Application

KEL PLUS AUTOMATIC NITROGEN / PROTEIN ESTIMATION SYSTEM

NITROGEN AND ITS IMPORTANCE

Nitrogen is a necessary component of all living things. Amino acids are the building blocks of proteins. When Amino acid was discovered it was found that each amino acid contained Nitrogen.

Fertilizers which have Ammonia and Urea as key components are found to contain Nitrogen. Nitrogen components like TNT, Nitrocellulose etc. are key compounds in Explosives. Heterocyclic Nitrogen Compounds are important in production of Plastic, Rubber and Paint. The purity of water is evaluated in knowing the Nitrogen content of water. Nitrogen has proved to be an excellent indicator. For example the BTU potential of a sample of coal can be calculated based on the N2 content.

KJELDAHL NITROGEN METHOD & ITS HISTORY

Nitrogen determination has a long history in the area of analytical Chemistry. Johan Kjeldahl first introduced Kjeldahl Nitrogen method in 1883 at meeting of the Danish Chemical Society. As a Chairman of Chemistry department of the Carlsberg laboratorium near Copenhagen, Kjeldahl was assigned, to scientifically observe the processes involved in beer making. While studying proteins during production he developed a method of determining Nitrogen content that was faster and more accurate than any method available at that time.

An Italian Scientist by name Dumas developed a method in 1830 which dealt with the Pyrolytic distruction of sample with the Nitrogen being reduced to Nitrogen gas. From the volume of Nitrogen gas produced the Nitrogen content of the sample is calculated. But Dumas method was too slow and gave incomplete results too. There are number of secondary methods for determination of protein or nitrogen. But all have to be caliberated against a standard method and with no other option the standard method is Kjeldahl.

EVOLUTION OF KJELDHAL METHOD

The Kjeldhal 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 125 years, the basic principles introduced by John Kjeldhal endure even today. From 1889 onwards several Chemists have made improvements in the method. These include the use of metalic Catalyst which produced a faster and greater recovery of Nitrogen, addition of pottasium Sulphate which raised the digestion temperature and shortened the digestion time, etc.

Later another Scientist – a Scandinavian agricultural Chemist by name Rozen Mosseberg’s invented the block digestor for more economical, accurate, safe, efficient and speedy way of Analysis, based on Kjeldahl Chemistry.

EVOLUTION OF KJELDHAL METHOD

The Kjeldhal 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 homogenous sample in concentrated sulphuric acid and digestion catalyst.The end result is an ammonium Sulphate solution.

DISTILLATION:- Distillation involves adding base to the acid digestion mixture to convert NH4+ to NH3.This is followed by boiling.Finally NH3 gas is condensed and trapped in a receiving solution (H3BO3)

TITRATION:- Quantifying the amount of Ammonia ions in the receiving solution the percentage of Nitrogen can be calculated.

Sampling & Preparation:

Representative sample is essential if analytical data are to be meaningful. The samples must be also homogeneous. The necessity for consideration of homogeneity increases as sample size for analytical procedure decreases. To assure homogeneity samples are usually dried and ground prior to analysis. In case of liquid samples, samples are collected in new or acid washed glass polythene bottles. They should be analysed as quickly as possible. If immediate analysis is not possible samples can be preserved for Kjeldhal digestion by acidifying to PH 1.5 to 2.0 with concentrated sulphuric acid and storing at 4°C. For analysis of Nutrients in plant materials previous wet digestion of sample is recommended. Rapid changes in the nitrate and ammonia contents of soil samples occur after removal of the samples from field or pot, because of the increased aeration and rise in temperature. It is, therefore, desirable that the extraction of nitrates and ammonium follow the collection of the samples closely.

Instrumentation :

Very early Kjeldhal digestion and distillation were performed using stone fume hoods and gas mantles as a heat source. In 1920’s this was replaced by classical Macro Kjeldhal digestion and distillation apparatus using Kjeldhal flask from 500-800ml volume.Latter variation in equipment made use of Aluminium heating blocks designed to accept number of straight digestion tubes at once simultaneously. The latest advancement in block digestion techniques by Kjeldhal procedure gave birth to automatic Nitrogen and protein analyzer in the world market from countries like Sweden, Germany, Japan, etc which made pioneering efforts for automation.

The advance Microbrilliant automatic Nitrogen /Protein Analysis system is a Pollution free and Environmental friendly system. The Hazardous acid fumes liberated during digestion are efficiently dissolved in water through giant aspiration filter pump and sent to the drain without polluting the atmosphere. The compact design of automatic block digestion with built in exhaust fume manifold system avoids the use of large fume hoods or Cupboards, saving installation cost as well as space. The instrumentation includes automatic Microprocessor based temperature controller with various built in safety features to enable a clean and a safe operation.The second stage of experiment feeds the digested sample in an automatic distillation system.The system can handle hot digestion tubes directly without the need of transferring the sample.This avoids manual spillage error during transfer, which would otherwise happen in a conventional traditional system. The Distillation process is fully programmable with precise automation and the entire process of sample dilution, reagent addition, steam injection, Boric acid addition, residue removal, water refilling, goes unhindered with a single start operation and it takes not more than 3 to 6 minutes for the completion of distillation.The third stage of the equipment a titration phase is simple and easy to handle with conventional glass burette titration system by any user. Alternatively the user may go in for a fully automatic distillation system with online integrated Auto Titration system with PC interface.

