Qualitative Method: |
Principle: These Preservatives Are Separated By Steam Distillation, Extracted Into Ether From The Acid Solution And The Ethereal Extract Is Examined By Tlc. |
Reagents: · 50% Hcl (V/V) · Diethyl Ether (Peroxide Free): The Absorbance Characteristics Of This Must Be Checked And If Necessary The Ether Petroleum Ether Or Cyclohexane May Be Used To Replace Ether As Long As The Absorbance In The Range 220 – 290 Is Acceptably Low E.G. By Use Of Spectroscopically Pure Grade · Tlc Developing Solvent-Ethanol: Ammonia (9:1) · Standard Solution: 1% Benzoic Acid And Sorbic Acid In Ethyl · Peroxide- Ferric Chloride Spray Regent: Mix Equal Volume Of Freshly Prepared 2% Ferric Chloride And 5% Hydrogen Peroxide Solution. · Thiobarbituric Acid Spray Reagent: 2% Solution In Water Of 2- Thiobarbituric Acid. · Solid Magnesium Sulphate Heptahydrate · 1m H2so4 · 1m Naoh · Tlc Plates And Silica Gel G. |
· Procedure: Place A Weighed Portion, Usually 25 -50 Gm Of Sample In A One Litre Steam Distillation Flask, Add 100 Gm Of Magnesium Sulphate And 100 Ml 1m H2so4. Steam And Distill As Rapidly As Possible, Collecting About 450 Ml In 10 Minutes Steam- Distillate In The Flask Containing 10 Ml 1 M Sod Hydroxide Solution. Heating The Flask Containing The Sample May Result In A Coloured Or Impure Distillate. Add 15 Ml 1 M Sulphuric Acid To The Distillate And Dilute To 500 Ml. Extract And Aliquot (100 Ml) With Three Or Four 25 Ml Portions Of Diethyl Ether Or Other Solvent. Combine The Extracts And Wash Them With A Few Ml Of Water Dry The Combined Solvent Layer Over Anhydrous Sodium Sulphate And Reduce To 1 Ml At The Lowest Temperature Possible. Use Of A Rotary Evaporator Is Preferable Spot 20 µl Or Less On The Silica Gel G Tlc Plate Along With Standard Solution. Develop For About 10 Cm Using Developing Solvent. Air Dry The Plate And Spray With Peroxide-Ferric Chloride Reagent. Benzoic Acid Shows As A Mauve Coloured Spot (Rf 0.5) And Sorbic Acid May Be Distinguished As A Yellow Coloured Spot Slightly Below It. Further Spraying With Tba Solution And Heating At 100ºc For 5 Min, Sorbic Acid Appears As A Pink Spot A Little Below Benzoic Acid |
Sorbic Acid – Quantitative Method: |
· Uv Spectrophotometric Method: (Applicable To Fresh Dairy Products, Cottage Cheese, Ricotta And Mozzarella Cheese, Sour Cream And Yoghurt) |
· Principle: Sorbic Acid Is Extracted From The Sample Using The Solvent Mixture Of Diethyl Ether And Petroleum Ether (1 : 1) And Absorbance Of The Extract Is Measured At 250 Nm. Sorbic Acid In Another Aliquot Is Destroyed With Permanganate And Absence Of The Peak At 250 Nm Is Taken As Confirmation Of The Presence Of Sorbic Acid In The Sample |
· Apparatus : Spectrophotometer |
Reagents : · Metaphosphoric Acid Solution: Dissolve 5 Gm In 250 Ml Water And Dilute To 1 Litre With Alcohol. · Mixed Ethers: Petroleum Ether And Anhydrous Diethy1 Ether (1+1). · Potassium Permanganate Solution: Dissolve 15 Gm In 100 Ml · Sorbic Acid Standard Solution (1 Mg/Ml): Dissolve 100 Mg And Make Up To 100 Ml With Mixed · Working Standard Solution: Dilute 5 Ml To 100 Ml With The · Reference Solution: Shake 10 Ml Of Mixed Ethers With 100 Ml Of Phosphoric Acid Solution And Dry The Supernatant Ether Layer With Anhydrous Sodium |
· Procedure : |
· Homogenise The Sample (Cheese And Related Products) By Cutting Into Small Pieces Using A Food Chopper Or By Shredding It Over A Sieve. With Creamed Ottage And Similar Cheeses Place 300 – 600 Gm Of Sample At 150 C In A 1 Litre Cup Of A High Speed Blender And Blend For The Minimum Time (2 – 5 Minutes) Required To Obtain A Homogeneous Mixture Accurately Weigh About 10 Gm Of The Prepared Sample, In A High Speed Blender, Add Enough Phosphoric Acid To Yield A Total Of 100 Ml Of Liquid In The Mixture. Blend For One Minute And Immediately Filter Through Whatman No.3 Paper. Transfer 10 Ml Of Filtrate To A 250 Ml Separator Containing 100 Ml Of Mixed Ethers And Shake For One Minute. Discard The Aqueous Layer And Dry The Ether Extract Over 5 Gm Of Anhydrous Sodium Sulphate And Read The Absorbance At 250 Nm Against Reference Solution. Determine The Concentration Of Sorbic Acid From The Standard Curve Prepared As Follows |
