Methods of quantitative and qualitative analysis of carbohydrates in agricultural crops

Analysis of the carbohydrate composition of plants is necessary to assess the nutritional value of feed, predict the shelf life of vegetables, and control crop ripening. Different groups of carbohydrates — from simple sugars to structural cellulose and lignin — require specific approaches in the laboratory. Accurate data help the agronomist to timely adjust cultivation technology, harvesting schedules, and product storage regimes.

Analysis of simple sugars and pectins

The determination of simple (reducing) sugars is based on their ability to be easily oxidized and reduce other compounds. In laboratory practice, Fehling's solution is most often used for this — an alkaline mixture of copper sulfate with Rochelle salt. Due to the presence of Rochelle salt, copper hydroxide in an alkaline medium does not precipitate, forming a complex compound. The reagent oxidizes the aldehyde and ketone groups of sugars, resulting in the formation of a copper(I) oxide precipitate.

The amount of copper(I) oxide formed, which is used to judge the sugar concentration in the sample, is determined by the volumetric method:

1. The copper(I) oxide precipitate is treated with ferric sulfate or ferric ammonium alum in an acidic solution, converting copper into the oxidized form and reducing the iron.
2. The amount of ferrous iron formed is determined by titration with potassium permanganate in an acidic medium.

For the rapid determination of the total amount of all soluble sugars, it is convenient to use a rapid method based on a color reaction with phenol. It includes the following sequential stages:

1. Extraction of soluble sugars from plant material with ethyl alcohol.
2. Removal of alcohol by evaporation and subsequent dissolution of the dry sugar residue in water.
3. Addition of phenol and concentrated sulfuric acid to the aqueous solution to color the mixture yellow-orange.
4. Determination of the color intensity of the solution using a photoelectric colorimeter or spectrophotometer.

Accurate qualitative and quantitative analysis of individual sugars is carried out by gas-liquid, ion-exchange, or liquid chromatography methods. Quantitative determinations are also performed by ionometry using highly selective enzyme electrodes.

For the analysis of pectic substances, a volumetric method is used, which consists of three stages:

• Extraction: water-soluble substances are extracted with water, insoluble ones — with hydrochloric acid and ammonium citrate.
• Precipitation: after saponification, pectic substances are precipitated from the solution as copper complexes.
• Analysis: the content of pectic substances is calculated by the amount of bound copper, which is determined by the volumetric method.

Methods for determining starch and structural carbohydrates

Methods for starch analysis are based on its enzymatic or acidic cleavage to glucose, or on the assessment of the color intensity of the starch-iodine complex. In laboratory practice, three approaches are most common.

The first approach is the acid hydrolysis method with preliminary extraction of starch. It includes the following stages:

1. Extraction of starch from plant material with perchloric acid and its precipitation as an iodine complex.
2. Chemical decomposition of the formed iodine complex.
3. Hydrolysis of the released starch to glucose.
4. Determination of the amount of glucose obtained by the volumetric method with Fehling's solution and subsequent calculation of the starch content.

When using the second (volumetric) method, starch is transferred into solution by heating in a calcium nitrate solution and precipitated as an iodine complex. Then, the following reactions are carried out:

1. Oxidation of starch in an acidic medium with potassium dichromate to carbon dioxide and water.
2. Destruction of excess potassium dichromate by the addition of potassium iodide.
3. Titration of the released free iodine with hyposulfite.
4. Calculation of starch content based on the amount of titrant that reacted.

The colorimetric method for starch analysis includes the following steps:

1. Dissolution of starch in a hot calcium nitrate solution.
2. Precipitation with iodine in the presence of potassium iodide and calcium nitrate until a dark blue compound is formed.
3. Dissolution of the obtained precipitate in a sodium hydroxide solution.
4. Dilution of the mixture with water to a specific volume.
5. Conducting a color reaction with iodine in an acidic medium.
6. Measurement of the optical density of the solution and calculation of concentration using a graph.

• Concentration of calcium nitrate for dissolution — 80 %
• Iodine content in the starch complex — 14–16 %
• Wavelength for colorimetry — 580–610 nm

Hemicellulose. The analysis of this polysaccharide is based on the quantitative determination of reducing sugars formed after acid or alkaline hydrolysis of plant tissue. The calculation is carried out using special conversion factors.

Cellulose. The fiber content is determined by the gravimetric method. To do this, hydrolysis of all easily soluble carbohydrates of the sample is performed, after which the non-hydrolyzable residue is dried and weighed.

Lignin. The substance is separated from other components of plant tissues through chemical treatment. The purification procedure includes the sequential exposure of a sample to selective reagents to remove associated carbohydrates and proteins.

• treatment with 1% acetic acid to remove sugars and organic acids;
• treatment with acetone or a mixture of ethyl alcohol and ether (1:1) to remove chlorophyll, lipids, and resins;
• treatment with 72% sulfuric acid to remove cellulose and hemicellulose;
• washing the residue with distilled water;
• oxidation of the lignin preparation with potassium dichromate in the presence of sulfuric acid;
• determination of the excess potassium dichromate by titration with Mohr's salt or the iodometric method.