Determination of total carbohydrates by anthrone method. Carbohydrates are the important components of storage and structural materials in the plants. Recently, Morse4 and Morris5 have described the use of anthrone for the quantitative estimation of carbohydrates. This method is both quicker and. The precise method of carbohydrate isolation depends on the carbohydrate type, solution as this would cause an underestimation of the carbohydrate content. The Anthrone method is an example of a colorimetric method of determining.
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Carbohydrates are one of the most important components in many foods. Carbohydrates may be present as isolated molecules or they may be physically associated or chemically bound to other molecules.
Individual molecules can be classified according to the number of monomers that they contain as monosaccharidesoligosaccharides or polysaccharides. Molecules in which the carbohydrates are covalently attached to proteins are known as glycoproteinswhereas those in which the carbohydrates are covalently attached to lipids are known as glycolipids.
Some carbohydrates are digestible by humans and therefore provide an important source of energy, whereas others are indigestible and therefore do not provide energy. Indigestible carbohydrates form part of a group of substances known as dietary fiberwhich also includes lignin. Consumption of significant quantities of dietary fiber has been shown to be beneficial to human nutrition, helping reduce the risk of certain types of cancer, coronary heart disease, diabetes and constipation.
As well as being an important source of energy and dietary fiber, carbohydrates also contribure to the sweetness, appearence and textural characteristics of many foods. It is important to determine the type and concentration of carbohydrates in foods for a number of reasons. Monosaccharides are water-soluble crystalline compounds.
They are aliphatic aldehydes or ketones which contain one carbonyl group and one or more hydroxyl groups. Most natural monosachharides have either five pentoses or six hexoses carbon atoms. Commonly occurring hexoses in foods are glucose, fructose and galactosewhilst commonly occurring pentoses are arabinose and xylose.
The reactive centers of monosaccharides are the carbonyl and hydroxyl groups. Disaccharides consist of two monomers, whereas trisaccharides consist carbohdyrate three. Oligosaccharides containing glucose, fructose and galactose monomers are the most commonly occurring in foods. The majority of carbohydrates found in nature are present as polysaccharides. Polysaccharides containing all the same monosaccharides are called homopolysaccharides e.
A large number of analytical techniques have been developed to measure the total concentration and type of carbohydrates present in foods see Food Analysis by Nielssen or Food Analysis by Pomeranz and Meloan for more details. The carbohydrate content of a food can be determined by calculating the percent remaining after all the other components have been measured: Nevertheless, this method can lead to erroneous results due to experimental errors in any of the other methods, and so it is usually better to directly measure the carbohydrate content for accurate measurements.
The amount of preparation needed to prepare a sample for carbohydrate analysis depends on the nature of the food being analyzed. Aqueous solutions, such as fruit juices, syrups and honey, usually require very little preparation prior to analysis. On the other hand, many foods contain carbohydrates that are physically associated or chemically bound to other components, e.
In these foods it is usually necessary to isolate the carbohydrate from the rest of the food before it can be analyzed. The precise method of carbohydrate isolation depends on the carbohydrate type, the food matrix type and the purpose of analysis, however, there are some procedures that are common to many isolation techniques. For example, foods are usually dried under vacuum to carbohydratr thermal degradationground to a fine powder to enhance solvent extraction and then defatted by solvent extraction.
Monosaccharides and oligosaccharides are soluble in alcoholic solutions, whereas proteins, polysaccharides and anthrrone fiber are insoluble.
The soluble components can be separated from the insoluble components by filtering the boiled solution and collecting the filtrate the part which passes through the filter and the retentante the part retained by the filter. These two fractions can then be dried and weighed to determine their concentrations.
In addition, to monosaccharides and oligosaccharides various other small molecules may also be present in the alcoholic extract that could interfere with the subsequent analysis e.
It is usually necessary to remove metbod components prior to carrying out a carbohydrate analysis. This is commonly achieved by treating the solution with clarifying agents or by passing it through one or more ion-exchange resins. Prior to analysis, the alcohol can be removed from the solutions by evaporation under vacuum so that an aqueous solution of sugars remains.
Chromatographic methods are the most powerful analytical techniques for the analysis of the type and concentration of monosaccharides and oligosaccharides in foods. Carbohydrates are separated on the basis of their differential adsorption characteristics by passing the solution to be analyzed through a column. Carbohydrates can be separated on the basis of their partition coefficients, polarities or sizes, depending on the type of column used.
HPLC is currently the most important chromatographic method for analyzing carbohydrates because it is capable of rapid, specific, sensitive and precise measurements. In addition, GC requires that the samples be volatile, which usually requires that they be derivitizedwhereas in HPLC samples can often be analyzed directly. HPLC and GC are commonly used in conjunction with NMR or mass spectrometry so that the chemical structure of the molecules that make up the peaks can also be identified.
Carbohydrates can also be separated by electrophoresis after they have been derivitized to make them electrically charged, e. A solution of the derivitized carbohydrates is applied to a gel and then a voltage is applied across it.
Anthrone Method for Determination of Carbohydrate
The carbohydrates are then separated on the basis of their size: A number of chemical methods used to determine monosaccharides and oligosaccharides are based on the fact that many of these substances are reducing agents that can react with other components to yield precipitates or colored complexes which can be quantified.
