eChapters

Principle

The residue remaining from the hemicellulose determination is digested with 72 percent sulphuric acid at room temperature. The lignin and acid insoluble  ash are retained in the filter crucible while the digested carbohydrate passes through. The lignin is separated from acid insoluble ash by ashing the residue.

Equipment

    1.    Acid digestion apparatus. A glass tray about 18 cm x 30 cm, 4 cm deep will hold twelve, 100 ml Griffen beakers. Another tray of the same size can be inverted over the beaker.
    2.    Filter crucibles. Same as described for cell wall determination.
    3.    Filtering apparatus. Same as described for cell wall determination.

Limitation of Weende Method

The partition of carbohydrate into fiber (CF) and nitrogen free extract (NFE) is presumed to represent a separation of less digestible starch and sugars. However, in 20 to 30 percent feeds listed by Morrison, the NFE is less digestible than the CF. This comes about for several reasons, the most important of which is the CF method (successive boiling with dilute sulphuric acid and sodium hydroxide) does not recover all the fiber and large portions of fibrous constituents  are extracted into the NFE. The most  important of these fractions are lignin and hemicellulose are dissolved by both acid and alkali. The basic error of the NFE concept is the assumption that if constituents are soluble they are digestible. Lignin, the rigid component of wood, not only is indigestible but lowers the digestibility of substances with which it is associated.

Another portion of the indigestible part of NFE arises from an artifact in the calculation of faecal NFE, where it is presumed that faecal nitrogen is protein (N x 6.25). Actually, 80-90 percent faecal nitrogen is NPN and is composed of bacterial residue in which the ratio of organic matter to nitrogen is about 1:4. Thus, part of the faecal NFE arises by an underestimation of the organic matter associated with faecal nitrogen.

Introduction

As per statement of Dr. Henneberg of Weende Experiment Station (Göttingen, Germany) the carbohydrates are divided into two groups; crude fiber and nitrogen free extract. The nitrogen free extract, which  comprises the sugars, starch and a large part of the material classed as hemicellulose, is determined by difference. It is represented by the figure obtained when the sum of the water, ash, protein, fat and crude fiber of a feed is subtracted from 100. Since the figure is determined by difference instead of directly, it includes the cumulative errors of the other determinations and thus is not an exact value.

Introduction

Wet ashing is suitable for the determination of Ca, Cu, Fe, Mg, Mn, K, Na, Se and Zn in biological samples and may be applicable to the determination of other elements as well.

The organic matter of the sample is oxidized with concentrated nitric acid and perchloric acids. The acids are partially removed by volatilization and the solube mineral constituents remain dissolved in nitric acid. Any silica present is dehydrated and made insoluble.

Introduction

Wet ashing is suitable for the determination of Ca, Cu, Fe, Mg, Mn, K, Na, Se and Zn in biological samples and may be applicable to the determination of other elements as well.

The organic matter of the sample is oxidized with concentrated nitric acid and perchloric acids. The acids are partially removed by volatilization and the solube mineral constituents remain dissolved in nitric acid. Any silica present is dehydrated and made insoluble.

Definition
The residue of incineration at 550-600ºC is the crude ash.

Reagent
Dilute hydrochloric acid -approximately 5 N prepared from concentrated hydrochloric acid. Dissolve 445 ml conc. hydrochloric acid (about 36 percent HCl) in one litre distilled water. 

Procedure for Total Ash Estimation
Take a vitreosil crucible in oven at 100ºC for six hours, remove and keep it in a desiccator and weigh up to constant weight. About 5-10 g of sample is taken in the weighted vitreosil crucible. The material in the crucible is charred on a low flame and the crucible is then kept in muffle furnace and temperature is allowed to raise to 600ºC and kept constant for two hours. It is removed on cooling and kept in a desiccator  and weight of crucible is taken.

1. Ammonia Nitrogen by Distillation Method
Method I (For Animal Feeds)
Reagents
    1.    Mangnesium oxide (carbonate free)
    2.    Standard sodium hydroxide 0.25 N
    3.    Standard sulphuric acid 0.5 N
    4.    Methyl red indicator

Weigh accurately 2 to 4 g  of the prepared sample. Shake it with water and filter. Wash the residue thoroughly with water. Transfer the filtrate to the distillation flask and dilute to about 200 ml with water. Add about 5 g of the magnesium oxide, Connect the flask to the condenser by means of the connecting bulb tube and distill about 100 ml of liquid into the receiver containing standard sulphuric acid and methyl red indicator solution, Titrate the contents of the receiver with the standard sodium hydroxide solution. Carry out a blank determination using all reagents in the same quantities but without the material to be tested.

