Kjeldahl nitrogen analysis as a reference method for protein determination in dairy products

Measurement of total nitrogen by Kjeldahl analysis is the historical reference method for determination of the protein content of dairy products and is used for both calibration and validation of alternative methods for protein determination. Accurate evaluation of alternative methods is not possible if there is large uncertainty regarding the reference values. When Kjeldahl analysis is used to establish reference values, the performance of the Kjeldahl testing must be verified and within established expectations. Advice is given for Kjeldahl system optimization, evaluation of test results, and trouble-shooting. Techniques for successful Kjeldahl nitrogen analysis of dairy products other than milk are discussed.     

全自动凯氏定氮仪测定化肥中的含氮量

建立全自动凯氏定氮仪检测化肥中氮含量的分析方法。称取总氮量在0.03~0.3 g范围的样品,经消解仪消解及全自动凯氏定氮仪碱化蒸馏,以硼酸为接收液,用0.15~0.20 mol/L的盐酸标准溶液进行自动滴定。该方法加标回收率为99.0%~101.5%,测定结果的相对标准偏差小于1.0%(n=6)。与国标GB/T 8527–2010法测定结果无显著性差异,全自动凯式定氮仪法的准确度、精密度均优于国标方法,而且所用试剂少、分析时间短,满足实验室快速检测大批量化肥中含氮量的需要。     

A rapid method for the determination of organic nitrogen and phosphorus based on a single perchloric acid digestion

Results of nitrogen determinations obtained by perchloric acid digestion of organic matter under reflux were found to be equivalent to those obtained by the conventional sulfuric acid Kjeldahl method. A rapid determination of nitrogen and phosphorus was thus possible after a single perchloric acid digestion. Nitrogen was determined by nesslerization and phosphorus by a conventional molybdenum blue technique. The method devised was found satisfactory for a great variety of natural products and pure organic compounds, including materials containing all kinds of tertiary nitrogen.     

Rapid estimation of global sugars by UV photodegradation and UV spectrophotometry

The UV/UV method developed for the measurement of specific wastewater pollution parameters (ammonium, Kjeldahl nitrogen, total phosphorus) is adapted to the determination of sugars in fruit juices and soft drinks. The procedure is based on the UV spectrophotometric detection of by-products of UV photodegradation of carbohydrates. Time of analysis is greatly reduced by using specific pH conditions and diluted samples.     

Bestimmung von stickstoff in refraktärmetallen—I molybdän und wolfram

This is a report on round-robin tests concerning the determination of nitrogen traces in molybdenum and tungsten, which were carried out by the refractory metals group of the chemistry section of the Gesellschaft Deutscher Metallhütten- und Bergleute. The following methods were used: classical and modified Kieldahl methods (in particular, a newly developed micro-Kjeldahl method), inert-gas fusion, vacuum fusion and ultrahigh-vacuum diffusion extraction. As the nitrogen values obtained by the first round-robin test exhibited considerable scatter, possible shortcomings of the various Kjeldahl methods, as well as the quantitative nitrogen recovery by the extraction methods, were carefully examined. The second round-robin test then gave the following results: the nitrogen content of the investigated molybdenum sheet was 2 ppm, the content of the tungsten sheet 0.5 ppm. Classical Kjeldahl methods are not applicable to the determination of nitrogen contents below 10 ppm. Frequently, detection limits of Kjeldahl methods range considerably above 10 ppm, mainly because of contamination by reagents and/or the laboratory atmosphere. The micro-Kjeldahl method and the hot-extraction and fusion-extraction methods are capable of determining nitrogen contents down to 1 ppm at best. The only method available for nitrogen determinations in metals at sub-ppm levels is by ultrahigh-vacuum diffusion extraction. The good agreement between the nitrogen values obtained for molybdenum by the micro-Kjeldahl method and by the various extraction methods is the most reliable proof of the quantitative nitrogen recovery by the extraction methods.     

Potential for chlorate interference in ion chromatographic determination of total nitrogen in natural waters following alkaline persulfate digestion.

Determination of total nitrogen in aqueous samples after thermal potassium peroxydisulfate (persulfate) digestion is a commonly used alternative to the tedious Kjeldahl procedure. When ion chromatography is used to quantify the nitrate formed during digestion, there is a potential for interference from a chlorate peak if the digested sample initially contained chloride in concentrations close to or greater than the concentration of nitrogen. It was determined that this interference can be avoided either by using chromatographic conditions which cleanly resolve the nitrate and chlorate peaks (e.g., the Dionex AG9-HG column) or by using digestion reagent concentrations chosen to maintain a high pH throughout the digestion. The second alternative is not a viable option for investigators using a single digestion for both total nitrogen (TN) and total phosphorus (TP) analysis.     

