|
|||||||||||||||||||||||||||||||||||||||
Analysis of monosaccharides and oligosaccharides
Chromatographic methods are the most powerful analytical techniques for the analysis of the type and concentration of monosaccharides and oligosaccharides in foods. Thin layer chromatography (TLC), Gas chromatography (GC) and High Performance Liquid chromatography (HPLC) are commonly used to separate and identify carbohydrates. 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 derivitized, whereas 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.g., by reaction with borates. A solution of the derivitized carbohydrates is applied to a gel and then a voltage is applied across it. The carbohydrates are then separated on the basis of their size: the smaller the size of a carbohydrate molecule, the faster it moves in an electrical field.
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. Titration Methods
Gravimetric MethodsThe Munson and Walker method is an example of a gravimetric method of determining the concentration 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 sample. The concentration of precipitate present can be determined gravimetrically (by filtration, drying and weighing), or titrimetrically (by redissolving the precipitate and titrating with a suitable indicator). This method suffers from the same disadvantages as the Lane-Eynon method, neverthless, it is more reproducible and accurate. Colorimetric MethodsThe Anthrone method is an example of a colorimetric method of determining the concentration of the 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 620 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.
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: (i) allowing the reaction to go to completion and measuring the concentration of the product, which is proportional to the concentration of the initial substrate; (ii). measuring the initial rate of the enzyme catalyzed reaction because the rate is proportional to the substrate concentration. Some examples of the use of enzyme methods to determine sugar concentrations in foods are given below: D-Glucose/D-FructoseThis method uses a series of steps to determine the concentration of both glucose and fructose in a sample. First, glucose is converted to glucose-6-phosphate (G6P) by the enzyme hexakinase and ATP. Then, G6P is oxidized by NADP+ in the presence of G6P-dehydrogenase (G6P-DH).
The amount of NADPH formed is proportional to the concentration of G6P in the sample and can be measured spectrophotometrically at 340nm. The fructose concentration is then determined by converting the fructose into glucose, using another specific enzyme, and repeating the above procedure. Maltose/SucroseThe 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-glucosidase, and 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.g., lactose, galactose and raffinose.
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 polarimetry, refractive index, IR, and density. PolarimetryMolecules 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 extent of polarization is related to the concentration of the optically active molecules in solution by the equation a = [a]lc, where a is the measured angle of rotation, [a] is the optical activity (which is a constant for each type of molecule), l is the pathlength and c is the concentration. The overall angle of rotation depends on the temperature and wavelength of light used and so these parameters are usually standardized to 20oC and 589.3 nm (the D-line for sodium). A calibration curve of a versus concentration is prepared using a series of solutions with known concentration, or the value of [a] is taken from the literature if the type of carbohydrates present is known. The concentration of carbohydrate in an unknown sample is then determined by measuring its angle of rotation and comparing it with the calibration curve. Refractive Index
DensityThe density of a material is its mass divided by its volume. The density of aqueous solutions increases as the carbohydrate concentration increases. Thus the carbohydrate concentration can be determined by measuring density, e.g., using density bottles or hydrometers. This technique is routinely used in industry for determination of carbohydrate concentrations of juices and beverages. InfraredA material absorbs infrared due to vibration or rotation of molecular groups. Carbohydrates contain molecular groups that absorb infrared radiation at wavelengths where none of the other major food constituents absorb consequently their concentration can be determined by measuring the infrared absorbance at these wavelengths. By carrying out measurements at a number of different specific wavelengths it is possible to simultaneously determine the concentration of carbohydrates, proteins, moisture and lipids. Measurements are normally carried out by measuring the intensity of an infrared wave reflected from the surface of a sample: the greater the absorbance, the lower the reflectance. Analytical instruments based on infrared absorbance are non-destructive and capable of rapid measurements and are therefore particularly suitable for on-line analysis or for use in a quality control laboratory where many samples are analyzed routinely. More sophisticated instrumental methods are capable of providing information about the molecular structure of carbohydrates as well as their concentration, e.g., NMR or mass spectrometry.
Immuoassays are finding increasing use in the food industry for the qualitative and quantitative analysis of food products. Immunoassays specific for low molecular weight carbohydrates are developed by attaching the carbohydrate of interest to a protein, and then injecting it into an animal. With time the animal develops antibodies specific for the carbohydrate molecule. These antibodies can then be extracted from the animal and used as part of a test kit for determining the concentration of the specific carbohydrate in foods. Immuoassays are extremely sensitive, specific, easy to use and rapid. back to top of
page |