True protein nitrogen is also termed a Albuminoid nitrogen in animal feeds and fodders and human foods. If material (Such as seeds, seed residue or oil cake) is rich in alkaline phosphates, 1-2 ml of 10 percent solution of potash alum is added to decompose them. If this is not done Cu3 (PO4) and free alkali may be formed and the protein-copper precipitate may partially dissolve in the alkaline liquid.

Principle

The nitrogen of protein and other compounds is transformed into ammonium sulphate by acid digestion with boiling sulphuric acid. the acid digest is cooled, dilute with water, and made strongly basic with sodium hydroxide. The released ammonia is distilled into a boric acid solution or standard sulphuric acid solution. When boric acid is used to collect ammonia then is the titrated with standard sulphuric acid or standard hydrochloric acid. When standard sulphuric acid is used to collect ammonia then it is titrated with standard sodium hydroxide solution.

Apparatus and Reagents

    1.    Soxhlet extraction asembly. This consists of 3 parts (a) condenser at the top, (b) the soxhlet or extractor in the middle, and (c) the receiver flask at the bottom. The 3 parts are assembled by means of their grounds glass interchangeable joints (Fig. 1).

    2.    Extraction thimble.

    3.    Petroleum ether (B.P. 40ºC to 60ºC)

    4.    Constant temperatue bath or six heater hot plate with temperature controlling device.

Calorimetry

The process of measuring  the amount of heat generated by the combustion of a substance is known as calorimetry. The first successful calorimeter using oxygen under pressure for the combustion of substance in a closed vessel was devised by Berthelot in 1881. Subsequent development by Mahler in 1992, Atwater in 1899, and Parr in 1912, served to improve and to reduce the cost of the original Berthelot apparatus by variations in mechanical details.

The dry matter content of feed samples and other materials is expressed on three aspects as fed, partially dry, and dry.

As fed refers to the feed as it is consumed by the animal, the term as collected is used for materials which are not usually fed to the animal, that is, urine, faeces, etc. If the analysis on a sample is affected by partial drying the analyses are made on the as fed or as collected sample. Similar terms: Air dry, that is hay: as received: fresh; green and wet.

Partially dry, reports to a sample of “as fed” or “as collected” material that has been dried in an oven (usually with forced air) at a temperature usually about 60ºC of freeze-dried and has been equilibrated with the air; the sample after these processes would usually contain more than 88 percent dry matter (12 percent moisture); some materials are prepared in the way so that they may be sampled, chemically analysed and stored. This analysis is referred to as “partial dry matter” percent of “as fed” or “as collected” sample. The partially dry sample must be analysed for dry matter (determined in an oven at 105ºC) to correct subsequent chemical analyses of the samples to a “dry” basis. This analysis is referred to as “dry , percent of partial dry sample similar term - “Air dry” is also used.

Dry matter is generally defined as the constant  weight a sample  attains when heated at 100ºC (occasionally 150ºC). This definition , suitable for inert materials such as sand, has three main lacunae when applied to biological materials.

    1.    In these materials, water is present in various states ranging from extraneous moisture  as rain or dew, through water present in cell sap, water present in various physico-chemical and chemical combinations.

    2.    Biological materials contain active respiratory enzymic systems which continue to function during the early stages of drying; in fact, as the material heats up, activity will be enhanced until it is stopped either  by denaturation of the enzymes or by desiccation. Besides altering the chemical composition of the material, such activity will  lead to loss of dry matter.

    3.    Most biological materials contain organic  compounds which are volatile at 100ºC and which are, therefore, lost on drying. This leads to an under-estimation of dry matter content, for example, in the case of faeces, silage and molasses.

A biological sample received in the laboratory should be processed as below:

Fresh Green Sample: During metabolism trial period it is always advisable to take a sample of fresh green fodder daily so as to  avoid chemical composition variation from place-to-place in a field. Take about two and half kilo sample from the bulk, cut the whole plant including the stem and stalks with a pair of scissors into small pieces and mix thoroughly. Weigh about 100g accurately in the aluminium tray 23156cm approximately for estimation of moisture. It is always recommended to weigh the sample immediately in the laboratory so as to avoid the losses of moisture.

