Isolation and Characterization of Non-Saponifiable and Saponifiable Lipids from Calf’s Brain Abella, Angelica Balmes, Angela
- 3B8 Bustillo, John Paul
Dalupang, Audrey Figueroa, Cyreen
Introduction
What is a Lipid? •a heterogeneous class of naturally occurring organic compounds classified together on the basis of common solubility properties.
● hydrophobic (insoluble in water =nonpolar) , but soluble in aprotic organic solvents ● Amphipathic in nature
Functions: 1. Cell membrane component a. Phospholipids (Glycerophospholipids and Sphingolipids) b. Glycolipids (Sphingolipids) 2. Energy storage and Insulation Triacylglycerol 3. Signaling molecules
Calf’s Brain (Beef brain) Brain tissues contains high amount of lipid content Contains complex lipids such as phosphatides, cerebrosides, sphingosines and cholesterol It also contain 17% grams of fat and 4% of saturated fat Brain tissues are rich in saponifiable and non-saponifiable lipids which are important to life processes.
Saponifiable Lipid ● Simple Lipids-fatty acid ester of different alcohols and carries no other substance (Example: fatty acids and triglycerides) ● Complex Lipids-esters of fatty acids with alcohol and contain additional groups ○ Phosphorylated- containing a phosphoester group (Example: glycerophosphatides, sphingosine phosphatides) ○
Non-phosphorylated- do not contain phosphoester group (Example: Sphingosine glycosides)
Non-saponifiable Lipid ● Does not contain ester linkages ● Not hydrolyzed by sodium hydroxide or other basic compounds ● Example: cholesterol and prostaglandins
Gravity Filtration ● Method used either for the removal of solid impurities from an organic solution or for isolation of organic solid through the use of filter paper and funnel ● Hot Gravity Filtration ○
Used to separate insoluble impurities from a hot solution
○
Prevents the crystals to form in the filter funnel which then blocks the filtration process
Cholesterol - A type of sterol (modified steroid) which serves as the biosynthetic precursor needed to synthesize steroid hormones, bile acids, and vitamin D. - Synthesized by all animals - Lack of ester functional group makes it non-saponifiable
Glycerophospholipid - A type of lipid wherein two fatty
acids are attached by an ester linkage to the 1st and 2nd carbon of glycerol while a highly polar/charged group is attached to the 3rd carbon by a phosphodiester linkage
- Forms the lipid bilayer of cell membranes:
Phosphate group = polar head
Fatty acids = non-polar tails
- Most glycerophospholipids are identified through “X” head group attached to the phosphate group
Sphingolipid -
a class of lipids with a polar head group and two nonpolar tails
-
The core of a sphingolipid is an amino alcohol called sphingosine
-
Abundant in Central Nervous System
-
They also serve as adhesion sites for extracellular proteins and play important roles in signal transmission and cell recognition
Lecithin
- Also known as phosphatidylcholine - It is a phospholipid which consists of glycerol, two fatty acids, a phosphate group and choline. - serves a structural role in cell membranes
Galactocerebroside - A type of cerebroside consisting of a ceramide* with a galactose residue at the 1hydroxyl moiety - It is also composed of sphingosine and fatty acid *ceramide: the amino group of sphingosine is attached to the fatty acid by an amide linkage
CHEMICAL TESTS
Liebermann-Burchard Test - Test for the presence of Cholesterol Salkowski Test - Test for the presence of Cholesterol Phosphate Test - Test for the presence of Phosphate Kraut’s Test - Test for the presence of Choline Ninhydrin Test - Test for the presence of free amino groups and secondary amines Molisch Test - Test for the presence of Carbohydrates
Objectives
Objectives ❏ To isolate lipids from calf’s brain and to separate it into phosphorylated and non-phosphorylated lipids ❏ To characterize the isolated lipids and standards using various chemical tests
Methodology
A.Isolation of Cholesterol Calf’s Brain - Part A
➢ ➢ ➢ ➢
Filtrate (Cholesterol) Evaporate using steam bath Cool over ice bath Filter and collect the crude product Dissolve in (1:3) MeOH:CHCl3
Residue - Part B
B. Isolation of Glycerophosphatides Residue - Part B ➢ ➢ ➢ ➢ ➢
Filtrate (Glycerophosphatides) ➢ Concentrated over steam bath ➢ Add 30mL acetone ➢ Decant and dissolve the precipitate in 10mL (1:3) MeOH:CHCl3
Wash with acetone Transfer to small beaker Add 30mL hexane Stir occasionally for 30 minutes Filter
Residue - Part C
C. Isolation of Sphingosine Phosphatides and Sphingosine Glycosides Residue - Part C ➢ ➢ ➢ ➢
Filtrate (discard)
Transfer to a small beaker Add 50mL EtOH Boil over water bath Filter
Precipitate (Sphingosine) ➢ Dissolve in 5mL (1:3) MeOH:CHCl3
Characterization: Liebermann-Burchard Test
Characterization: Salkowski Test
20
Characterization: Test for Phosphate
-ignite over a free flame until all organic matter is burned away
-heat 65 oC
Characterization: Kraut’s Test
Characterization: Ninhydrin Test
Characterization: Molisch Test
Results and Discussion
Isolation of Non-saponifiable from Saponifiable Lipids from Calf’s Brain Trituration - A process used to purify crude chemical compounds containing soluble impurities. Recrystallization - Used to purify organic compounds - The principle behind recrystallization is that the amount of solute that can be dissolved by a solvent increases with temperature.
