Chemistry: analytical and immunological testing – Nitrogen containing – Total nitrogen determined
Reexamination Certificate
1999-07-29
2001-09-11
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Nitrogen containing
Total nitrogen determined
C436S163000
Reexamination Certificate
active
06287868
ABSTRACT:
The present invention relates to a process for determination of nitrogen, primarily Kjeldahl nitrogen, using an equipment which substantially comprises a multiple step working order. The working order comprises, besides sample preparation, an pre-treatment, such as digestion, of a sample of a nitrogen containing material, followed by distillation and determination/analysis of the nitrogen content, determination of to said nitrogen content directly related amounts of one or more compounds and/or determination of to said nitrogen content directly related analytical parameters. Said determination/analysis is suitably performed gravimetrically, volumetrically and/or chromatographically. More specifically, the process of the present invention refers to a new sequence of the substeps of said distillation, whereby a number of surprising advantages, such as increased operational security and increased analytical accuracy, are obtained.
The Kjeldahl method for determination of nitrogen was introduced in 1883 and has since been subject to substantial development efforts with the aim of improving said method. Determination of nitrogen according to Kjeldahl is today one of the most frequently used analytical methods for analysis of raw materials and final products within for instance the food and forage industry. The chemical principle behind the Kjeldahl method can in short be said to comprise a digestion step carried out at a high temperature, whereby a nitrogen containing sample, such as a protein, in the presence of an acidic reagent, usually comprising sulphuric acid, and a catalyst is transformed into ammonium ions. The sample is subsequently cooled and alkali is added to the digested sample, whereby said ammonium ions are transformed into gaseous ammonia and recovered as such during a distillation. The amount of distilled ammonia is finally determined, for instance titrimetrically. In modern high capacity laboratories are nowadays instruments designed to automatically perform said digestion, distillation and analysis normally used. These instruments provide increased capacity and operational security as well as release operators time due to extended unattended operations.
A typical working sequence when determining nitrogen according to Kjeldahl and using a commercially available equipment is digestion, distillation and titration and can be exemplified as follows. A prepared sample of a nitrogen containing material is weighed into a 100-1000 ml digestion tube in an amount of 0.1-0.5 g followed by addition of 5-20 ml of an acidic reagent, normally concentrated sulphuric acid, and 3-9 g of a catalyst/salt mixture. The catalyst/salt mixture can for instance comprise potassium salts, such as potassium sulphate, in combination with copper and/or selenium and optionally a peroxide, such as hydrogenperoxide. The digestion tube is normally a cylindrical tube (a so called Kjeldahl tube) made of heat resistant glass. Digestion is normally performed at a temperature of 300-600° C., such as 400-500° C. or preferably ~420° C., during for example 45-120 minutes. The digestion involves a chemical reaction wherein bonded nitrogen or nitrogen otherwise included in the material sample is transformed into ammonium ions. The material sample with added reagent and salt/catalyst is sometimes put on hold before distillation. A certain degree of digestion (chemical reaction) occurs, despite the lack of heating, during this hold. A certain amount, such as 2-15 ml, of acid is normally present in the digestion mixture after completed digestion. The digested sample is normally a liquid (acidic solution), but acidic salt crusts are sometimes formed during cooling of the sample. The digestion conditions can within certain limits be adjusted to avoid formation of said salt crusts. The use of high amounts of acid normally result in a liquid solution also after cooling. The disadvantages using high amounts of acid are obvious and comprise increased energy consumption, increased raw material costs, increased costs for waste disposal, hygienic problems related to handling of concentrated acid and reduced analytical accuracy and operational security. The tube containing the digested sample is subsequent the digestion transferred to a distillation equipment and most often diluted with for instance water. The dilution is primarily made to reduce the acid strength in the digested sample. The dilution is in simpler equipment made manually and in more sophisticated equipment by means of an automatic control system. Alkali, such as sodium or potassium hydroxide, is now added to increase the pH value from ~0 to more than 10, preferably ~12. Heat is developed when alkali is added to the sample, which despite the dilution is strongly acidic, and the solutions are normally allowed to mix, at least partly, before distillation. Distillation, most often an steam distillation, is then commenced and obtained ammonia containing distillate is recovered, suitably in an acidic and/or complexing medium such as boric acid. Determination of ammonia, protein and nitrogen, that is Kjeldahl nitrogen, is performed titrimetrically, so called acid-base titration.
Standard methods, such as ISO, CEN, SIS, AOAC, etc., for determination of Kjeldahl nitrogen are normally adapted to manual handling of samples. These methods basically follow above disclosed procedure, including the substeps of the distillation step. The same is valid for control systems used in commercially available automatic equipment. The process steps and substeps is always performed in below sequence:
a) Sample pre-treatment, normally digestion,
b) Distillation comprising the substeps:
a) Dilution,
b) Alkali addition,
c) Distillation, such as steam distillation,
c) Determination/analysis.
Above procedure performed according to for instance any of the standard methods comprises a number of drawbacks and hazards. Dilution with water means that the operator mixes the digested and highly acidic sample with water. The hazards of mixing water and strong acids are well known, such formation of layers and major differences in concentration between said layers. Acid containing salt crusts formed during the digestion are only to a minor degree influenced by water addition. It takes a substantial amount of time to dissolve said salt crusts under conditions existing in a digestion and/or distillation tube. Addition of alkali is in commercially available Kjeldahl equipment normally performed in a closed system, by means of for instance a pump, during 15-30 seconds. The degree of mixing is dependent on for instance the pump used for alkali addition. Added alkali is normally slowly descending towards the bottom of the tube, forming interfacial layers, and if a salt crust is formed during the digestion towards the liquid layer beneath the salt crust. Addition of alkali is uncertain and a heavy heat generating reaction may occur between acid and alkali. The alkali solution has a tendency to form a layer beneath the water diluted sample solution, why a large excess of alkali will be added before reaction between alkali and acid occurs. The heat generated by reaction between alkali and acid is proportional to the amount of acid remaining in the digested sample. The subsequent step, the distillation step, is normally commenced 5-30 seconds after the alkali addition. The acid containing digested sample solution and the alkali solution arc during said 5-30 seconds mixed to a certain degree through mainly diffusion processes in the interface between these solutions. Inlet of steam for steam distillation implies a rapid mixing of the acidic sample and the alkali with pendant increased temperature. A large acid excess or the presence of salt crusts may give rise to a heavy and even hazardously heavy reaction. Alkali and/or sample may escape the tube due to tube cracking or activation of safety lock gates and the like. These risks are well known to users of commercially available Kjeldahl equipment, why many users dilute the sample manually whereby a more efficient mixing can be obtained which reduces th
Browdy and Neimark
Cole Monique T.
Foss Tecator AB
Warden Jill
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