The methodology of analysis may vary slightly based on the sample behaviour. One may choose a 100ml microdigestion system for samples which are not violent in nature. High Protein samples create high effervescence and frothing and hence require a Macro digestion system of 250ml capacity. The Soil Samples have very low Nitrogen content and hence one needs at least 2 to 5gms of sample for analysis for which a Macrodigestion system is a must. A Technical expert or Chemist would recommend the appropriate use of catalyst, temperature of digestion, acid salt-ratio and so on. The only reliable method of determination of protein is through determination of Nitrogen by formula conversion i.e. 1mg nitrogen equals 6.25 mg protein. In other words 100mg of protein contains 16mg Nitrogen. Hence Nitrogen value multiplied by 6.25 factor gives percentage of protein. Thus the only standard method of determination of protein is through Kjeldhal Nitrogen method.

Common Problems

Like any other method, the Kjeldhal procedure is subject to problems with methodology, technique and equipment. Understanding the principles behind the Kjeldahl process and using appropriate quality control procedures should prevent most problems from occurring. When developing a new or modifying an existing method, one should proceed which caution and experimental verification.

When nitrogen results differ from those expected, knowledge of common problems makes finding and correcting the error easier.

Common problems include the following: Sample size and type, Incompatibility with amounts and types of acid, salt or catalyst Used Samples, standards and reagents. Inadequate or excessive distillation, Dilution volume or aliquot volume; foaming or bumping during digestion, uneven digestion, Inadequate or excessive sample temperature, precipitation of salt or caking of digested sample, leaks in apparatus connections, Inadequate sodium hydroxide addition, Improper standardisation Instrumental and calculation errors, etc.

Thanks to the invention of Kjeldahl technique by the great Danish Scientist, Johan Kjeldahl for estimation of Nitrogen, for, there is no doubt since 1883 till now there has been no other technique to supersede Kjeldahl Chemistry inspite of several advancement in Science and Technology

Chemistry Of Socs Plus

Oils and Fats are the triglycerides of fatty acids in which one molecule of glycerol has combined with three molecules of long chain fatty acids with the Elimination of water. In addition to triglycerides, the oils and fats may contain a total of 2% free fatty acids, and sterols, phospholipids coloring material and hydrocarbons.

An oil is a liquid at the ordinary temperature whereas a fat is usually a solid.

Extraction is a physical separation method used when the substance to be extracted is soluble in a solvent. The more soluble the substance the faster the extraction process.

There are two methods of separation.

Chemical.

Physical

Chemical separation results after a chemical separation. Precipitation is an example of a chemical separation. Physical separation results from the use of mechanical methods. Extraction and volatilization are examples of physical separation.

Solvent Extraction is a physical separation using a separating funnel. The liquid extraction method used water and non-miscible organic solvent. The substance to the extracted (Solute) on use have a greater affinity for the organic solvent than the water.

This is expressed as the distribution coefficient K where
K = CS/CW
CS - Solubility of the Solute in Solvent.
CW - Solubility in Water.

K must have a high numeric value for an efficient extraction to be completed.

In 1879 German Chemist, Franz Von Soxhlet developed the technology for the first time. The condensed solvent is continually contacting the sample, the solutes affinity for the solvent will continue until the extraction is complete.

The solvent flask now contains solvent as soluble. In gravimetric analysis the solvent is driven off. The solute must be removed and weighed for analytical measurement.

Chemistry Of Fibra Plus

Chemistry Of Crude Fibre

Crude fibre refers to the residue of a feed that is insoluble after successive, boiling with dilute acid and alkali. This method was originally proposed at the weende experiment station. Hence this method on determination of crude fibre is known as Weende s method of determination of crude fibre.

Crude fibre is the portion of the total carbohydrate of a food that is resistant to the acid and alkali treatment.

The Weende scheme of proximate analysis is to separate the total carbohydrate into two categories viz. crude fibre and Nitrogen free extractives. Nitrogen free extractives represents the soluble sugar starches,glycogen,some fraction of hemicellulose and of course other water soluble components like water soluble vitamins etc.

As per Weende method, crude fibre is the fraction of carbohydrate that remains after treatment with acid and alkali.

Effect of acid and alkali treatment on a fat free food

Constituent	Treatment	Treatment
	1.25% H2 SO 4	1.25 % NaOH
Protein	Partial extraction	Complete extraction
Starches and sugars	Hydrolysis and extraction	.........
Hemicellulose	variable extraction	Extensive but variable extraction
Cellulose	variable extraction	Extensive but variable extraction
Lignin extraction	slight	Extensive but highly

But this method any insoluble material after the acid and alkali digestion will be made up of (1)all original cellulose (2) variable proportion of hemicellulose (3) a small but highly variable portion of lignin.

Crude fiber

In the Weendes method when the sample is subjected to acid and alkali digestion, we obtain a residue comprising ash (mineral matter) of the feed and the resistant fraction of carbohydrate. When the residue is ignited the organic matter gets oxidized leaving the inorganic residue or ash. Thus the difference in weight of the residue before and after ashing gives the weight of crude fiber.

Weight of the sample	-W gm
Residue (crude fiber + mineral matter)	-WI gm
Ashing at 600 c Organic matter Oxidised
Ash remains	-W2 gm
Crude fiber	= W1-W2.
% of crude fibre	= W1-W2/ W X 100