This document discusses separating and identifying lipids by electrophoresis. It explains that electrophoresis uses an electric field to separate charged particles, with cations moving toward the cathode and anions toward the anode. Lipids are composed of glycerol bonded to hydrocarbon chains and other molecules. Lipoproteins are complexes of lipids and proteins that transport lipids in the bloodstream. Electrophoresis can separate lipoprotein fractions based on their protein and lipid content. Density gradient centrifugation further separates lipoproteins by density. Various lipid fractions can be quantified through gel electrophoresis.

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Separation and
Identification of
Lipids by
Electrophoresis
Present ed To: Prof . Sana Zulf iqar
Presented by: Aafia Aslam

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Introduction
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• Electrophoresis is the
technique of separation.
• Electrophoresis literally
means “running in the
electric field.”

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Principle
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• A charged particle in solution will migrate towards one of the electrodes
when placed in the electric field.
• Used for separation of ionizable substances in a sample.
• Cations or positively charged particles move towards cathode.
• Anions or negatively charged particles move towards anode.
• When a voltage is applied across the electrodes, it generates a potential
gradient. This potential gradient apply a force on charge bearing
molecules. And drive a charged molecule towards an electrode. A
frictional resistance oppose this movement of charged molecules .

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Lipids
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• Lipid is a macrobiomolecule that is soluble in nonpolar
solvents.
• Lipids are composed of a glycerol molecule bonded to
long hydrocarbon chain (can be single or multiple) and,
depending on the lipid, to other molecules, such as a
phosphate group (phospholipids).
• Lipids have many different biological functions such as
fuel molecules as storage source of energy,
• Structural building blocks for phospholipids and
glycolipids,
• Covalent attachments to guide molecules to specific
membrane locations, and intracellular messengers.

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Lipoproteins
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• Spherical complexes of lipids &
proteins.
• Transport various lipids & fat soluble
vitamins to & from tissues.
• Contain core of hydrophobic lipids
surrounded by hydrophilic lipids &
proteins.

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Introduction to Separation and Identification
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• The plasma lipoproteins can be separated by electrophoresis, along with the
other plasma proteins. In fasting serum or plasma, the two most prominent
lipoprotein bands are in the α1 and β fractions.
• They are designated as α- and β-lipoproteins. A weaker band, the pre–β-
lipoproteins, moves slightly ahead of the β-lipoproteins. The chylomicrons,
found only after a fatty meal, do not move upon electrophoresis.
• Density gradient centrifugation separates the lipoproteins according to their
protein/lipid ratio. Nonpolar lipids have densities near 0.9 g/cm3.
• For lipoprotein particles, the densities increase from 0.95 g/cm3 in the most
lipid-rich particles to well above 1.0 g/cm3 in the protein-rich types.
• Based on their order of density and protein content, we can distinguish
chylomicrons, very-low-density lipoprotein (VLDL), low-density lipoprotein
(LDL), and high-density lipoprotein (HDL). The correspondence of these density
classes to the electrophoretic separation

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Extraction and
Identification of Lipids
S u b t i t l e
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Separation and Identification of Lipids by Electrophoresis
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• Highly purified yeast lipid particles with an enrichment factor of 700 to 800 for
triacylglycerols, steryl esters, and Erg6p over the homogenate were prepared from
cells grown to the late logarithmic phase.
• Nonpolar lipids were extracted with 2 volumes of diethyl ether.
• The organic phase was withdrawn, residual diethyl ether was removed under a stream
of nitrogen, and proteins were precipitated from the aqueous phase with
trichloroacetic acid at a final concentration of 10%.
• SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out.
• Various lipid fractions (LDL, HDL, VLDL) can be quantified by gel electrophoresis.
• 0.975(very low density lipoproteins, VLDL), 0.986(low density lipoproteins, LDL) and
0.965(high density lipoproteins, HDL) and for triglyceride levels 0.994(VLDL),
0.963(LDL) and 0.959(HDL) respectively.

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By weight percentage of components.
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Lipoprotein separation
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• Lipid particle proteins (LP) were separated by SDS-
PAGE, reisolated from the gel.
• ST, standard proteins

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Thank You!