22 Separation and analysis of lipids

Prof. M. N. Gupta

1. Objectives

•  To learn about important parameters like Lovibond colour, iodine number, solid fat content and acetyl number
•  To learn about how lipids are isolated and purified
•  To learn about isolation of vegetable oils, milk fats and fish oils
•  To learn about the analysis of lipids by HPLC, GC etc.

2. Concept Map

3. Description

By now it may be clear that even simple lipids, fats and oils, when initially obtained contain a large number of other compounds/materials. So, is the case with complex lipids. We have already briefly discussed how lecithin preparations of different purity levels are utilized by different industries.

Separation/purification and analysis go hand in hand. Analysis is used to monitor efficiency of the purification methods. Analysis is also part of the quality control of the finished product.

So, we will look at the analytical aspects first and then the various separation methods.

While all classes of lipids have their importance, fats and oils occupy a central place. The long chain fatty acids of fats and oils are also part of other glycerolipids like many phospholipids.

Hence many parameters developed by oil industry and regulatory agencies to assess oil quality have wider applications. The number of double bonds are important not just for fats/oils!

As by definition, lipids are soluble in organic solvents. So, solvent extraction has been traditionally very important in the isolation and purification of lipids.

For some classes of lipids, some specific protocols may be used. Here, we will discuss only general methods. It may however be added that supercritical fluids have also been used in some cases. It is possible that with time, we will see greener options emerging in this area.

Let us start with the analysis of oils and fats. Oils/fats are obtained from natural sources. Whether this source is of plant or animal origin, the crude oil base is bound to vary in composition. So, separation/purification or refining protocols are designed to be robust enough to factor this in.

Same goes for the analysis. Whatever method we follow should be able to take care of the interference from both known level/unknown levels of “impurities”. Not all analytical procedures can necessarily handle this by running controls.

As edible oil industry is a mature industry, methods for determining every considerable parameters have been developed. Professional organisations who have developed standard methods include American Oil Chemists Society (AOCS), The Association of official Analytical Chemists (AOAC) and the American Society for Testing Materials (ASTM).

For illustrative purposes, let us look at the routine quality control tests which are carried out for soybean oil. More or less similar tests are carried out for other edible oils.

Vegetable oils (as oils of plant origins are called in the industry) when extracted are coloured. Carotenoids and chlorophylls are the main pigments but many other compounds also contribute in different oils.

Lovibond colour measures the red and yellow colours mostly. In this method, a 52.5 inch column of the oil/melted fat is used as the sample and its colour compared in a tintometer against red and yellow lovibond colour glasses. These glasses are calibrated according to AOCS tintometer colour scale. Worldwide, British Standard 684 is used as a colour scale.

Oils which are dark and contains impurities of colours other than red and yellow are unsuitable for measurement by this method. Olive oil, soybean oil (if extracted from green beans), canola oil contain enough chlorophyll to often impart green colour to these colours.

In many cases, if polyphenols are present, oils generally develop brown colour and it is difficult to get any reliable estimate. The obvious solution is to develop spectrophotometric methods but no standard has been developed so far.

Fat analysis committee of AOCS has provided various standards and the best match is determined. Drying oils are generally graded by Gardner scale which uses 1963 Glass standards based upon solutions of inorganic salts. The scale reads 1 for the lightest colour and 18 for the darkest.

Iodine value:

Also called iodine number, this reflects unsaturation in the fatty acid composition. Most double bonds of fatty acids react quantitatively with Br2 or IBr at room temperature in acetic acid or methanolic solution. The quantity of halogen remaining can be measured iodometrically. Amount of iodine in g which is required to react with 100 g of lipid is called its iodine value/number.

The iodine number of saturated fatty acids is zero. Oleic acid, linoleic acid and linolenic acid have iodine numbers of 90, 181 and 274 respectively.

Iodine numbers are more quickly obtained by measuring refractive index and correlating the value with standards of known iodine numbers. As refractive index changes with temperature, refractometers with temperature control must be used.

Titration equivalent weight:

This estimates the average molecular weight of the fatty acids present in the oil. Titration of alcoholic solution of fatty acid mixtures with standard alkali using phenolpthalein as indicator determines the end point.

