c




 


 

Upon collecting the Column Chromatography, the fourth member is tasked to use the eluate for qualitative
analysis of lipids. The first eluate was from a 9:1 mixture of petroleum ether and ethyl ether, the second eluate
from 5mL 5% methanol from dichloromethane and the last eluate was procured from CH2Cl2:CH3OH:H2O (1:3:1).
Two tests were performed, these are test for ester and acrolein test. Under the test for ester, both eluates
numbers 1 and 3 exhibited a positive result: burgundy coloration. It is now certain that eluates 1 and 3 both
contains an ester functional group. Under the acrolein test, only eluate number 2 produced the expected pungent
burnt fat odor, the positive result. Eluates 1 and 3 on the other hand did not produce any noticeable odour. The
molecule acrolein was responsible for the pungent odour in eluate 1, which meant it definitely had glycerol prior to
the experiment. By performing these qualitative test, the group was able to characterize which eluates were
saponifiable and non-saponifiable.




 
Carbohydrates are the active energy source of
the body. Our body has a limited capability to
store carbohydrates but it can convert
carbohydrates into inert energy storage
molecules called lipids. Our body burns these
reserved energy when carbohydrates are all
spent. Lipids also play another functional use by
insulating the body and help maintain its
temperature. 1A lipid is a fat-soluble molecule. To
put it another way, lipids are insoluble in water
but soluble in at least one organic solvent. The
other major classes of organic compounds
(nucleic acids, proteins, and carbohydrates) are
much more soluble in water than in an organic
solvent. The water insolubility of lipid is due to its
structure, the polar groups are much smaller
than its non-polar portions. 2These non-polar
portions provide water repellent or hydrophobic
property,? hydrophobic or amphipathic small
molecules that may originate entirely or in part
by carbanion-based condensations of thioesters.
3
Unlike carbohydrates, lipids do not possess a
unique functional group to characterize
themselves from other compounds using
4
chemical tests. Lipids are chemically
heterogenous mixtures that are further classified
as saponifiable and non-saponifiable lipids.
Saponifiable lipids are capable of alkaline
hydrolysis of esters of fatty acids to form glycerol
and sodium salt of fatty acids commonly known
as soap, examples of these are triglycerides,
phospholipids, glycolipids, sphingolipids and the
waxes. Thus, the presence of the esters in
glycerol is basis for saponification. There are also
non-saponifiable lipids such as cholesterol.
1
Acrolein test is a test for the presence
of glycerin or fats. Fatty acids are further
characterized based on length of fatty acid,
degree of saturation or unsaturation, and ability
to be synthesized in the diet.
5
The yolk comprises an approximate 30% portion
of the egg and contains about 4.5 grams of fat.
All the fat soluble vitamins(A,D,E and K) are all
found on the egg yolk. Egg yolk is a source of
lecithin, an emulsifier and surfactant. The yellow
color is caused by lutein and zeaxanthin which
are both yellow and orange is called
xanthophylls.
Ñ
Test for Ester
Using the eluates obtained from the Column
Chromatography. 0.5mL of Ethanol:1-
butanol(3:1) was added to 10 drops of the
eluates in separate test tubes. 2 drops of 2M
NH2OH and 3M NaOH were sequentially added,
the solution was mixed well and stood for 5
minutes undisturbed. 2 drops of 6M HCl and 1
drop 5% FeCl3‡6H2O in 0.1M HCl was added and
again, the solution was mixed. Samples with
esters produced a burgundy color.
Acrolein Test
A pinch amount of KHSO4 was added to 10 drops
of eluates in a test tube. This test tube was
heated in a boiling water bath and the odor
produced was carefully observed. The burnt fat
odor indicated the presence of glycerol.

  

     

 
At this stage, the orthoester has a choice:
Test Visible results reforming the carbonyl can be accompanied by
Ester Burgundy color expulsion of either hydroxide or the alkoxide. The
former leads back to the starting materials and is
Glycerol(Acrolein Test) Burnt Fat odor
unproductive (explaining why saponification is in
fact an equilibrium). On the other hand,
expulsion of the alkoxide generates a carboxylic
acid:

          
  
  
  

 
 
  

  
   
1 Burgundy color(+) ---
2 --- Burnt fat odor(+)
3 Burgundy color(+) ---
 The alkoxide is more basic than the conjugate
   base of the carboxylic acid, and hence proton
 
  transfer is rapid:
This test determines the presence of an ester
fuctional group in a solution. The group has
performed this test in the laboratory to know
whether the analyte lipid is saponifiable or non
saponifiable. The principle behind this test is
based on 1The reaction known as saponification,
it is the way most of the soaps are manufactured. Saponification to the sodium salt of myristic acid
Refluxing an ester with aqueous NaOH produces takes place with NaOH in water. The acid itself
can be obtained by adding dilute hydrochloric
an alcohol and the sodium salt of the acid.
acid.
O O
|| H2 O ||
R ± C ± OR' + NaOH ====== R ± C ± O± Na+ + R'OH
This reaction is essentially irreversible because
carboxylate ion is inert towards nucleophilic
substitution. Below is a more detailed illustration
of the overall reaction of ester as it is being
presented in a step by step fashion.
6 burning fat, it can be produced by heating
The mechanism by which esters are cleaved by
base involves nucleophilic acyl substitution. The
hydroxide anion adds to (or "attacks") the
carbonyl group of the ester. The immediate
product is an orthoester:
 
3
Acrolein is the simplest unsaturated aldehyde.
It is produced widely but is most often
immediately reacted with other products due to
its instability and toxicity. It has a piercing,
disagreeable, acrid smell similar to that of
glycerol to 280 Celsius or in this case, by reacting
potassium bisulfate with glycerol. 5The test that
differentiates between cholesterol and lecithin is
the acrolein reaction. When lipids containing
glycerol are heated in the presence of potassium
hydrogen sulfate, the glycerol is dehydrated,
forming acrolein, which has an unpleasant odor.
Further heating results in polymerization of
acrolein, which is indicated by the slight
blackening of the reaction mixture. Both the
pungent smell and the black color indicate the
presence of glycerol and therefore fat and/or
lecithin. Cholesterol gives a negative acrolein
test.

Illustrated below is the conversion of glycerol into

acrolein using potassium bicarbonate. Acroleine
can be characterized as a substance that
produces a burnt fat odor.





 
 
1
Boyer, Rodney(2006). Concepts in Biochemistry,
3rd edition. John Wiley and Sons(Asia) Pte.Ltd
2
David, Nelson (2008)Lehninger Priciples of
Biochemistry 5th Edition, W.H. Freeman & Co.
3
Ninfa, A., Ballou, D., Benore M.(2005).
Fundamental Laboratory Approaches for
Biochemistry and Biotechnology 2nd Ed., B1.1.1-
B1.1.27, John Wiley & Sons, Inc.
4
Laboratory Manual in General
Biochemistry(2010), C and E Publishing, Inc.

  

5
http://www.cerlabs.com/experiments/10875404
472.pdf
6
www.wikipedia.com