Lipid Identification and Chromatography
[IMAGE]Lipids are the single class of biological macromolecules which
do not contain polymers. Molecules of carbohydrates and proteins for
example can be made of sometimes thousands of monomers, each monomer
linked to the next by a covalent bond. However, a lipid is made of a
very small number of parts in comparison, the smallest being made of
only four smaller molecules
: one glycerol and three fatty acids
shown below. The glycerol molecule is comprised of three carbon
atoms, each of which is bonded to a hydroxyl group. The fatty acids
are hydrocarbons with a carboxyl group, and their carbon skeletons are
saturated with hydrogen. The glycerol will always remain the same in
every lipid, but it is the number and size of the fatty acids that
gives a lipid its variation. A lipid is formed when the hydroxyl
group of the carboxyl group and a hydrogen atom from the hydroxyl
group of the glycerol dehydrate in a process known as esterification.
The bond that is formed is called and ester bond. A completed lipid
has three fatty acids attached to the glycerol, and so it is called a
triglyceride, the most common form of lipid.
Because none of the atoms in the hydrocarbon chains are more
electronegative than any other, a lipid is non-polar, giving it the
well-known property of repelling water. However, some lipids are
unsaturated, meaning that there are some hydrogen atoms missing from
the fatty acid chains, and so some double bonds are formed between the
carbons. This forms a kink in the chain, meaning that molecules of
lipids cannot fit together as easily as when they are straight
chains. This differing formation creates the difference between solid
butter and liquid oils, for example. In the body, fats are used
mainly for energy storage, its long-term food reserves being stored in
adipose cells, which enlarge and shrink as fat is removed for use or
deposited for storage. However, fats can also be used for insulation
and cushioning against external forces.
Although all lipids are hydrophobic, some parts of some lipids known
as phospholipids are hydrophilic, which proves very useful for cells
in the body. Phospholipids have only two fatty acids chains linked to
a glycerol molecule instead of three, which leaves one hydroxyl group
free to form a covalent bond with a phosphate group. As the oxygen
atoms in the phosphate group are more electronegative, they form a
partial negative charge, and are in turn hydrophilic. Phospholipid
bilayers (two layers in opposite direction) are what make up cell
membranes, the phosphate group part of the molecule being in contact
with water on each side of the cell, meaning that water cannot get in
or out of the cell due to the hydrophobic fatty acid tails.
Phospholipids create a spherical structure when surrounded by water so
that the hydrophobic tails are at the centre. This means that
proteins such as some hormones may be transported to the cell through
water and be kept from that water through the layer of fatty acid
tails surrounding it, with the hydrophilic phosphate groups
surrounding the fatty acids.
Chromatography is a technique used to distinguish chemicals when they
are in a mixture. Different solutes, from a dye to an amino acid,
have different solubilities in different solutes, meaning that some
solutes will dissolve more in a solvent than other solutes will. The
process works by placing a sample of the solute on a piece of
chromatography paper and allowing the solvent to soak up the paper.
The result will be that the further up the paper a solute moves, the
more soluble it is in the solvent, whereas the less soluble a solute
is in that particular solvent, the lower up the paper it will move.
This procedure can be used to distinguish between substances and
recognise their properties with regard to certain solvents in order to
make discoveries about their function or similar.
A particular solute will always move the same relative amount of
distance to the total distance the solvent has moved up the paper in
that particular solvent. For a different solvent the relative
distance will be different, as solutes have different solubilities in
different solvents. The calculation used to obtain this relative
distance is: distance moved by the solute/distance moved by the
solvent (solvent front). This gives the Rf (Relative Fraction or
* To understand the biochemical tests for lipids.
* To understand the use of chromatography.
Refer to method sheet, any changes have been corrected on the sheet in
1. Olive oil will not dissolve in water.
2. Propan-1-ol will dissolve in water, crating s slimy or stringy
effect under observation.
3. Olive oil dissolves in propan-1-ol.
4. When solution of olive oil and propan-1-ol is added to the solution
of propan-1-ol and water, an emulsion is created (solid suspended in
water) and a cloudy white colour is formed; the solute has dissolved.
Grease Spot Test
The paper turned translucent when olive oil was placed upon it.
Solvent Front (cm)
0.672 and 0.793
0.776 and 0.879
0.759 and 0.966
0.776 and 0.983
0.776 and 0.996
N.B. The colours in the Rf section correspond to the colours observed
on the chromatography paper.
An emulsifying agent is a substance that is soluble in both oil and
water, allowing the two to mix. Therefore if a lipid, such as olive
oil which is made of unsaturated fats, is dissolved in an emulsifying
agent, such as propan-1-ol, the solution will be able to dissolve in
water. This is exactly what the results reflect, and so it was the
alcohol that allowed the oil to dissolve in the water. The prpan-1-ol
is able to do this of its chemical structure. It is an alcohol with a
hydrocarbon skeleton and a hydroxyl group ‘head’, which is
hydrophilic. When the propan-1-ol is added to the olive oil, the
olive oil’s triglyceride structure is broken down, which in turn means
that although the fatty acid tails are still hydrophobic, they are
balanced out by the hydrophilic nature of the hydroxyl groups with
constitute the head of the propan-1-ol, meaning that the solution will
dissolve in water, which the results confirm.
Grease Spot Test
As the paper turned translucent when olive oil was placed upon it,
this indicates that the olive oil contains lipids.
The results of the experiment do seem to be accurate, as the colours
found in each of the samples are generally true at face value. The
colours found in the Green sample, for example, were found to be
yellow and blue, there being more blue in the mixture than yellow.
This composition and rough ratio of less yellow to more blue are true
to the colours that come together to form the colour green. The
colours found to make the Yellow sample also seem accurate, as they
are very similar. However, the ratio of more orange to yellow dye in
the mixture is surprising, especially as yellow is a primary colour
and it would be expected to simply have one result of yellow, as the
Red sample does of red. The X sample was found to contain more blue
than red, and the Y sample was found to contain a mixture of more blue
than yellow. This is where chromatography can be very useful, as it
shows which solutes are in a mixture, which there are more of in a
mixture, and which solutes dissolved more in that mixture. Using the
Relative Front calculation was also very useful, as it meant that no
matter how long the experiment lasted for, the relative solubility and
amounts of solute would be the same for each sample. This can be very
useful when identifying mixtures in biological compounds, as a
particular molecule will have a particular Rf in a particular
solvent. For example, when a protein is being looked at, and
chromatography is used to separate out the amino acids in order to
identify them, certain amino acids will always have the same Rf in a
particular solvent, and therefore they can be easily identified.
From the results of this test, it may be concluded that a lipid, such
as olive oil, is able to dissolve in water when an alcohol, such as
propan-1-ol, is added to it. This is important when identifying
lipids in a substance.
Grease Spot Test
From this test, it can be concluded that a substance is a lipid or
contains a lipid if it is able to turn paper translucent.
From this test, it can be concluded that the solutes which have
dissolved more in the dye or sample move furthest up the paper, and
that those solutes which have dissolved less in the sample will move
less up the paper, separating out the mixture into its components and
amounts of components.