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  Section: Plant Lab Protocols
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Methodology for Lipids

Estimation of volatile acids
Volatile fatty acids are water-soluble and they can be distilled at  atmospheric pressure. These fatty acids are intermediates in the process of methane production and many other allied reactions. These acids can be removed by steam distillation, in spite of high boiling points, because of their high vapor tension. This group includes water-soluble fatty acids having up to six carbon atoms.


Straight distillation or steam distillation after acidifying the sample with 1 + 1 H2SO4 results in the evaporation of volatile acids which on condensation yield liquefied volatile acids. These acids could be titrated against 0.1N alkali with phenolphthalein indicator to find out the amount of volatile fatty acids.
The steam distillation method is more tedious, requires at least 4h to complete, but results in the recovery of 92% to 98% volatile acids from sewage sludge. The steam distillation is conducted on a MgSo4 saturated, acidified, sludge-free liquor produced by chemical treatment with ferric chloride and filter-aid followed by vacuum filtration. The effects of varying concentrations of dissolved solids and mineral acids during distillation are avoided.

Dilute Sulphuric Acid 1 + 1: Mix one volume of conc. H2SO4with one volume of water.
Ferric Chloride Solution: Dissolve 82.5g FeCl3 6H2O in one liter distilled water.
Diatomaceous-Silica Filter Aid
Magnesium Sulphate (MgSO4,7H2O)
Standard Sodium Hydroxide, titrant 0.1N: Dissolve 4g sodium hydroxide in 1L of distilled water and standardize against any standard acid.
Phenolphthalein Indicator: 0.1% in 95% alcohol.

Adjust the sample containing volatile fatty acids (sludge and water) 200 to 1,000mL to pH 3.5 with (1 + 1) H2SO4.
Add 6mL of FeCl3 solution/L sample (equivalent to 500mg/L).
Add 50g filter aid/L, and mix well.
Filter by suction, using a Buchner funnel containing a filter paper freshly coated with a thin layer of filter-aid.
Wash the residue thoroughly three or four times with water, and adjust the filtrate to pH 11 with NaOH solution.
Concentrate by evaporation to 150mL and cool in a refrigerator.
Adjust the cooled filtrate to pH4 with dilute H2SO4 and add it quantitatively and quickly to the distilling flask.
Add magnesium sulphate to a slight excess of saturation.
Apply heat with a small flame to the flask until rapid evolution of volatile acids commences. This will prevent excessive increase in the volume of the mixture.
Steam distill slowly so that about 200mL of distillate is collected in 25min.
Increase the rate of distillation and continue until a total of 600mL is collected.
Titrate the distillate against 0.1N NaOH using phenolphthalein indicator.

mg/L volatile acids as acetic acid =
mL 0.1N NaOH x 6,000
mL sample
(molecular weight of acetic acid is 60)


1. Rand, M C, Greenberg, A E and Taras, M J (eds) (1976) In: Standard Methods for the Examination of Water and Waste Water (14th Ed) American Public Health Association Washington p 529.
2. Olmslead, W H, Whitaker, W M and Duden, C W (1930) J Biol Chem 85 109.

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