Isolation of Non-saponifiable from Saponifiable Lipids from Calf’s Brain Acetone - Used to separate the non-phosphorylated lipids from phosphorylated lipids - Non-phosphorylated lipids are dissolved in acetone solution because they are both polar - Less reactive to the lipid components compared to alcohol 95% Hot EtOH - Forms a concentrated solution - Cholesterol’s solubility is directly proportional to the temperature - Selective solvent for sphingolipids
Isolation of Non-saponifiable from Saponifiable Lipids from Calf’s Brain Chloroform-Methanol solvent mixture (MeOH:CHCl3) • dissolves/ miscible with lipids. Moreover, polar lipids dissolve in methanol. • Chloroform = nonpolar solvent used in dissolving lipids • Methanol = polar component used for further isolation of remaining compounds in the lipids Hexane • good solvent only for lipids of low polarity • Its main use is to extract neutral lipids from mixtures of water with alcohols • With a fairly high volatility and a low sensible heat it is relatively easy to remove from the solids and oil with low energy use. • The low boiling point of hexane (67°C / 152°F) and the high solubility of oils and fats in it are the properties exploited in the solvent extraction process. • Used as a selective solvent of glycerophosphatides to separate them from sphingolipids
Isolation of Non- Saponifiable and Saponifiable Lipids from Calf’s Brain ● Hexane Solvent Extraction
➢ Solvent Extraction is a process which involves extracting oil fro materials by treating it with a low boiler solvent as opposed to by mechanical pressing methods (such as expellers, hydraulic p
➢ This method recovers almost all the oils and leaves behind onl residual oil in the raw material.
➢ Because of the high percentage of recovered oil, solvent extrac the most popular method of extraction of oils and fats.
Test for: Cholesterol Reagents: Acetic anhydride and Sulfuric acid Principle: Acetylation of the hydroxyl group of cholesterol located at C-3 then reaction with concentrated sulfuric acid Positive Result: Emerald green solution
Liebermann-Burchard Test
A. Liebermann-Burchard Test (+)Emerald green solution STANDARDS GROUP
C
GP
SP C
LECITHIN
1
-
+
-
+
-
2
+
+
-
+
-
3
-
+
-
+
-
4
+
-
-
+
-
5
-
+
+
-
-
GLC
A. Liebermann-Burchard Test (+)Emerald green solution STANDARDS GROUP
C
GP
SP C
LECITHIN
6
+
+
-
+
-
7
-
-
-
+
-
8
+
+
-
+
+
9
+
+
-
+
-
10
+
+
-
+
+
GLC
Mechanism
POSITIVE: CHOLESTEROL & GALACTOCEREBROSIDE
Test for: Cholesterol Reagents: concentrated Sulfuric acid Principle: Addition of sulphuric acid reacts with cholesterol resulting to red biocholestadien disulfonate then dehydration forming a bisteroid Positive Result: red interphase
Salkowski’s Test
B. Salkowski’s Test (+) Red interphase STANDARDS GROUP
C
GP
SP C
LECITHIN
1
+
-
-
+
+
2
-
+
-
+
+
3
+
+
-
+
+
4
-
+
-
+
+
5
-
+
-
+
+
GLC
B. Salkowski’s Test (+) Red interphase STANDARDS GROUP
C
GP
SP C
LECITHIN
6
+
+
-
+
+
7
+
+
-
+
+
8
+
+
-
+
+
9
-
+
-
+
+
10
+
+
-
+
+
GLC
Mechanism
POSITIVE: CHOLESTEROL & GALACTOCEREBROSIDE
Test for: Phosphate Reagents: fusion mixture, 3M HNO3, 2.5% Ammonium molybdate Principle: Oxidation reaction, conversion of organic form to inorganic phosphate; Precipitation reaction Positive Result: yellow precipitate
Phosphate Test
C. Phosphate Test (+) Yellow precipitate STANDARDS GROUP
C
GP
SP C
LECITHIN
1
-
-
2
-
+
3
-
+
4
-
+
5
-
+
GLC
C. Phosphate Test (+) Yellow precipitate STANDARDS GROUP
C
GP
SP C
LECITHIN
6
-
-
7
-
+
8
-
+
9
+
+
10
-
-
GLC
Mechanism
POSITIVE: GLYCEROPHOSPHATIDE & LECITHIN
Test for: presence of choline Reagents: Kraut’s reagent, Potassium iodide + Bismuth subnitrate and Nitric acid Principle: Complexation reaction of choline with bismuth potassium iodide Positive Result: dark orange or red precipitate