Chemical changes occur during oven-drying because of the direct effect of high temperature, or of enzymic or bacterial changes during the early stages of drying, or of loss of volatile consitituents. Feeds containing molasses should be ground in a mortar.

Dried materials should be ground to pass 1 mm sieve. Samples expected to moisture during the treatments are usually predried. If wet samples are used directly in the assay, sufficient large quantity has to weighted out. In the first stage, roughages about 250-1000g are used, somewhat dependent upon homogeneity and moisture content of the wet material. These samples are weighted into trays and dried in draft oven at 60-70ºC. The dry samples are then again weighed, determined the moisture content. The trays are then left to equilibrate in air at room temperature for two days. before a second reweighing of the trays with samples. Then the sample is milled and filled into airtight containers and labelled as dry samples.

General Views about Weende’s System of Analysis

A team of scientists under the leardship of Henneberg and Stohmann of (University of Göttingen, Germany) developed proximate principles analysis system in the sixties of last century at Weende’s Experimental Station (Germany), located at the University of Göttingen. Main components of different fractions in the proximate analysis of foods are moisture, ash, crude protein, ether extract, crude fiber, and nitrogen-free-extract. These workers emphasized that carbohydrates could be grouped into: (1) the starches and the sugars, and (2) coarse fibrous fractions. Later on an insoluble residue after boiling the fat free food sample first with dilute acid and then with dilute alkali was obtained based on the acid stomach digestion and the subsequent alkaline intestinal digestion of consumed food. At the end of digestion insoluble organic residue was denoted as crude fiber.

The Metric System

At present metric system is followed to calculate final results in analysis work. This system is quite simple in nature as well as in principle. The units of the metric system are: (1) the  metre (linear measure), (2) the litre (measure of capacity), and (3) the gram (measure of mass.)

One litre is the volume of pure water at 4ºC and 760 mm pressure which weigh 1 kilogram (kg). One litre equals 1000.027 cubic centimetres (Cm3) for most work, the units “millilitre”, “cubic centimetre” and used interchangeably.

At present there are three different temperature scales in common use. These are: the Fahrenheit (F), used chiefly in English-speaking countries. the centigrade (C), used universally in scientific work and the Absolute (T) or Kelvin (K) scale, differing from the centigrade only in the position of the zero point, its divisions being of the same size. its zero point is -273ºC. The kelvin scale is used especially in dealing with very high or very low temperature and in computations involving the laws of gases. The following formulae can be used for converting one scale to another:

Normal Solution in Analytical Chemistry

A normal solution is that one that contains one equivalent weight expressed  in grams (one gram equivalent weight) to be dissolved substance per litre of solution, or one gram-milliequivalent weight per millilitre.

In the case of acid a 1N solution contains 1.008 gram of replaceable hydrogen per litre of solution. A 1N solution of a base is one that contains 17.008 of hydroxyl per litre. A 1N solution of a precipitating agent contains a weight of precipitating ion equivalent to 1.008 hydrogen. The quantity of pure reagent necessary per litre of 1N solution of a precipitating solution is calculated by dividing the gram molecular weight by the valency of the precipitating ion. In case of normal solution of oxidising and reducing agents, the amount is calculated by dividing the gram molecular weight by the total valency change in the ion concerned.

Appendix - 1

Differential Diagnosis of Poisons based on Clinical Signs

Chemical warfare involves using the toxic properties of chemical substances to kill, injure or incapacitate an enemy.

Chemical warfare is different from the use of conventional weapons or nuclear weapons because the destructive effects of chemical weapons are not primarily due to any explosive force. The offensive use of living organisms (such as anthrax) is considered to be biological warfare rather than chemical warfare; the use of nonliving toxic products produced by living organisms (e.g., toxins such as botulinum toxin, ricin, or saxitoxin) is considered chemical warfare under the provisions of the Chemical Weapons Convention. Under this Convention, any toxic chemical, regardless of its origin, is considered as a chemical weapon unless it is used for purposes that are not prohibited (an important legal definition, known as the General Purpose Criterion)