The determination of microgram quantities of organic nitrogen

A method is descriibed for the determination of microgram quantities of nitrogen in a wide variety of materials. This method involves the Kjeldahl procedure for the conversion of nitrogen to ammonium or amine sulphate. Digestion of the sample with concentrated sulphuric acid and catalyst, and, if necessary, pretreatment with hydriodic acid and red phosphorus, are followed by steam-distillation into a dilute acid solution. The final determination depends on the reaction of ammonia, in a suitably buffered solution, with sodium hypobromite reagent, and measurement of the excess reagent by titration of the iodine liberated by it from acidified potassium iodide, with a standard $\text{N}\text{500}$ sodium thiosulphate solution, using sodium starch glycollate indicator.Since sample size is relatively unrestricted and the final titration can measure 5 micrograms of nitrogen, the sensitivity of the method is very high.Reference is made to existing sub-micro methodes which, although sensitive, are time-consuming and specific in application.     

Fast and selective determination of total protein in milk powder via titration of moving reaction boundary electrophoresis

In this paper, moving reaction boundary titration (MRBT) was developed for rapid and accurate quantification of total protein in infant milk powder, from the concept of moving reaction boundary (MRB) electrophoresis. In the method, the MRB was formed by the hydroxide ions and the acidic residues of milk proteins immobilized via cross‐linked polyacrylamide gel (PAG), an acid‐base indicator was used to denote the boundary motion. As a proof of concept, we chose five brands of infant milk powders to study the feasibility of MRBT method. The calibration curve of MRB velocity versus logarithmic total protein content of infant milk powder sample was established based on the visual signal of MRB motion as a function of logarithmic milk protein content. Weak influence of nonprotein nitrogen (NPN) reagents (e.g., melamine and urea) on MRBT method was observed, due to the fact that MRB was formed with hydroxide ions and the acidic residues of captured milk proteins, rather than the alkaline residues or the NPN reagents added. The total protein contents in infant milk powder samples detected via the MRBT method were in good agreement with those achieved by the classic Kjeldahl method. In addition, the developed method had much faster measuring speed compared with the Kjeldahl method.     

Repeatability and reproducibility of determination of the nitrogen content of fishmeal by the combustion (Dumas) method and comparison with the Kjeldahl method: interlaboratory study

Ten fishmeal samples (hidden duplicates of 4 meals plus 2 high-protein meals as a Youden pair), tryptophan, and nicotinic acid were analyzed by 18 laboratories using the Dumas method. Thirteen of the laboratories also analyzed the same 12 samples using their current Kjeldahl method. Recoveries (+/-SR) of tryptophan and nicotinic acid were 99.3+/-1.04 and 98.8+/-2.11% by Dumas and 97.1+/-3.03 and 74.6+/-26.76% by Kjeldahl. The Dumas method gave significantly greater values (P < 0.001) than the Kjeldahl method. For fishmeals, Kjeldahl N = 0.989 of Dumas N (P < 0.001). A similar proportionate difference (0.984 of Dumas N) was observed with tryptophan. Most laboratories failed to determine nicotinic acid correctly by Kjeldahl. For fishmeals, the relative standard deviations for repeatability and reproducibility were for Dumas 1.48 and 2.01% and Kjeldahl 1.62 and 2.37%, respectively. A single analysis conducted in 2 laboratories should not differ by more than 5.63% of the mean value when measured by Dumas or by more than 6.64% by Kjeldahl. It is concluded that with fishmeal, Dumas gives a more reliable measure of organic nitrogen than Kjeldahl, and, therefore, Dumas should be the method of choice.     

微波消解凯氏定氮法快速测定酱油中全氮

研究了凯氏定氮法测定酱油中全氮时样品的微波消解方法,进行了微波消解条件的选择及消解结果精密度试验,并与国家标准方法对比,验证了方法的准确度。试验结果表明,消解完全仪需20min,相对标准偏差均小于2%（n=7）,回收率范围为96%～103%,经t检验,微波溶样法与国家标准凯氏定氮法的测定结果无显著性差异。     

Simultaneous Determination of Total Nitrogen and Metal Elements in Tobaccos by High Performance Ion Chromatography