Mean Molecular Weight = mg of fatty acid / mEq of alkali

Acetyl Number:

This estimates the presence of free –OH groups in the fat/oil or fatty acid. Acetic anhydride is used to acetylate all such groups. Acetyl number is the number of mg of KOH required to neutralise acetic acid obtained from 1 g of acetylated sample.

Solid fat content (SFC) or solid fat index (SFI) is a useful parameter if the intended application of fat requires plasticity. Margarine and shortenings are two such end products. SFI uses dilatometry which measures volumes at series of temperatures: 10°, 21.1°, 26.7°, 33.3° and 40 ° C. SFC is measured by pulsed nuclear magnetic resonance.

FFA content represents the amount of free fatty acids present in the lipid. This is simply determined by titration with a standard alkali. FFA analysis of a refined oil indicates the efficiency of alkali refining and deodorization steps in the refining process.

Higher FFA in a frying oil lowers the smoke point of the oil which is an undesirable trait. As mentioned elsewhere, if the oil is to be used for production of esters with alkali as a catalyst, formation of soaps becomess a hindrance.

In fact soap analysis measures the soap formed by titrating with alkali. This not only measures how much alkali refining has succeeded, it also reflects upon the efficiency of water washing step in the refining process.

Alkali refining consists of continuous mixing of sodium hydroxide solution of a known concentration with the crude oil. At 65-90 °C, soap coagulates with formation of water phase (soap stock) and refined oil phase.

Centrifugation generally does not remove all soap and other impurities from the oil phase. Hence, this oil phase is washed with hot water and centrifuged again. The mixing time of alkali with caustic solution is generally 3-10 min at 20-40 °C. The temperature is quickly raised to separate coagulated soap.

In some countries in Europe and elsewhere, an alternative short mix method is used. The mixing is only for 1-15 secs at 80 -90° C. This process is often repeated to remove all free fatty acids.

Water washing can be done once or twice and can even be carried out in countercurrent mode. Economics and equipment availability decides some of these choices.

Often, alkali refining is preceded by treatment with phosphoric acid to deal with non hydratable phosphatides which are present in some oils including soybean and canola.

These non hydratable phosphatides are Ca2+ and Mg2+ salts. The hydratable phosphatides, of course are removed by addition of soft water and removing precipitated materials. Water is removed from the degummed oil.

Purification of Lipids

The source of the lipid material to be extracted can be an animal tissue, plant tissue or even microbial.

Nevertheless, isolation of lipid follows some common approaches and common concerns.

It is advisable to keep the sample at low temperature (less than -25°C is a good choice) and in an oxygen free/inert atmosphere. The lipids containing unsaturated bonds, as we know, are prone to oxidation.

For the same reason, butylated hydroxy toluene (BHT) is added in the range of 1-10 mg/L of the extracting solvent. Extraction with the organic solvents is invariably the first step in lipid isolation.

Keeping the temperature low also minimizes damage to the lipids by enzymatic or non-enzymatic routes.

This is especially important for plant tissues which are rich in both polyphenols and polyphenol oxidases.

It must be remembered that even in the frozen form, enzymatic degradation cannot be totally stopped.

This is best appreciated by recollecting that even frozen food have shelf life.

Some of the approaches which have been tried for inactivating endogeneous enzymes are: exposing the tissues to boiling water or steam or even microwaving the sample.

The solvent chosen for extraction depends upon the nature of the lipid to be extracted. Generally, mixture of solvents, rather than a single solvent is tried. Extracting with CHCl3: CH3OH: H2O (in the proportion of 1:2:0.4) is reported to extract practically all kinds of lipids.

The amount of water present in the sample should be factored in while preparing the solvent mixture.

These solvents mixed as above form a single phase mixture.

The lipid material is homogenized in the above solvent mixture. The rest of the solids separated from the extracted lipids remain in the solvent mixture.

Adding either of the three liquids leads to separation of the extracted samples into two phases. Upper aqueous phase has any non-lipid material which got extracted.

The lower CHCl3 containing phase has all the extracted lipids. This phase is washed once with water to further remove any adhering non lipid material.

Phospholipids etc which are more polar in nature can partition into the water phase. This can be avoided by using salt solutions (at all above steps) in place of water.

