PRESERVATIVES

BENZOIC ACID
HYDROXYBENZOATE
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BENZOIC ACID

 White solid
 Food, beverages, toothpastes, mouthwashes, dentifrices, cosmetics and
pharmaceuticals
 Fruit juices, soft drinks, pickles, barbecue sauces and salad dressings
 Inhibitory effect on the growth of yeast
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Additive Permitted in or upon Maximum level of use

Jam, juice,jelly, packed,

pickles, tomato paste,
puree

Benzoic acid Meat and meat by 1000 ppm

product, fish, poultry
meat

Margarine
4 QUALITATIVE TEST

Sample is shaken with ether after acidifying with dil.H2SO4

Separate the watery ether layer, add more solvent and wash with water

Shake the ether layer with dilute ammonia

Transfer the layer into another separator, acidify and extract

Pour equal volume of ether extract into two porcelain dish and evaporate off
 Residue + conc. H2SO4 + Potassium nitrate

5 heat
Examine the residue by dissolving in water Acids-----m-dinitrobenzoic acid
FeCl3
Salmon colour precipitate of ferric benzoate Dilute with water and make alkaline with ammonia

Transfer into a test tube and boil

cool
add ammonium sulphide

reddish brown ring

COMBINED
6 EXTRACTION AND SEPARATION PROCEDURE
Food is homogenised with water

slurry is applied to a chromatographic paper

Buffer solution, sodium bicarbonate solution and NaOH solution are applied to the paper

Food line is moistened with dil HCl

End of the paper is dipped into diethyl ether

 ether soluble acids at bicarbonate line and parahydroxybenzoates at sodium hydroxide line
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examine under UV lamp

extraction with acidified ethanol

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QUANTITATIVE ESTIMATION
• DIRECT METHOD
foods containing benzoic acid is shaken out with ether after acidifying with dil. H2SO4

purified with dil.NaOH and treating with permanganate at 60ºC

excess permanganate is then reduced with SO2

Dissolve the residue with acetone and water

Titrate with 0.05M NaOH using Hph as indicator

9 CALCULATION

122 x titre value x dilution x 1000 x ml of 0.05M NaOH

Benzoic acid (ppm)=
weight of sample x aliquot taken
 SEPARATION METHOD
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For viscous liquid employ steam distillation with phosphoric acid, purification by permanganate
To a distillation flask add sample, water and excess salt

add phosphoric acid and rapidly steam

Distil the content into large porcelain basin containing 1M NaOH

Wash down and separate the alkaline distillate

Decolourise with S02 + dil.H2SO4 to dissolve the precipitated MnO2

 Transfer to a cylinder, saturate with salt and extract the acid with ether
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Transfer the extract to a test tube and evaporate off the solvent

Wash down any deposit present and evaporate off

The residue is sufficiently pure to be weighed

The solution may be then assayed by TLC, UV spectrophotometry, colorimetry, GLC or

HPLC
 UV SPECTROPHOTOMETRIC METHOD
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Benzoic acid is extracted from sample using diethyl ether

Absorbance is measured at 272nm, 267.5nm, 276.5nm in the UV region

From the corrected absorbance and calibration graph, the amount of benzoic acid is determined
 HPLC METHOD
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Chromatographic condition
• Detector - UV-Visible
• Wavelength - 230nm
• Flow rate - 1ml/min
• Mobile phase - Acetonitrile: phosphate buffer (40:60)
• Injection volume - 20µL
• Run time - 20mins
• Retention time - 5-6mins
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CALCULATION
A C
Benzoic acid (ppm)= x
slope W

A - Peak area of benzoic acid

C - dilution factor
W - Weight of sample in gm
15 4-HYDROXYBENZOATES (PARABEN)

• Esters of p-hydroxy benzoic acid

• Low toxicity and effective antimicrobial activity
• Used in processed vegetables, baked goods, fats and oils, sugar substitutes, frozen
diary
• 1000 ppm
 QUALITATIVE TEST
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MILLON’S REAGENT
Dissolve 3ml of mercury in cold fuming nitric acid and dilute with equal volume of water

Millon’s reagent give rose red colour with hydroxybenzoates

 TLC METHOD
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PRINCIPLE
The sample is acidified and extracted with diethyl ether. The concentrated ethereal extract is subjected to
TLC. Using U.V or Denige’s reagent for visualization
PROCEDURE
Add sulphuric acid to sample and grind with sodium sulphate

Grind the sample with ether and decant the ether.

Filter the ether extract, evaporate and dissolve the residue in methanol

Spot 20 μL along with standard

 View the plate under UV (254 nm).
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Para-hydroxy benzoates show black spots.

Mark any quenched area lightly with a pin and spray lightly with Denige’s reagent.

P-hydroxy benzoate gives a white spot

Heat at 100°C for 5 min and spray lightly with 2% sodium nitrite solution.

Appearance of red spots

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QUANTITATIVE METHOD

PRINCIPLE
Sample are hydrolysed using alkali and extracted with diethyl ether after acidification of the
sample
After re-extraction with NaOH from ether colour is developed with Denige’s reagent and the absorption
is read at 518nm
PROCEDURE
20 sample + water
adjust the pH to 7.5 with NaOH
Heat at 50ºC for 30mins

Add potassium ferrocyanide and mix

Add ZnSO4, mix and dilute to 100ml with water

Filter, take 50ml of the filtrate and add dil. H2SO4

extract with diethyl ether and wash the extract with water
 add Hph and shake with NaOH
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wash with water and combine the alkaline extract and make upto 10ml

take 5ml of above solution and add Denige’s reagent

heat
cool and add aq.Sodium nitrite and allow to stand for 45mins

measure the absorbance of pink colour at 518nm

Prepare a calibration graph

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REFERENCE

• David Pearson, The Chemical analysis of foods, Food additives, seventh

edition , Churchil livingstone , Edinburgh London,1976
• Manual of methods of analysis of food by fssai ,Government of India,2015
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