A simple method, developed primarily for simultaneous determination of total nitrogen and inorganic cations by high performance ion chromatographic (HPIC), was optimized for digestion of flue‐cured tobaccos, and compared with the traditional Kjeldahl method and atomic absorption spectrometry (AAS). Nitrogen determination by either Dumas method or Kjeldahl method is time‐consuming and tedious. Metal elements determination by either inductively coupled plasma‐mass spectrometry (ICP‐MS) or AAS may be more expensive and requires specialist equipment. The use of HPIC to simultaneous determine total nitrogen as ammonium ion and metal elements as inorganic cations after sample digestion significantly improves the speed of the analysis compared with the conventional methods. The cation‐exchange column and suppressed conductivity detector was used for determination of ammonium and inorganic cations in the presence of the elevated levels of sulfuric acid found in digested sample. The propsoed digestion method was accurate and precise, and required little investment. The determination of ammonium and inorganic cations was linear from 15 pg·L^?1 up to 25 ng·L^?1. The results obtained by the HPIC method were compared with those for the conventional methods approach for the determination of total nitrogen and metal elements. The application of the HPIC method is also demonstrated for a variety of other plant samples matrices.     

Determination of the total nitrogen content of hard, semihard, and processed cheese by the Kjeldahl method: collaborative study

The objective of this collaborative study was to determine interlaboratory performance statistics for a modified and optimized version of AOAC Method 920.123 for the determination of the total nitrogen content of hard, semihard, and processed cheese by Kjeldahl analysis. Details included addressing the issues of material homogeneity, test portion size (1 g), quantitative transfer (weighing on to filter paper), ensuring system suitability (nitrogen recoveries), and using AOAC Method 991.20 as the basis for nitrogen analysis. Fifteen laboratories tested 18 pairs of blind duplicate cheese materials with a crude protein content between 18 and 36%. Materials represented hard, semihard, and processed commercial cheeses with a wide range of composition. Statistical performance parameters expressed as crude protein (nitrogen x 6.38), g/100 g, with invalid and outlier data removed were mean = 26.461, repeatability standard deviation (Sr) 0.111, reproducibility standard deviation (S(R)) = 0.153, repeatability relative standard deviation (RSDr) = 0.42%, reproducibility relative standard deviation (RSDR) = 0.58%, repeatability (r) = 0.312, and reproducibility (R) = 0.428. The interlaboratory study results were acceptable and comparable to those for the milk Kjeldahl nitrogen method on a relative nitrogen basis. The Study Directors recommend that this modified method for the determination of total nitrogen in hard, semihard, and processed cheese by Kjeldahl analysis be adopted First Action as an improved method to replace Method 920.123.     

Measurement of protein in serum and rodent embryos by spectrophotometry at 215 and 225 nm

The Waddell quantitative method for total protein estimation was slightly modified and applied to sera and rodent embryos, and also compared to the Lowry method. It was not necessary to employ the Kjeldahl method to determine an instrument correction factor; reference sera were used to establish a standard curve. The results from this simplified Waddell method were reproducible from day to day with a high degree of accuracy. Comparative data obtained by total protein determination in three commercially available reference sera demonstrated a sensitivity in the modified Waddell method of 4.8 μg/ml with a precision of >96% and an accuracy of >93%. The overall precision in the determination of the total protein by the method in Day 12 rat embryos was >91% as compared to >86% in the Lowry method.     

Continuous propagation ofKluyveromyces fragilis in cheese whey for pollution potential reduction

A 25-L working volume, upright cylindrical fermenter made of stainless steel was used to investigate cheese whey yeast fermentation for pollution potential reduction. The effluent total and soluble chemical oxygen demand, total and volatile solids, and total Kjeldahl and ammonium nitrogen concentrations were significantly affected by the hydraulic retention time, air flow rate, and mixing speed. The system removal efficiencies were 15.90-58.61%, 25.20-69.33%, 12.43-49.90%, 9.22-51.77%, 1.66-10.06%, and 54.82-72.22% for total chemical oxygen demand, soluble chemical oxygen demand, total solids, volatile solids, total Kjeldahl nitrogen, and ammonium nitrogen, respectively, depending on the hydraulic retention time, air flow rate, and mixing speed used.     

A comparison of the Kjeldahl and Dumas methods for the determination of protein in foods,using data from a proficiency testing scheme

Both the Kjeldahl and the Dumas methods for the determination of protein in foodstuffs are currently in use, but the empirical nitrogen factors used to convert the determined nitrogen content to protein content are based on the Kjeldahl method alone. Non-equivalence between the two methods could therefore result in some laboratories reporting an incorrect protein content. We report here a study using data accumulated over several years in the results of a proficiency testing scheme. On average the Dumas method provided results that were relatively higher by about 1.4% than the Kjeldahl method, but the difference between the methods depended on the type of foodstuff. The methodology of looking for bias between analytical methods is critically discussed.     