The CHCl3 phase is dried by treatment with anhydrous Na2SO4. The solvent (CHCl3) thereafter can be removed by evaporation by any of the usual devices.

Selective extraction of neutral lipids from the dried mass can be carried out by extraction with cold anhydrous acetone. This extraction procedure does not extract phospholipids. Dry acetone of low water content is available from good vendors like Aldrich. This low water grade acetone can be further dried by placing over molecular sieves.

As has been mentioned before, some free fatty acids are always present in a fat/oil. These fatty acids can be isolated by treating the tissue or liquid extract by dilute alkali. Both aqueous and methanolic solutions have been used.

Most of the vegetable oils are obtained by mechanical processing and/or extraction from seeds or by pressing of fruits like olive or olive palm.

Among the solvents (for solvent extraction), hexane is invariably used. Hexane is considered a volatile organic compound (VOC) which hurts the ozone layer. However, despite numerous efforts industry has failed to find a good substitute for hexane.

From time to time, some greenner options like aqueous oil extraction and aqueous enzymatic oil extraction have been tried. Adequate improvement in these methods by assistance from microwaves and ultrasonication have also been reported.

Economic considerations and reluctance of the industry to invest in development of these greener options have contributed to hexane being continued to be used in the oil industry.

Lipids from various sources

The milk lipids contain ~97% triglycerides and small amount of phospholipids and sterols which originate from membranes of the milk fat globule. Over 400 different fatty acids are detected in the milk fat with estimated number of triglycerides in thousands.

Oxo- and hydroxy fatty acids present in milk fat are responsible for forming flavoring ketones, aldehydes and lactones. Short chain fatty acids released if fat globules are damaged (during processing and in transfer through pipelines) cause the unpleasant smell in raw milk.

Obtaining milk fat involves separation of fat globules, churning, aggregation of fat globules, separating the butter milk and stabilization of water-in-oil emulsions. Milk fat is liked because of its high and pleasant organoleptic sensations like flavour and texture.

Fish oil is mostly a by product of fish mill industry. It is produced by passing steam over raw fish. The oil is separated from water. Fish oil is known for high degree of double bonds, >50 different fatty acids (C14-C24) and above all for the presence of ω-3 and ω-6 fatty acids.

Fish oils contain unsaponifiable lipids like sterols, hydrocarbons, glyceryl ethers, pigments, vitamins and oxidized oil. Industrially fish body oil and fish liver oil is distinguished. Cods and pollack have large livers and are sources of liver oils. In other cases, it is the fish flesh which is the source of the oil.

When required as an ingredient or supplement (like ω-3 fatty acids), it refined by processes similar to vegetable oils: neutralization of free fatty acids, bleaching, winterization and deodorization.

Adsorption and Normal phase liquid Partition Chromatography

Silica gel as a chromatographic media has been widely used for analysis of lipids. TLC, low pressure liquid chromatography and HPLC formats have exploited the differences in the number and nature of the polar functional groups in lipids.

Silica as a medium has silanol groups which can be free or H-bonded. Silica has layers of water of hydration. To ensure variability in results, it is necessary to remove all but very tightly bound water layer. This dictates choice of the solvent for running the column.

Reversible damage to silica columns can occur if polar impurities accumulate. In such cases, the column should be washed with a negative gradient of polar solvents. Solvents containing water at pH <2 or > 7.5 can also dissolve the silica from the surface and would irreversibly damage the column.

In the normal phase chromatography, silica can be bonded with some organic moieties like diol, nitro, nitride and alkyl or aryl cyano. Such bonded phases are known to decrease “tailing” of the peaks.

It should be noted that even such columns do contain variable amounts of silanol groups which remain and contribute to the separation process.

In all HPLC methods, the choice of the detector is based upon not only the compounds to be separated but also by the chice of eluting solvents. UV detectors are useful if the lipid contains conjugated double bonds or aromatic rings or oxidised lipids as a result of hydroperoxidation. Derivatization techniques like benzoylation of glycolipids or converting fatty acids to aromatic esters have been used to make UV detection possible.

RI detectors and evaporative light scattering detectors are of more general use.

