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Monday, May 26, 2014

Estimation of free fatty acids

Fatty acids are carboxylic acids with a hydrocarbon chain. They have a chain of even number of carbon atoms, generally 4-28. Fatty acids are of Two Types:

  1. Saturated Fatty acids
  2. Unsaturated Fatty acids
Saturated fatty acids have carbon - carbon single bond, whereas unsaturated fatty acids have carbon-carbon double bond.
Structure of Fatty Acid

Fatty acids are usually derived from triglycerides or phospholipids. when they are not attached to other molecules it is called as Free Fatty acids.

A small quantity of free fatty acids is usually present in oils along with the triglycerides. The free fatty acid content is known as acid number/acid value. It increases during storage. The keeping quality of oil therefore relies upon the free fatty acid content.


The free fatty acid in oil is estimated by titrating it against KOH in the presence of phenolphthalein indicator. The acid number is defined as the mg KOH required to neutralize the free fatty acids present in 1g of sample. However, the free fatty acid content is expressed as oleic acid equivalents.

  • 1% phenolphthalein in 95% ethanol.
  •  0.1N potassium hydroxide
  • Neutral Solvent: Mix 25mL ether, 25mL 95% alcohol and 1mL of 1% phenolphthalein solution and neutralize with N/10 alkali.
  1. Dissolve 1-10g of oil or melted fat in 50mL of the standard solvent in a 250mL conical flask.
  2. Add a few drops of phenolphthalein.
  3. Titrate the contents against 0.1N potassium hydroxide.
  4. Shake constantly until a pink color which persists for fifteen seconds is obtained.

Acid value (mg KOH/g) = (Titrate value x Normality of KOH x 56.1) / Weight of sample (g)

The free fatty acid is calculated as oleic acid using the equation
1mL N/10 KOH = 0.028g oleic acid.

Saturday, May 3, 2014

Mechanism of Interaction of Ethidium Bromide with DNA

Ethidium Bromide (EtBr) is an intercalating agent widely used in molecular biology labs as a fluorescent dye for nucleic acid gel electrophoresis .Ethidium Bromide is very sensitive in detecting DNA bands on gel.

etbr structure
Structure of Ethidium Bromide (EtBr)

Mechanism of Interaction of Ethidium Bromide with DNA

Ethidium Bromide contains tricyclic phenanthridine ring system that is able to interact with stacked base pairs of double stranded DNA. Ethidium is capable of forming close van der Walls contacts with the base pairs and due to that it can bind to the hydrophobic interior of the DNA molecule. The peripheral phenyl and ethyl groups projects into the major groove of DNA helix.

intercalation of EtBr with DNA

Approximately one ethidium bromide molecule will get intercalated per 2.5 base pairs, there will be a 20 fold increase in the fluorescence when intercalated. Absorption maxima of EtBr is in the UV range, upon excitation EtBr emits orange light with a wavelength of 605 nm.

EtBr Stained DNA gel / Agarose gel
EtBr Stained Gel

Ethidium Bromide can bind to helical regions formed by  intrastrand base pairing in RNA and in heat denatured or single stranded DNA. The fluorescent yield of DNA-Ethidium Bromide dye complex is many fold higher than the free dye, so it is advantageous in visualizing the DNA on gels. small amounts of DNA ~10ng can be detected using EtBr staining. Ethidium Bromide stock solutions are prepared at a concentration of 0.25  - 1.0 microgram /mL for gel staining.

Ethidium Bromide is considered as a potent mutagen,  it is called as intercalating agent since it can intercalate into the DNA and can interfere with DNA replication and Transcription.

Safer Alternatives
There is still controversies regarding the toxicity of Ethidium Bromide. Even then some researchers prefers to use Ethidium Bromide for nucleic acid staining. But there are some safer alternatives to Ethidium Bromide, here are the commercially available safer dyes: 
  • SYBR Safe, 
  • Gel Red