The concentration of carbohydrate can be determined gravimetrically, spectrophotometrically or by titration. Non-reducing carbohydrates can be determined using the same methods if they are first hydrolyzed to make them reducing. It is possible to determine the concentration of both non-reducing and reducing sugars by carrying out an analysis for reducing sugars before and after hydrolyzation. Many different chemical methods are available for quantifying carbohydrates.
Most of these can be divided into three catagories: An example of each of these different types is given below.
The Lane- Eynon method is an example of a tritration method of determining the concentration of reducing sugars in a sample. A burette is used to add the carbohydrate solution being analyzed to a flask containing a known amount of boiling copper sulfate solution and a methylene blue indicator. The reducing estimatino in the carbohydrate solution react with the copper sulfate present in the flask.
Determination of Carbohydrate by Anthrone Method
Once all the copper sulfate in solution has reacted, any further addition of reducing sugars causes the indicator to change from blue to white. The volume of sugar solution required to reach the end point is recorded.
The reaction is not stoichemetricmetgod means that it is necessary to prepare a calibration curve by carrying out the experiment with a series of standard solutions of known carbohydrate concentration.
The disadvantages of this method are i the results depend on the precise reaction times, temperatures and reagent concentrations used and so these parameters must be carefully controlled; ii it cannot distinguish between different types of reducing sugar, and iii it cannot directly determine the concentration of non-reducing sugars, iv it is sucseptible to interference from other types of molecules that act as reducing agents.
The Munson estiamtion Walker method is an example of a gravimetric method of determining the estomation of reducing sugars in a sample. Carbohydrates are oxidized in the presence of heat and an excess of copper sulfate and alkaline tartrate under carefully controlled conditions which leads to the formation of a copper oxide precipitate:. The amount of precipitate formed is directly related to the concentration of reducing sugars in the initial anrhrone.
7. ANALYSIS OF CARBOHYDRATES
The concentration of precipitate present can be determined gravimetrically by filtration, drying and weighingor titrimetrically by redissolving the precipitate and titrating with a suitable indicator.
This method suffers from the same disadvantages as the Lane- Eynon method, neverthlessit is more reproducible and accurate. The Anthrone method is an example of a colorimetric method of determining the concentration of cqrbohydrate total sugars in a sample.
Sugars react with the anthrone reagent under acidic conditions to yield a blue-green color. The sample is mixed with sulfuric acid and the anthrone reagent and then boiled until the reaction is completed. The solution is then allowed to cool and its absorbance is measured at nm. There is a linear relationship between the absorbance and the amount of sugar that was present in the original sample.
This method determines both reducing and non-reducing sugars because of the presence of the strongly oxidizing sulfuric acid. Like the other methods it is non- stoichemetric and therefore it is necessary to prepare a calibration curve using a series of standards of known carbohydrate concentration. The Phenol – Sulfuric Acid method is an example of a colorimetric method that is widely used to determine the total concentration of carbohydrates present in foods.
A clear aqueous solution of the carbohydrates to be analyzed is placed in a test-tube, then phenol and sulfuric acid are added.
Carbohdyrate solution turns a yellow-orange color as a result of the interaction between the carbohydrates and the phenol.
The absorbance at nm is proportional to the carbohydrate concentration initially in the sample. The sulfuric acid causes all non-reducing sugars to be converted to reducing sugars, so that this method determines the total sugars present.
This method is non- stoichemetric and so it is necessary to prepare a calibration curve using a series of standards of known carbohydrate concentration. Analytical methods based on enzymes rely on their ability to catalyze specific reactions.
These methods are rapid, highly specific and sensitive to low concentrations and are therefore ideal for determination of carbohydrates in foods. In addition, little sample preparation is usually required. Liquid foods can be tested directly, whereas solid foods have to be dissolved in water first.
There are many enzyme assay kits which can be purchased commercially to carry out analysis for specific carbohydrates. Manufacturers of these kits provide detailed instructions on how to carry out the analysis. The two methods most commonly used to determine carbohydrate concentration are: Some examples of the use of enzyme methods to determine sugar concentrations in foods are given below:.
This method uses a series of steps to determine the concentration of both glucose and fructose in a sample. The amount of NADPH formed is proportional to the concentration of G6P in the sample and can be measured spectrophotometrically at nm. The fructose concentration is then determined by converting the fructose into glucose, using another specific enzyme, and repeating the above procedure.
The concentration of maltose and sucrose disaccharides in a sample can be determined after the concentration of glucose and fructose have been determined by the previous method.
The maltose and sucrose are broken down into their constituent monosaccharides by the enzyme a- glucosidase: The concentrations of glucose and fructose can then be determined by the previous method. The major problem with this method is that many other oligosaccharides are also converted to monosaccharides by a – glucosidaseand it is difficult to determine precisely which oligosaccharides are present.
This method is therefore useful only when one knows the type of carbohydrates present, but not their relative concentrations. Various other enzymatic methods are available for determining the concentration of other monosaccharides and oligosaccharides, e. Many different physical methods have been used to determine the carbohydrate concentration of foods. These methods rely on their being a change in some physicochemical characteristic of a food as its carbohydrate concentration varies.
Commonly used methods include polarimetryrefractive index, IR, and density. Molecules that contain an asymmetric carbon atom have the ability to rotate plane polarized light.
A polarimeter is a device that measures the angle that plane polarized light is rotated on passing through a solution. A polarimeter consists of a source of monochromatic light, a polarizer, a sample cell of known length, and an analyzer to measure the angle of rotation. The overall angle of rotation depends on the temperature and wavelength of light used and so these parameters are usually standardized to 20 o C and