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  Method of analysis of lipids, Biology tutorial

Introduction:

Similar to other macromolecules, lipids have their physicochemical properties and can be analyzed qualitatively or quantitatively. Physical and chemical properties of lipids are essentially thus of the composition of lipids. Insolubility of lipids in water makes it have a special approach to isolation/extraction different from other macromolecules such as, carbohydrates and proteins.

Physical properties of lipids:

i) Lipids are insoluble in water but are soluble in organic solvents like ether and chloroform.

ii) They are oily or greasy organic substances .

iii) Lipids are found in plant and animal tissues where they can be extracted from.

iv) Lipids can be solid or liquid at room temperature.

v) They have characteristics melting point that is influenced by nature of fatty acids contained in the lipid. The more C-chain length and degree of saturation of a fatty acid, the higher its melting point.

Chemical properties of lipids:

Lipids like (Neutral fats) are vulnerable to acid, alkaline or enzyme hydrolysis. Enzymes which catalyze hydrolysis of lipids are known as esterases or lipases. Alkaline hydrolysis of the lipids is known as saponification.

1073_saponification.jpg

Reaction is irreversible and thus carboxylate ions combine with sodium (Na) or potassium (K) salt of alkali to create soap. Saponification of lipids is estimated by its saponification value which is the number of milligram of KOH needed to saponify 1gram of fat.

Polar lipids like (phospholipids) are ampiphatic (has a charged head group and hydrophobic tail) as such in aqueous systems polar lipids spontaneously disperse to create micelles in which hydrophobic tails are lucked or hidden inside micelle structure and polar heads are exposed to aqueous environments.

Acid value:

Acid value of fat can be stated as number of milligram of KOH needed to neutralize free fatty acids present in 1gram of fat. Every lipid sample has a characteristic acid value that is stated by number of and kind of fatty acid contained in it.

Iodine value:

Iodine value is another property of lipids that is stated as number of grams of iodine absorbed by 100g of lipid. The molecule of iodine adds across each double bond of unsaturated fatty acid. Iodine value provides the measure of degree of unsaturation of the lipid.

Functions of lipids:

  • Lipids are stored in tissues largely in the water free state and thus serve as reservoirs of energy.
  • Few lipids serve as structural components of membranes like phospholipids.
  • Few lipids act as intracellular signals like (phosphatidylinositols).
  • Lipids like biological waxes play significant role in giving the water barrier for insects, birds and other animals like sheep. Biological waxes find the variety of application in pharmaceutical, cosmetic and other industries
  • Lipids like Gangliosides form very significant components of specific receptor sites on surfaces of cell membranes.
  • Lipids (phospholipids) play significant roles in architectures of membranes.
  • Lipids serve as good sources of fat soluble vitamins: A, D, E and K.

Analyses of lipids:

Because of the fact that lipids are insoluble in water, their extraction and subsequent fractionation needs use of organic solvents and some methods not usually in utilized in purification of water soluble molecules like proteins and carbohydrates.

Extraction of lipids:

Neutral lipids (triacylglycerols, waxes, pigments) are voluntarily extracted from tissues with ethylether, chloroform or benzene. Usually utilized extractant is the mixture of chloroform, methanol and water. Initial volume proportions (1:2:0.8) which are miscible are utilized in homogenization of tissues to extract all lipids. Lipids remain in chloroform layer and more polar molecules like protein and sugar partition in methanol/water layer.

Qualitative/quantitative analyses of lipids:

One very significant way of qualitatively analysis for lipids is by use of thin layer Chromatography (TLC) utilizing silica gel or silicic acid. The thin layer of silica gel is spread onto the glass plate, to which it adheres. The small sample of lipids dissolved in chloroform is applied near one edge of plate that is dipped in shallow container of the organic solvent or solvent mixture. All of which is surrounded within the chamber saturated with solvent vapor. As solvent rises on plate by capillary action, it carries lipids with it. Less polar lipids move farthest, as they have fewer tendencies to bind to silica gel. Lipids can then be detected after separation by spraying plate with the dye (rhodamine) which fluoresces when related with lipids, or by exposing plate to iodine fumes that provides the yellow or brown color with lipids having unsaturated fatty acids. Other chromatographic techniques like High Performance Liquid Chromatography (HPLC), Gas Liquid Chromatography (GLC) can be used in both quantitative and qualitative analysis of lipids. Colorimetry, methods which involve use of chromogenic reagents which could complex with lipids to provide colored complex which can be estimated by use of colorimeters are also available for quantitative estimation of lipids.

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