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PTT 202:
ORGANIC CHEMISTRY FOR BIOTECHNOLOGY
PREPARED BY:
NOR HELYA IMAN KAMALUDIN
[email protected]
Introduction of Lipids
Lipids
• Lipids are naturally occurring esters of long
chain fatty acids that are soluble in organic
solvents such as chloroform, diethyl ether or
hydrocarbons but are insoluble in water.
• The examples of lipids are fatty acids and their
various derivatives, steroids, prostaglandins,
and ‘fat-soluble’ vitamins A, D, E and K.
Classification of lipids
Simple
lipids
• The substances that contain only a long
chain fatty acid and an alcohol (which
may be either glycerol, along chain
alcohol, a sterol or one of the fat-soluble
vitamins).
• e.g: waxes, acylglycerols, sterol esters,
vitamin A,D, E and K.
Complex
lipids
• Acyl esters of either gkycerol or the long
chain amino alcohol, sphingosine, which
also contain a hydrophilic group such as a
phosphate ester of an organic alcohol, a
carbohydrate or a sulphate group.
• e.g: phospholipids, glycolipids,
Biosynthesis of terpenes
STEP 1
STEP 2
STEP 3
• Acetyl-coenzyme A, also known as activated acetic acid, is the biogenetic precursor
of terpenes. Two equivalents of acetyl-CoA couple to acetoacetyl-CoA, which
represents a biological analogue of acetoacetate.
• Following the pattern of an aldol reaction, acetoacetyl-CoA reacts with another
equivalent of acetyl-CoA as a carbon nucleophile to give β-hydroxy-βmethylglutaryl-CoA.
• Followed by an enzymatic reduction with dihydronicotinamide adenine
dinucleotide (NADPH + H+) in the presence of water, affording (R)-mevalonic
acid.
• Phosphorylation of mevalonic acid by adenosine triphosphate (ATP) via the
monophosphate provides the diphosphate of mevalonic acid which is
decarboxylated and dehydrated to isopentenylpyrophosphate
(isopentenyldiphosphate, IPP).
Biosynthesis of terpenes
STEP 4
• The latter isomerizes in the presence of an isomerase containing
SH groups to γ,γ-dimethylallylpyrophosphate.
STEP 5
• The electrophilic allylic CH2 group of γ,γdimethylallylpyrophosphate and the nucleophilic methylene
group of isopentenylpyrophosphate connect to
geranylpyrophosphate as monoterpene.
STEP 6
• Subsequent reaction of geranyldiphosphate with one equivalent
of isopentenyldiphosphate yields farnesyldiphosphate as a
sesquiterpene (Figure 1).
Biosynthesis of
terpenes
Figure 1: Scheme of the biosynthesis of
mono- and sesquiterpenes.
Biosynthesis of cholesterols
• Acetyl-CoAs are converted to 3-hydroxy-3-methylglutaryl-CoA
(HMG-CoA)
Step 1
Step 2
• HMG-CoA is converted to mevalonate
• Mevalonate is converted to the isoprene based molecule, isopentenyl
pyrophosphate (IPP), with the concomitant loss of CO2
Step 3
Step 4
Step 5
• IPP is converted to squalene
• Squalene is converted to cholesterol (Figure 2).
Biosynthesis of cholesterols
Figure 2: Pathway for cholesterols biosynthesis
Separation of lipid mixtures
Column
techniques
Thin-layer
chromatography
Solvent
extraction
Gas-liquid
chromatography
Separation
methods
Solvent extraction
Extraction of lipids from biological materials using a variety of organic
solvents
• Different classes of lipid can be extracted selectively on the basis of their
solubility in different organic solvents.
• This may be achieved by the addition of a solvent that will effect either the
precipitation or the extraction of the lipids of interest.
• An example of the former is the precipitation of high concentrations of
phospholipids with cold, dry acetone, and of the latter, the extraction of fatty
acids into ether or heptane at an acid pH.
Fractionation of lipids classes based on polarities
• The crude fractionation of lipid classes with differing polarities is done by
partitioning of different lipids between two immiscible solvents.
• The effectiveness of the separation can be increased by conducting repeated
extractions in a carefully chosen solvent pair.
Solvent extraction
Examples of solvent systems for fractionation of
lipids
• Petroleum ether-ethanol-water system:
• Used to remove polar contaminants (into the alcoholic phase)
when interest lies in the subsequent analysis of neutral
glycerides, which may be recovered from the ether phase.
• Carbon tetrachloride-methanol-water system:
• Useful in the analysis of plant lipids for separating non-polar
carotenes and chlorophylls from more polar phospholipids
and glycolipids.
Column techniques
Adsorption chromatography
• Column used: Silicic acid (silica gel)
• Used to separate mixtures of lipids on a preparative scale.