Gas Liquid Partition Chromatography

For volatile or low boiling point lipid samples, GC is a convenient and frequently used technique. Again, silica is the material. Fused silica narrow bore column (0.1-0.3 mm internal diameter, 25-100 m in length) with inner walls coated with the liquid phase is used. The distribution coefficient of the lipid molecules between the liquid phase and the carrier gas dictates the separation.

The molecules partitioning in the gas phase travels with the gas down the column. Depending upon the differences in partition coefficient, different materials emerge out of the column at different retention times.

The parameters which influence retention times are : nature and flow rate of the carrier gas, column dimensions, column temperature and thickness of the liquid phase. Of these column length is less important as resolution is proportional to √l.

Many non volatile molecules can be derivatized and converted into more volatile substances. Again silylation chemistry is widely used for this purpose and various silylation reagents are commercially available.

Reverse Phase Liquid Partition Chromatography

Here the stationary phase is non polar and the mobile phase is relatively polar. This makes this mode of HPLC very valuable for separation of various lipid molecules of similar kind/class.

Some of the detectors used in GC are flame ionization detector, electron capture detector (especially useful for halogen containing lipids), mass spectrometers. Actually GC –MS has emerged as powerful even in lipid analysis.

Chain length of the fatty acid component and the number of double bonds present in the chain decides the distribution coefficient of a particular molecule between the two phases. Binding of lipids to the column involves London forces, polar dipole-induced dipole, dipole-dipole and proton acceptor-proton donor interactions.

The stationary phase for lipids most frequently used are chemically bonded long chain hydrocarbons to spherical silica particles of 3-10 μm. The most common is the so called C18 or octadecylsilyl (OOS) column.

For most lipid separation on such a column, acetonitrile or methanol is mixed with another modifier solvent. The latter is decided on the basis of nature of the lipid being separated.

The modifier organic solvent (mixed with either CH3CN or CH3OH) solvates the chain bonded to the silica. This stretches the chains. Solvents like THF (with a low dielectric constant) results in the formation of a kink in the chain. So, the solvent composition decides how much the chains are extended or are in kinks or gauche form.

Ion Exchangers

Some ionic liquids like some phospholipids have been separated on ion exchangers. Again, silica bonded with amine group (anion exchanger) or sulphonic acids (cation exchanger) has given good results. Organic solvents used in the mobile phase change the nature of the chromatography to mixed mode chromatography as adsorption and partition processes come into play.

Silver Ion Chromatography

This is complexation chromatography in which silver ions are part of the stationary phase. This is also called argentation chromatography. The technique is particularly useful in lipid analysis.

Silver ions interact with π-electrons of double bonds to form a polar complex. A lipid with larger number of double bonds obviously forms a stronger polar complex and is retained on the column more firmly.

These are essentially charge transfer complexes in which the C-atoms with double bonds are donors and silver ions are acceptor of electrons. The free olefinic bond and complexes are in dynamic equlibrium during the chromatographic process.

In TLC, silica gel is impregnated with silver nitrate molecules. The same approach can also work in HPLC. However, a more robust approach is to have silica bonded with a moiety like phenylsulfonate residue. The resulting ion exchanger binds silver ions.

As a result of steric factors, a double bond in the cis-configuration forms a more stable charge transfer complex with the silver ion. The relative position of double bonds if present also matter.

The most stable are chelates formed with double bonds separated by two methylene groups. Conjugated double bonds result in a less stable complex of the lipid molecule with silver ion.

Olefinic bonds form stronger complexes then acetylinic bonds. The complex stability is more at lower temperatures. The technique has been extensively used for separation of simple fatty acid mixtures. It is also useful for separation of triglycerides.

There is a whole area called “lipidomics” which deals with lipids: isolation, analysis, production and quality control. What we have discussed is just a broad overview to illustrate the kind of concerns which are there in the actual isolation, separation and analysis of lipids.

Today GC-MS, capillary electrophoresis-MS and many hyphenated techniques are available which allows one to analyse lipids in detail. Industry, however, goes by established standards and hence the classical parameters are still important and worth knowing.

Summary

•  Important parameters for lipids
•  Isolation and purification of lipids
•  Isolation of vegetable oils, milk fats ans fish oils
•  Analysis of lipids