Joseph H. Flynn Institute for Materials Research, National Bureau of Standards, Washington, D.C. 20234
IN THE DIFFERENTIAL SCANNING CALORIMETRIC method (DSC), the heat of transition of a pure substance is determined, from the area under the differential power-time curve. How one should draw the base line in determining the heat of transition has been either ignored (1-4, arbitrarily posited ( 5 , 6 ) , or based on the author’s conception of what is happening during the transition process (7-10). The uncertainty in how to draw the base line usually arises from the fact that the heat capacity of the sample after the transition is different from that before the transition. Generally since this change in heat capacity gives rise to heats quite small compared to the heat of transition, the method used to determine the base line may (1) E. S. Watson, M. J. ONeill, J. Justin, and N. Brenner, ANAL. CHEM.,36, 1233 (1964). (2) M. J. O’Neill, ibid., p 1238.
(3) A. P. Gray, “Analytical Chemistry,” R. S . Porter and J. R. Johnson, Ed., Plenum Press, New York, N.Y., 1968, p 209. (4) “Thermal Analysis News Letter” Analytical Division, PerkinElmer Corp.. Norwalk, Conn., Nos. 1-9 (1965-70). ( 5 ) H. G. McAdie, “Report of the Committee on Standardization,” International Confederation for Thermal Analysis, Abstracts of Third International Conference on Thermal Analysis, Davos, Switzerland, August 1971, p 11. (6) R. C. MacKenzie, Chairman, “Report of Nomenclature Committee,” ibid., p 3. (7) W. P. Brennan, B. Miller, and J. C. Whitwell, l i d . Eng. Clzem., Fundurn., 8, 314 (1969). (8) W. P. Brennan, PhD. Thesis, Princeton University, 1970. (9) G. Adam and F. H. Muller, Kolloid-Z. Z. Polym., 192, 29 (1963). (10) A. Engelter, ibitl., 205, 102 (1964). 408
not be critical within the limits of experimental accuracy for available instruments. However, advances in DSC instrumentation (11)should increase the precision of the heat measurements and thus make significant the correct determination of the base line. Furthermore, some cases do exist where the heat capacity of the sample before the transition differs considerably from its heat capacity after the transition. Generally these arise from a change in sample mass. Such transitions may involve the sudden evolution of a gaseous product, combination with a gaseous atmosphere, or vaporization of the sample itself (4). For such cases, the correct drawing of the base line is of importance. In this note we shall present arguments suggesting that the correct base line can be obtained by extrapolating the heat capacities of the initial and final temperature state to the thermodynamic transition temperature. The heat of transition is then the area under the differential power-time curve using this base line. It will be shown that this procedure gives heats which are largely independent of the kinetic processes of transition for transitions that occur at a single thermodynamic temperature. In the first part of the note we shall describe the traces from a DSC; then we shall describe the drawing of a base line when one does not have to worry about machine time constants. Finally, we shall include the effect of machine time constants in our discussion. (11) M. J. O”eil1 and A. P. Gray, “Design Considerations in Advanced Systems for Differential Scanning Calorimetry,” Proceedings, Third International Conf. on Thermal Analysis, Davos, Switzerland, 1971, to be published.
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