• Adsorbents used: Florisil (magnesium silicate), acid-washed
Florisil or alumina
• Useful for the isolation of glycolipids.
Factors that improved the separation
•
•
•
•
Complexity of the lipid mixture.
Dimension of the column.
Type of adsorbent used.
Choice of eluting solvents.
Column techniques
Principle of separation using column techniques
• The lipids are bound to the fine particles of the adsorbent by polar, ionic and
van der Walls forces.
• The most polar being held most tightly to adsorbent and separation takes
place according to the relative polarities of the individual lipids (Table 1).
• It is usual to elute the lipids from the column with solvents of increasing
polarity, when the least polar lipids will emerge in the early fractions.
Separation of diverse mixture of lipids
• The separation of lipids mixture into the three broad groups of neutral
lipids, glycolipids and phosphatides can be effected using a chloroformacetone-methanol solvent sequence.
• The collected fractions may be then separated into their individual
components by column or preparative thin-layer techniques.
Column techniques
Table 1: Lipid classes in order of
increasing polarity
Column techniques
Separation of
neutral lipids
mixture
• Column used: Silicic acid
• Solvent used: hexane containing increasing
proportions of diethyl ether (0-100%)
• The lipids are eluted in the following order.
Separation of
glycolipids and
sulpholipids
mixture
• Solvent used: chloroform-acetone (1:1) for
eluting glycolipids followed by acetone for
eluting sulpholipids
Separation
phospholipids
mixture
• Solvent used: chloroform-methanol (95:5)
• Increasing the proportion of methanol to
(50:50) resulting the following typical
sequence.
Column techniques
Separation of complex lipids on a large scale
• Techniques used:
• Ion-exchange cellulose chromatography with DEAE (diethyl-aminoethyl) or
TEAE (triethylaminoethyl)
• Fundamental of separation:
• Separation takes place primarily on the basis of ion-exchange of those lipids
with ionic groups, although non-ionic polar groups, e.g. hydroxyl, also exert an
influence owing to adsorption by the cellulose.
• The DEAE cellulose is used in the acetate form, and charged, non-ionic lipids
are eluted with chloroform containing varying proportions of methanol.
• Weakly acidic lipids (e.g.phosphatidyl serine) can be eluted with glacial acetic
acid and strongly acidic or highly polar lipids (e.g. phosphatidylglycerol,
phosphatidylinositol, sulphatides and sulpholipids) with chloroform-methanol
containing ammonium acetate and dilute ammonia.
Thin-layer chromatography
Silica gel thin-layer plates
• Function:
• Used to separate lipids on either a preparative or an analytical scale.
• Sometimes used to fractionate the lipids into classes prior to
removal from the plate and further analysis by GLC.
• Principle of separation:
• The polarity of the lipid determines the degree of adsorption to the
silica, the most polar being most strongly held.
• The mobility of the lipids during chromatography will be affected by
the polarity of the solvent;
• Increasingly polar solvents will break the adsorptive bonds with
greater efficiency, resulting in greater Rf values.
Thin-layer chromatography
Two-dimensional techniques
• This involves carrying out the chromatography in one solvent and after
drying the plate and turning it through 90˚, running it in another solvent.
• Necessary to resolve mixtures of complex polar lipids (e.g. phospholipids,
glycolipids, sphingolipids).
Silver nitrate chromatography
• The silver nitrate may be incorporated in the adsorbent slurry giving the
final concentration of about 5% in the dry plate.
• The silver ions bind reversibly with the double bonds in the unsaturated
compounds, resulting in selective retardation, and the lipids are separated
according to the number and configuration (cis or trans) of their double
bonds.
• This technique is extremely useful in fatty acids, mono-, di- and
particularly triacylglycerol analyses.
Separation of several lipids classes
using thin-layer chromatography
Non-polar
lipids
Fatty acids
Lipids
classes
Complex
lipids
Acylglycerols
Separation of non-polar lipids using
thin-layer chromatography
Solvent used
• Commonly used solvent systems:
• hexane/light petroleum-dietyl ether-acetic acid
(90:10:1)
• diisopropyl ether-acetic acid (98.5:1.5)
The sequence of mobility of lipids
• Cholesterol esters > triacylcerols > free fatty acids >
cholestorol > diacylglycerols > monoacylglycerols >
complex polar lipids (remain unmoved)
Separation of fatty acids using thinlayer chromatography
Methylation method for identification of fatty acids
• Applicable to both esterified and non-esterified fatty acids.
• Procedures: Heat the lipid sample for 2 h under a current of nitrogen at 80-90˚C
with 4% sulphuric acid in methanol. After cooling and addition of water, metyl
esters are extracted into hexane and extracts are dried over sodium carbonate and
anhydrous sodium sulphate.