Kraut’s Test
D. Kraut’s Test (+)Orange-red precipitate STANDARDS GROUP
C
GP
SP C
LECITHIN
GLC
1
-
+
-
-
+
-
2
-
+
-
-
+
-
3
-
+
-
-
+
-
4
-
+
-
-
+
-
5
+
+
+
+
+
+
D. Kraut’s Test (+)Orange-red precipitate STANDARDS GROUP
C
GP
SP C
LECITHIN
GLC
6
-
+
-
-
+
-
7
-
+
-
-
+
-
8
-
+
-
-
+
-
9
-
+
-
-
+
-
10
-
+
-
-
+
-
Mechanism
POSITIVE: GLYCEROPHOSPHATIDE SPHINGOSINE LECITHIN
Test for: Free amino groups and secondary amines Reagents: Ninhydrin Principle: Oxidative deamination followed by condensation Positive Result: blue-violet solution
Ninhydrin Test
E. Ninhydrin Test (+)Violet solution STANDARDS GROUP
C
GP
SP C
LECITHIN
GLC
1
+
+
+
+
+
-
2
+
+
-
+
+
-
3
-
+
+
-
+
-
4
-
+
+
-
+
-
5
-
+
+
-
-
+
E. Ninhydrin Test (+)Violet solution STANDARDS GROUP
C
GP
SP C
LECITHIN
GLC
6
+
+
+
-
+
-
7
-
+
-
-
-
-
8
+
+
+
-
+
-
9
-
+
-
-
+
-
10
-
+
-
-
-
-
Mechanism
POSITIVE: GLYCEROPHOSPHATIDE, SPHINGOSINE & LECITHIN
Test for: Carbohydrates Reagents: α-naphthol in ethanol, concentrated Sulfuric acid Principle: Dehydration forming furfural and its derivative Positive Result: violet interphase
Molisch Test
F. Molisch Test (+) Violet interphase STANDARDS GROUP
C
GP
SP C
LECITHIN
GLC
1
-
+
+
-
+
-
2
-
+
-
-
+
-
3
-
+
+
-
+
-
4
-
+
-
-
+
-
5
-
+
-
-
+
-
F. Molisch Test
(+) Violet interphase STANDARDS
GROUP
C
GP
SP C
LECITHIN
GLC
6
-
+
+
-
+
-
7
-
+
-
-
+
-
8
+
+
-
-
+
-
9
-
+
+
-
+
-
10
-
+
-
-
+
-
Mechanism
POSITIVE: GLYCEROPHOSPHATIDE, SPHINGOSINE, LECITHIN GALACTOCEREBROSIDE
IDEAL RESULT
TESTS
Cholesterol
Glycerophos phatide
Sphingosine
Lecithin
Galactocereb roside
LiebermannBurchard Test
+
-
-
-
+
Salkowski
+
-
-
-
+
Phosphate
N/A
+
N/A
+
N/A
Kraut’s
-
+
+
+
-
Ninhydrin
-
+
+
+
-
Molisch
-
(-/+)
+
+
+
Conclusion
Through trituration with different solvents, various types of lipids in a calf’s brain were extracted. Cholesterol’s lack of phosphorylation and polarity were observed when it was isolated and dissolved via acetone. In addition, hexane’s properties allowed it to be isolated through solvating with hexane. Lastly, hot ethanol, a specific solvent for sphingosine was used to isolate sphingosine. In all of the procedures used in isolating the various lipids, methanol:chloroform mixture was used for its preferrable properties as a solvent.
Based from the experiment, it can be inferred that the extracted cholesterol and standard cholesterol produced positive results in the Liebermann-burchard test and Salkowski test due to the presence of sterols.
Furthermore, it can be inferred that both lecithin and glycerophosphatides yielded positive results in the phosphate test due to the presence of phosphate groups.
In addition, lecithin tested positive in the kraut’s and Ninhydrin test due to the presence of choline as well as free amino groups.
Lastly, only galactocerebroside produced a positive result in the Molisch test due to the presence of a carbohydrate in its head group.
References: Cantarow, A. & Schepartz, B. (1962). Biochemistry, 3rd edition. Philadelphia: WB Saunders Company. Chawla, R. (2003). Practical Clinical Biochemistry: Methods and Interpretations. India: Jaypee Brothers Medical Publishers (P) Ltd. Christie, W. (1993). Advances in Lipid Methodology- Two. Dundee: Oily Press. Clark, J. (1964). Experimental Biochemistry. San Francisco: WH Freeman and Company. Domodoran, G. (2011). Practical Biochemistry. New Delhi: Jaypee Brothers Medical Publisher (P) Ltd. Krishnaswamy, N.R. (2003). Chemistry of Natural Products: A Laboratory Handbook. India: Universities Press Private Ltd. Yoder, C. (2016). Recrystallization. Retrieved on November 02, 2016 from http://www.wiredchemist.com/chemistry/instructional/laboratory-tutorials/recrystallization
