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A Guide To Kjeldahl Nitrogen Determination Methods and Apparatus An Industry Se

A Guide To Kjeldahl Nitrogen Determination Methods and Apparatus An Industry Service Publication Forward This booklet has been developed to serve as a technical companion in the selection and use of Kjeldahl nitrogen determination methods and apparatus. This material has been prepared with the help of Kjeldahl chemists, consul- tants and Labconco engineers. It will not teach you to perform the Kjeldahl procedure but will help you understand steps in the process. This generic presentation is designed to help educate persons unfamiliar with Kjeldahl methodology and answer frequently asked questions raised by Kjeldahl users. 3 History Nitrogen determination has a long history in the area of analytical chemistry. Johan Kjeldahl first introduced the Kjeldahl nitrogen method in 1883 at a meeting of the Danish Chemical Society. As chairman of the chemistry department of the Carlsberg Laboratorium near Copenhagen, Kjeldahl was assigned to scientifically observe the processes involved in beer making. While studying proteins during malt produc- tion, he developed a method of determining nitrogen content that was faster and more accurate than any method available at the time. His method used simple equipment and could be performed by an inexperienced technician. Applications Since 1883, the Kjeldahl method has gained wide acceptance and is now used for a variety of applications. Kjeldahl nitrogen determinations are performed on food and beverages, meat, feed, grain, waste water, soil and many other samples. The method has been refined and tested for a wide variety of substances and approved by various scientific associations including: AOAC International (formerly the Association of Official Analytical Chemists) Association of American Cereal Chemists American Oil Chemists Society Environmental Protection Agency International Standards Organization United States Department of Agriculture What is the Kjeldahl method? The Kjeldahl method is a means of determining the nitrogen content of organic and inorganic substances. Although the technique and apparatus have been altered considerably over the past 100 years, the basic principles introduced by Johan Kjeldahl endure today. The Kjeldahl method may be broken down into three main steps: Digestion - the decomposition of nitrogen in organic samples utilizing a concentrated acid solution. This is accomplished by boiling a homogeneous sample in concentrated sulfuric acid. The end result is an ammonium sulfate solution. Distillation - adding excess base to the acid digestion mixture to convert NH4+ to NH3, followed by boiling and condensation of the NH3 gas in a receiving solution. Titration - to quantify the amount of ammonia in the receiving solution. The amount of nitrogen in a sample can be calculated from the quantified amount of ammonia ions in the receiving solution. The Digestion Process A general equation for the digestion of an organic sample is shown below as one basic example: Organic N + H2SO4 ¨ (NH4)2SO4 + H2O + CO2 + other sample matrix by-products A number of interrelated digestion conditions determine the rate of reaction and the completeness of the breakdown of nitrogen to ammonium sulfate. Among these are heat input to the acid digestion mixture, amount of inorganic salt added to elevate the acid boiling temperature, reflux rate of H2SO4 in the neck of the digestion flask, length of digestion, and catalyst addition. Adjusting any one of these factors has an influence on the others. Proper digestion conditions for a given sample matrix are achieved through establishing a bal- ance of these factors in a controlled and repeatable fashion. In addition, if the sample contains nitrate or nitrite nitrogen, it is possible to chemically pretreat the digest to include or exclude this nitrogen source from the analysis as desired in a particular situation. Acid Considerations Sulfuric acid has been used alone for the digestion of organic samples. (As a convenience to reduce bumping of the digestion mixture, Alundum boiling chips or pumice are often added.) The amount of acid required is influenced by sample size and relative amount of carbon and hydrogen in the sample, as well as amount of nitrogen. A very fatty sample consumes more acid. Also, heat input and digestion length influences the amount of acid loss due to vaporization during the digestion process. Remember that a Kjeldahl flask is essentially a bulb with a condenser neck off to the side for refluxing of the acid. Heat Input and Digestion Length Typically the heating elements used for Kjeldahl diges- tions have variable settings. Heat input is frequently specified as “that setting which brings 250 ml of water at 25° C to a rolling boil in 5 minutes.” Initially an organic sample usually chars and blackens. The reaction may at first be very vigorous depending on the matrix and the heat input. With organic decomposition the digestion mixture gradually clears as CO2 evolves. Metallic ions might tint the clear digestion mixture. Note that solution clearing in itself is not an indication that all organic nitrogen has been broken down. Digestion length must be determined by recovery studies on known materials of similar matrix if a new method is being developed. 