Argentation thIn-layer chromatography
• Useful procedure for the separation of metyl esters of fatty acids.
• Solvents used: hexane-dietyl ether (9:1) but the ratio of (4:6) is used for
separation compounds with more than two double bonds.
• Saturated fatty acids have the highest RF values, which decrease with the
increasing degree of unsaturation.
• For a particular acid, the trans isomer usually travels ahead of its corresponding cis
isomer.
Separation of acylgylcerols using thinlayer chromatography
Separation of mono-, di- and triacylglycerols
• Plates: Silver nitrate silica plates
• Solvent: chloroform-acetic acid (99:0.5)
Separation of monoacylglycerols and diacylglycerols
• Plates: Borate-impregnated plates
• Solvent: chloroform-acetone (96:4)
Separation of triacylglycerols containing compounds with widely differing number of double
bonds.
• Plates: silver nitrate impregnated plates
• Solvent:
• For mixture containing up to four double bonds: hexane-dietyl ether (80:20) or
chloroform-methanol (99:1)
• For mixture containing higher degrees of unsaturation: dietyl ether or chloroformmethanol (96:4)
• For mixture containing highly unsaturated: chloroform-methanol-water (65:25:4)
Separation of complex lipids using thinlayer chromatography
Separation of phosphoglycerides
• Plates: silver nitrate impregnated plates
• Solvent: chloroform-methanol-water (65:25:4)
• Additive: acetic acid useful to effect separation of neutral
phosphoglycerides from acidic phosphoglycerides (phosphatidyl serine
and phosphatidyl inositol).
• Modification to improve quality of separation:
• Addition of sodium carbonate or ammonium sulphate in the
preparation of silica gel
• Preliminary removal of the simple lipids to the top of the plate by
running it in acetone-hexane solvent (1:3).
• When the extract is rich in acidic phosphoglycerides , it is
preferable to use a solvent containing ammonia.
Gas-Liquid Chromatography
GLC is a very useful technique in lipid analysis, particularly
for the separation of very similar compounds within
classes.
It is useful for both quantitative and qualitative analysis and also in
the investigation of lipid structure.
Full structural analysis of lipid will offer necessitate
further analysis of the collected column effluent for a
single GLC peak.
Separation of several lipids classes
using gas-liquid chromatography
Wax esters
Acylglycerols
Cholesterol
esters
Lipids
classes
Fatty acids
Separation of acylgylcerols using gasliquid chromatography
Separation of acylglycerols
• It may be separated as intact molecules by GLC after the preparation of
acetate or trimetylsilyl (TMS) derivatives to reduce the polarity and prevent
acyl migration.
• Separation of monoacylglycerols:
• TMS ether derivatives of monoacylglycerols are sufficiently volatile to be
analysed on a polyester column, which permits separation on the basis of
chain length and degree of saturation of fatty acid component.
• Separation of diacylglycerols and triacylglycerols:
• Non-polar stationary phase which thermally stable up to 350˚C are
required.
• Principle of separation:
• The compounds are separated solely on the total number of fatty acid
carbon atom present, known as carbon number, and those differing in their
carbon number by two can be resolved.
• The temperature required for the separation of diacylglycerols is approx.
270-310˚C; higher and lower temperature are required for triacylglycerols
and monoacylglycerols, respectively.
Separation of wax esters using gasliquid chromatography
Wax esters have similar relative molecular masses to
diacylglycerols and are eluted under comparable gas
chromatographic conditions.
Alternatively, alcohol and fatty acid moieties can be
released by saponification and their metyl esters
subjected to a GLC.
Separation of cholesterol esters using
gas-liquid chromatography
These can be analysed using the columns applicable
to triacylglycerols.
It may be possible with temperature programming to separate
compounds that differ by only one carbon atom whereas
isothermal analysis will normally resolve compounds with the
same carbon number but which contain fatty acids with
different degrees of unsaturation.
Separation of fatty acids using gasliquid chromatography
Separation of fatty acids
• Column: polar or non-polar stationary phase
• Non polar column:
• Have either saturated paraffin hydrocarbon (Apiezon grease) or silicone
greases as the liquid phase.
• Separation takes place largely on the basis of the relative molecular mass
(chain length) and greases are useful for resolving mixtures of saturated
fatty acids, differentiate between saturated and unsaturated components
with the same chain length.
• Polar column:
• Liquid phase usually the heat-stable polymers of ethyleneglycol and dibasic
acids, succinic or adipic.
• Fatty acids are separated on the basis of both chain length and the degree
of unsaturation and some columns are capable of resolving fatty acids
with the same chain length but different number of double bonds.
• The saturation fatty acids shows the shortest retention times followed by
themonoenoic, dienoic, etc.
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