404
separation in chromatography they are adsorption, normal-phase liquid partition,
liquid- liquid or gas-liquid partition (in which reversed-phase chromatography is a
special case), ion exchange, gel-permeation and chiral-phase chromatography.
HPLC or TLC is generally used to separate each of the various simple lipid classes
from the complex lipids. The latter may be further fractionated by adsorption or ionexchange chromatography or by combinations of both, until the necessary separations are achieved. The following are the three types of chromatography that is
preferred in lipid analysis.
Thin Layer Chromatography is used to find concentration of different lipid
groups. The TLC plate is prepared with suitable adsorbent and kept in proper solvent. A drop of sample is placed on one end and let to flow. The plate after separation to different fractions is compared to standard plates to identify the lipids. GC,
MS, NMR are used for further analysis of the spots (McClements 2003). The use of
TLC has decreased considerably after the advent of TLC. However, the use of TLC
for instant result is still carried out with the help of pre-coated plates. It is cheaper
than HPLC. However, care during experimentation is required (Christie 2011).
Different types of phospholipids cannot be separated by this method (Zaima et al.
2011). TLC is used mainly to separate and determine the concentration of different
types of lipid groups in foods, such as tri, di, mono—acylglycerols, phospholipids
and cholesterol. TLC plate is coated with a suitable absorbing material and placed
into an appropriate solvent. This method involves spotting a small amount of the
lipid sample on TLC plate. Over a period of time, due to capillary force the solvent
moves up the plate and separates into various lipid fractions depending on their
affinity for the absorbing material. At the end of the separation process dye is
sprayed on the plate so as to make the spots visible. Identification of the lipids is
done by comparing the distance that the spots move with that of known composition
of standards. Further analysis of the scraped off spots can be done by using techniques such as GC, NMR or mass spectrometry. This is a cheap and rapid method
for analyzing various lipids in fatty foods.
High Performance Liquid Chromatography is now a preferred method for lipid
analysis because of its versatility over TLC at operating at room temperature and
hence can be used for analysis of labile groups which cannot be determined by
using GC (Christie 2011).
Gas Chromatography is the preferred method for analysis of trans fatty acid. It
can also be used for triglycerides and fatty acids; however, methylation is necessary
(Greenfield and Southgate 2003). Before carry out gas chromatography for fatty
acids that are non-volatile, it is imperative that the lipids are made into volatile fatty
acid methyl esters (FAME) by saponification and methylation (McClements 2003).
Gas - liquid chromatography (GLC) can only be used for compounds which can be
volatilized without decomposition. In practice this normally limits its application to
molecules with molecular mass of less than 800 Da. Lipids such as phosphoglycerides which do not remain intact are not amenable to this method. Perhaps the most
important use for the lipid biochemist is in the analysis of the fatty acid composition
of lipids or tissues. In addition, gas chromatography may be used for steroid, glycerol, sphingosine, inositol and carbohydrate analysis. Molecular species of partial
S. Asokapandian et al.
separation in chromatography they are adsorption, normal-phase liquid partition,
liquid- liquid or gas-liquid partition (in which reversed-phase chromatography is a
special case), ion exchange, gel-permeation and chiral-phase chromatography.
HPLC or TLC is generally used to separate each of the various simple lipid classes
from the complex lipids. The latter may be further fractionated by adsorption or ionexchange chromatography or by combinations of both, until the necessary separations are achieved. The following are the three types of chromatography that is
preferred in lipid analysis.
Thin Layer Chromatography is used to find concentration of different lipid
groups. The TLC plate is prepared with suitable adsorbent and kept in proper solvent. A drop of sample is placed on one end and let to flow. The plate after separation to different fractions is compared to standard plates to identify the lipids. GC,
MS, NMR are used for further analysis of the spots (McClements 2003). The use of
TLC has decreased considerably after the advent of TLC. However, the use of TLC
for instant result is still carried out with the help of pre-coated plates. It is cheaper
than HPLC. However, care during experimentation is required (Christie 2011).
Different types of phospholipids cannot be separated by this method (Zaima et al.
2011). TLC is used mainly to separate and determine the concentration of different
types of lipid groups in foods, such as tri, di, mono—acylglycerols, phospholipids
and cholesterol. TLC plate is coated with a suitable absorbing material and placed
into an appropriate solvent. This method involves spotting a small amount of the
lipid sample on TLC plate. Over a period of time, due to capillary force the solvent
moves up the plate and separates into various lipid fractions depending on their
affinity for the absorbing material. At the end of the separation process dye is
sprayed on the plate so as to make the spots visible. Identification of the lipids is
done by comparing the distance that the spots move with that of known composition
of standards. Further analysis of the scraped off spots can be done by using techniques such as GC, NMR or mass spectrometry. This is a cheap and rapid method
for analyzing various lipids in fatty foods.
High Performance Liquid Chromatography is now a preferred method for lipid
analysis because of its versatility over TLC at operating at room temperature and
hence can be used for analysis of labile groups which cannot be determined by
using GC (Christie 2011).
Gas Chromatography is the preferred method for analysis of trans fatty acid. It
can also be used for triglycerides and fatty acids; however, methylation is necessary
(Greenfield and Southgate 2003). Before carry out gas chromatography for fatty
acids that are non-volatile, it is imperative that the lipids are made into volatile fatty
acid methyl esters (FAME) by saponification and methylation (McClements 2003).
Gas - liquid chromatography (GLC) can only be used for compounds which can be
volatilized without decomposition. In practice this normally limits its application to
molecules with molecular mass of less than 800 Da. Lipids such as phosphoglycerides which do not remain intact are not amenable to this method. Perhaps the most
important use for the lipid biochemist is in the analysis of the fatty acid composition
of lipids or tissues. In addition, gas chromatography may be used for steroid, glycerol, sphingosine, inositol and carbohydrate analysis. Molecular species of partial
S. Asokapandian et al.