A simplified version of the total kjeldahl nitrogen method using an ammonia extraction ultrasound-assisted purge-and-trap system and ion chromatography for analyses of geological samples

The total Kjeldahl nitrogen (TKN) method was simplified by using a manifold connected to a purge-and-trap system immersed into an ultrasonic (US) bath for simultaneous ammonia (NH₃) extraction from many previously digested samples. Then, ammonia was collected in an acidic solution, converted to ammonium (NH₄⁺), and finally determined by ion chromatography method. Some variables were optimized, such as ultrasonic irradiation power and frequency, ultrasound-assisted NH₃ extraction time, NH₄⁺ mass and sulfuric acid concentration added to the NH₃ collector flask. Recovery tests revealed no changes in the pH values and no conversion of NH₄⁺ into other nitrogen species during the irradiation of NH₄Cl solutions with 25 or 40 kHz ultrasonic waves for up to 20 min. Sediment and oil free sandstone samples and soil certified reference materials (NCS DC 73319, NCS DC 73321 and NCS DC 73326) with different total nitrogen concentrations were analysed. The proposed method is faster, simpler and more sensitive than the classical Kjeldahl steam distillation method. The time for NH₃ extraction by the US-assisted purge-and-trap system (20 min) was half of that by the Kjeldahl steam distillation (40 min) for 10 previously digested samples. The detection limit was 9 μg g⁻¹ N, while for the Kjeldahl classical/indophenol method was 58 μg g⁻¹ N. Precision was always better than 13%. In the proposed method, carcinogenic reagents are not used, contrarily to the indophenol method. Furthermore, the proposed method can be adapted for fixed-NH₄⁺ determination.     

Determination of trace organic carbon and nitrogen in the presence of carbonates in anorganic bovine bone graft materials

The widespread concern of bovine spongiform encephalopathy (mad cow disease) has raised questions about the possibility of residual organic material in bovine-derived bone graft materials. Conflicting reports have shown both the presence and the absence of organic materials in bone derived products. This study compared residual organic levels in two commercial bone graft products: a bovine bone product treated chemically and at low temperature, and a bovine bone product treated at high temperature. In this study, organic carbon, the primary constituent of organic materials, is defined as carbon that is not liberated as CO₂ upon acidification (i.e. non-carbonate carbon). Three approaches to the determination were used: (1) organic carbon was calculated as the difference between total carbon determined by high temperature combustion and carbonate carbon determined by acid evolution of CO₂. (2) Organic carbon was determined by measuring total carbon by high temperature combustion of a sample that had been pretreated with acid to remove all carbonate. (3) The total organic carbon remaining in solution after acid dissolution was determined. In addition, organic nitrogen was determined as the difference between total Kjeldahl and ammonia nitrogen. Total nitrogen values were confirmed using an instrumental nitrogen analyzer. No detectable organic carbon or organic nitrogen was observed in the high temperature bone product. In the low temperature treated bovine product, however, approximately 2000 μg/g organic carbon was measured by all three methods, as well as 15 μg/g organic nitrogen.     

连续流动法同时测定污水中氨氮和总磷

采用连续流动分析法测定污水中氨氮、总磷的含量。比较了污水样品不同的稀释倍数对测定结果的影响。实验结果表明:氨氮和总磷的质量浓度在0.1~8 mg/L范围内线性良好,线性相关系数均为0.99997;测量结果的相对标准偏差分别为2.01%,0.84%(n=7);方法检出限分别为0.012,0.009 mg/L;质控样测量值均在标示值范围内;样品加标回收率为93.4%~101.1%。污水的洁净程度对测定结果影响较大。洁净度高的污水,直接测定与稀释后测定,测定值无显著性差异;而洁净度低的污水,直接测定与稀释后测定结果差异比较大。该法采用全谱直读CCD检测方式,灵敏度高,稳定性好,无光谱干扰,且支持氨氮和总磷同时测定,方便快捷,适合污水中氨氮和总磷的测定。     

腐植酸复合肥中的氮、磷、钾含量分析

采用凯氏定氮法、磷钼酸喹啉容量法、ICP-AES法分别分析腐植酸复合肥的氮、磷、钾含量。研究结果表明,在添加不同酸时,应使盐酸的离子浓度大于硫酸的离子浓度,从而消除硫酸盐对磷分析的影响;待测液的酸度控制在1.0~1.6 mol/L为较适宜条件。方法具有良好的准确度和精密度,适用于腐植酸复合肥中氮、磷、钾的精确分析。     

The determination of total nitrogen in plant materials with an automatic nitrogen analyser

An automatic analyser is used for the measurement of total nitrogen in chemical compounds and botanical material after the combustion method of Dumas in vacuum. The analyser is described and results for several chemical compounds and plant materials are given. The results have been statistically evaluated to compare the reproducibility and accuracy of the automatic analyser and the Kjeldahl method.