Chapter 18 Lipid analysis
Chapter 18.1 Thin layer chromatography
One o f the simplest and most effective methods for analysing oil in the fermentation broth proved to be TLC, (Thin Layer Chromatography). The main advantages o f TLC are all components can be visualised; the volume o f solvent used is small and hence it is economic and the process requires little instrumentation. This technique also gave a good indication of the presence of oil and oil breakdovm components within the fermentation sample. Therefore, results on a qualitative basis could be obtained relatively simply using the minimum o f equipment.
The separated lipid classes could be visualised and the resulting chromatograms kept as permanent record or samples could be recovered for further analysis. This method does not quantity the results although the intensity of the iodine stain was indicative o f the amount o f triglyceride present. The use of iodine stains the lipids brown/black but care must be taken since iodine will bind irreversibly to polyunsaturated fatty acids.
As mentioned previously, (chapter 13.1), lipid mixtures are separated by differential migration which gives each lipid component a characteristic mobility known as its R f value. This can vary due to the adsorbent used, temperature, solvent etc. The adsorbent used is variable. In this study, silica gel was used which is manufactured to contain pores o f a definite diameter such as silica gel 60, where the 60 denotes the pore size. At a given humidity the amount of water absorbed by the silica gel increases as pore size decreases. The water content of the silica gel determines the polarity of the adsorbent and hence its activity and chromatographic properties. For good separations the water content must be carefully controlled. Therefore, the silica gel on the TLC plates is normally activated by heating the plates before use at temperatures above 100°C to remove the water.
Chapter 18 Lipid analysis - discussion
Other adsorbents can be used such as Kieselgubr which is a diatomaceous compound based on a silicaceous material. However, since it is of fossil origin and non-synthetic its properties are more variable than silica gel and is more suited to reversed phase TLC. Aluminium oxide has also been used as an adsorbent for TLC o f lipids but has considerably less capacity than silica gel plus the oxide is of basic pH and can therefore, cause hydrolysis of esters. Thus, silica gel is the most suitable adsorbent for this method.
In its normal form, silica gel is a polar adsorbent and consequently polar lipids are more tightly adsorbed than non-polar lipids due to polar-polar interactions. In TLC separation of lipids using standard silica gel, the most non-polar lipids therefore migrate at the fastest rates, (high R f values) and the polar lipids at the slowest rates, (low Rf values). By increasing the polarity of the developing system the Rf values can be increased. The choice of a suitable solvent system is critical in the separation of lipid classes. It should be noted that complex polar lipids will remain at the origin irrespective o f the solvent system used (Touchstone, 1995).
In the TLC studies carried out, the lipids involved were simple neutral lipids and as such the most commonly used solvents are hexane, diethyl ether and acetic acid, (Henderson & Tocher, 1987), although other non-polar solvents may also be used. In this case the hexane was replaced by petroleum ether which may affect the Rf values of the more polar lipids although non-polar lipids will not readily be affected. The actual R f values obtained will vary depending on equipment as well as environmental conditions such as humidity as mentioned previously.
From the studies, the use of petroleum ether was suitable for the separation o f the components present in the fermentation samples although the resolution of the lipid classes could be variable. However, if the proportion o f diethyl ether is increased whilst the proportion of petroleum ether is decreased, the more polar lipids will migrate further from the origin and be defined more clearly. This may cause a loss o f resolution o f the least polar classes towards the top of the plate. Therefore, the choice of solvent(s) will be dependent on the lipids to be separated.
Chapter 18 Lipid analysis - discussion
It can also be observed that there were significant variations in the R f values o f the triglycerides especially with respect to tributyrin which cannot be totally explained by differences in polarity (table 13.1). This is because although lipid classes are given in a single R f value, some classes such as triacylglycerols are better represented by a range of values since in natural samples they frequently exhibit more than one band. This is due to their partial resolution into molecular species on the basis of fatty acid composition (Henderson & Tocher, 1989). The degree of spreading depends on the solvent system used. The separation o f individual lipid classes on the basis o f fatty acid composition can be achieved by reversed phase TLC using modified silica gel coated with silver nitrate. The silver ions form complexes with the double bonds of the fatty acid components.
There is some breakdown of the oil in its natural state since there are components which can be visualised with R f values of approximately 0.2-0.3. These resolved spots correspond with the diacylglycerols and also the monoacylglycerols. These can be difficult to resolve clearly using this solvent system but may be observed by using a solvent system consisting of benzene/propan-2-ol/water. However, there will be different levels of migration depending on the specificity of the diacylglycerols. For example a 1,3-diacylglycerol will have a R f value of approximately 0.45 whilst a 1,2- diacylglycerol will have an Rf value o f approximately 0.54 which is due differences in the structural chemistry causing variation in the polarity o f the components.
From running a TLC plate of standard fatty acids using the same solvent system it could be seen that again the resolution was not as clear as for the triglycerides and the resulting spots were harder to visualise even with bromophenol blue. Other stains can be used such as spraying the plates with a solution of 2',7'-dichlorofluorescein followed by 1% aluminium chloride in ethanol and 1% ferric chloride and then warming to 100°C after each spray. The fatty acids will give a rose-violet colour. However, the use of bromophenol blue will be sufficient in this study since only an approximate R f value is required for fatty acids as an indication of the oil breakdown.
Chapter 18 Lipid analysis - discussion
This use of TLC is a good qualitative method of lipid analysis and it can be used quantitatively by the use various methods such as scanning densitometry, elution of the separated components and weighing or by measuring the absorbance of the eluted samples. However, these tend to be inaccurate and as such a biochemical assay was investigated.