Info

a DPG = diphosphatidylglycerol; PA = phosphatidic acid. b PE = phosphatidylethanolamine; PS = phosphatidylserine. c PI = phosphatidylinositol; PC = phosphatidylcholine.

Adapted from Christie W.W. (1982) Lipid Analysis, 2nd edn, Pergamon Press, Oxford.

1.7.4 Thin layer adsorption chromatography can achieve very good separation of small lipid samples

In this technique a thin (usually 0.25 mm) layer is spread on a glass plate and separations are achieved by allowing appropriate solvents to rise up the plate by capillary action. Silica gel is the most usual adsorbent and samples, which are applied close to the edge of the plate, are fractionated due to their different adsorptions to the support phase and/or their solubility in the solvent. Very good separations can be achieved although the thickness of the layer means that only small amounts of lipids are usually analysed. When the plates have been run, they are dried and can be sprayed with various reagents to reveal individual lipid classes. If the spray reagent is non-destructive (e.g. 2',7'-dichloro-fluorescein, which causes all lipids to fluoresce) then the lipids can be recovered by scraping the appropriate area off the plate and eluting with a suitable solvent.

Complex lipid mixtures cannot always be separated by one-dimensional chromatography. In that case, either a preliminary fractionation on a column (to give say, neutral, glycolipid and phospholipid fractions) or two-dimensional thin layer chroma-tography (TLC) can be used. An example of the separation of lipid extracts from a variety of tissues by TLC is given in Fig. 1.3.

TLC offers a number of advantages over column chromatography. It is more rapid and sensitive, gives better resolution and is usually much quicker. Moreover, the apparatus required is minimal and, especially if plates are made in the laboratory, the technique is inexpensive. By incorporating various chemicals in the thin layer, special lipid separations can be made. For example, silver nitrate allows fatty acids (or more complex lipids) to be separated on the basis of their unsaturation. Silicone oil-silica gel TLC works on the basis of reverse-phase separation and can be used to fractionate fatty acid mixtures based on their hydrophobicity, with shorter chain or unsaturated components migrating faster. Boric acid impregnation allows separation of threo- or erythro-iso-

mers of vicinal diols or fractionation of molecular species of ceramides.

A number of useful chemical methods is available for detecting or quantifying lipid classes. In some cases, the specificity of the colours developed can be used to help with identifications. Also when a non-destructive fluorescent reagent is used, lipids can be quantified on the plates by a scanning fluorimeter - though, because different lipids have different fluorescence values, calibration curves must be made.

Specific chemical methods can be used to reveal phospholipids, glycolipids, sterols or their esters as well as compounds with quaternary nitrogens or vicinal diols. Particularly useful for many membrane extracts is the reaction of ammonium molybdate with inorganic phosphate released from phospholipids. The phosphomolybdic acid so produced is then reduced to give an intense blue colour. The method can be adapted to a spray reagent or, more often, used to detect as little as 1 p,g of phosphorus in scraped samples.

1.8 OTHER USEFUL METHODS

A detailed discussion of the wide variety of methods that are available to the lipid biochemist for identification and analysis is beyond the scope of this book. However, a few of the more widespread techniques are listed in Table 1.3.

1.9 SUMMARY

In contrast to carbohydrates, proteins and nucleic acids, lipids are defined on the basis of their physical properties (insolubility in water) rather than on the basis of consistent chemical features. For this reason, the student will need to learn and remember a wide range of different chemical types and their rather complex nomenclature.

Lipids can usually be extracted easily from tissues by making use of their hydrophobic characteristics. However, such extractions yield a complex mixture of different lipid classes which have to be purified further for quantitative analysis. Moreover, the crude lipid extract may be con-

Table 1.3 Some other methods for lipid analysis

Method

IR spectroscopy NMR spectroscopy

UV spectroscopy

Supercritical fluid chromatography

Mass spectrometry

Stable isotopes

Enzyme degradation

Identification of organic bases in phospholipids or frans-double bonds.

Widely used for lipid structure determination particularly identification and location of double bonds in fatty acids, functional groups (e.g. hydroxyl) on fatty acids and preliminary identification of glycerides, glycolipids and phospholipids. spectra give better quantitative data but 13C spectra provide more information on functional groups. 31P spectra are used often to assess solid/ liquid phase ratios of lipids.

Analysis of conjugated double bond systems, especially those formed by oxidation reactions.

Supercritical fluid chromatography uses a compressed gas (often C02) above its critical temperature and pressure to elute compounds from a column. Can be used at lower temperatures than GLC and more detectors are available than for HPLC.

Often linked to a gas chromatogram or HPLC. Has a wide variety of uses from the estimation of molecular weights (e.g. use of thermospray mass spectrometry on-line with an HPLC to quantify molecular species) to the location of double bonds in aliphatic chains or identification of functional groups. Different sample inlet systems (direct insertion, fast atom bombardment) and ion sources (electron or chemical ionization) are used depending on the lability of the sample and whether structural elucidation or detection and quantitation are the main aims.

This technique makes use of stable isotopes of H, C and O and mass spectrometry. It can be utilized for quantification of substances in mixtures (by isotope dilution), in metabolic experiments, to detect food adulteration and to determine sources of lipids in geological sediments or ecological samples.

Used to show nature of specific bonds and substituents in lipids. For example, the positional distribution of acyl groups can be determined or the presence of individual sugars or bases revealed. The stereo-specificity of most enzymes also allows the configuration of the target linkage to be demonstrated.

taminated by other hydrophobic molecules, e.g. by intrinsic membrane proteins.

Of the various types of separation, thin layer and column chromatography are most useful for intact lipids. High performance liquid chromatography (HPLC) is becoming increasingly used. With the development of the evaporative light-scattering detector (sometimes called the 'universal' detector) its utility has been considerably enhanced and HPLC has the twin advantages that it is usually used at room temperature and that lipid oxidation is much reduced compared to thin-layer methods.

A powerful tool for quantitation of the majority of lipids is gas-liquid chromatography (GLC). The method is very sensitive and, if adapted with capillary columns, can provide information with regard to such subtle features as the position or configuration of substituents along acyl chains. By using light-scattering detectors with HPLC, this method can also be made quantitative. By coupling GLC or HPLC to a radioactivity detector, then the techniques are also very useful for metabolic measurements.

Although research laboratories use generally sophisticated analytical methods such as GLC to analyse and quantify lipid samples, chemical deri-vatizations are often used in hospitals. For these methods, the lipid samples are derivatized to yield a product which can be measured simply and accurately - usually by colour. Thus, total triacylglycerol, cholesterol or phospholipid-phos-phorus can be quantified conveniently without bothering with the extra information of molecular species etc. which might be determined by more thorough analyses.

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