4 recovery. Salicylic acid followed by sodium thiosulfate has been used to pretreat the mixture to ensure complete reduc- tion. Other reduction schemes have been devised. Or other pretreatments have been used to prevent nitrates from being reduced at all during the charring process, leaving a clear digest with no contribution from nitrate ions. The Distillation Process The acid digestion mixture is diluted and made strongly alkaline with NaOH, liberating NH3 as follows: Salt Additions The problem with using sulfuric acid alone for diges- tion is very long digestion times result with many samples due to the slow rate of organic decomposition. The addition of an inorganic salt to the digest elevates the boiling point of the H2SO4. The solution temperature of concentrated sulfuric acid alone is about 330° C. Addition of a salt such as K2SO4 can elevate the solution temperature of the digestion mixture to 390° C or more, depending on the ratio of salt to acid. This significantly increases the rate of organic decomposition in the digestion mixture, shortening the length of time required for digestion. There are several precautions to keep in mind con- cerning salt addition. First, it is possible to raise the solution temperature of the digestion mixture too much. If the tem- perature goes much above 400° C during any phase of the digestion, volatile nitrogen compounds may be lost to the atmosphere. Remember that as acid is gradually consumed during the digestion process, for the various reasons mentioned above, the salt acid ratio of the digest gradually rises. This means that the hottest solution temperatures are attained at the end of the digestion. Heat input, consumption of acid by organic material and vaporization, salt/acid ratio, digestion length, and physical design of the Kjeldahl flask, are all interre- lated. Each has an effect on the final solution temperature. A second precaution is that if the salt/acid ratio is too high, a considerable amount of material will “salt out” upon cooling of the digest. Concentrated acid pockets can be con- tained within the cake. These can react violently when con- centrated base is added in the distillation process. A certain amount of salting out can be managed by diluting the digest with water while it is still somewhat warm, but not too hot. Catalyst Additions Several catalysts have been employed by Kjeldahl chemists over the years to increase the rate of organic break- down during the acid digestion. Mercuric oxide has been the most effective and widely used. However, mercury forms a complex with ammonium ions during digestion. The addition of sodium thiosulfate or sodium sulfide after digestion and before distillation will break the complex and precipitate mercuric sulfide. This is also important from a safety point of view, as mercury vapor might escape to the atmosphere during the distillation process. Because of environmental concerns over the handling and disposal of mercury, other catalysts are coming more into favor. Many methods employ copper sulfate. Titanium oxide and copper sulfate in combination have been found to be more effective than copper sulfate alone. Selenium is fre- quently used. Commercially prepared mixtures of potassium sulfate and a catalyst are available from laboratory chemical suppliers. Bulk custom mixtures are also available. Nitrate and Nitrite Reduction Kjeldahl digestions do not always recover all forms of nitrogen in a sample. Nitrate and nitrite ions in a sample must first be reduced prior to acid digestion for quantitative (NH 4) 2SO 4 + 2NaOH ¨ 2NH 3↑+ Na 2SO 4 + 2H 2O ammonium sulfate heat ammonia gas The Kjeldahl flask is attached to a water condenser and is heated to boil off the NH3 gas from the digest. The tip of the condenser is submerged in a flask of acidic receiving solution, either standard acid or boric acid solution, to again trap the distilled NH3 in receiving solution. Digestion Mixture Dilution The acid digestion mixture is usually cooled and diluted with ammonia-free water. As mentioned above, with digestion mixtures containing high salt/acid ratios, dilution prevents or minimizes caking. Sometimes this is done while the digestion mixture is uploads/Management/ guide-kjeldahl.pdf