Detection Of P-Hydroxy Benzoic Acid And Its Esters By TLC:

·       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

·       Apparatus :

·       Tlc Apparatus

·       Viewing Cabinet With Short Wave ( 254 Nm) Uv Light

Reagents :

·       Silica Gel G

·       Developing Solvent: Toluene: Methanol: Acetic Acid

·       2% Solutions In Diethyl Ether (Methyl, Ethyl Or Propyl Hydroxy Benzoates And The Free Acid).

·       Denige’s Reagent: Mix 5 Gm Of Yellow Mercuric Oxide With 40 Ml Water, Cool In Ice-Salt And Very Slowly Add Freezing Cold H2so4 (20 Ml) With Add Another 40 Ml Of Water.

·       Sulphuric Acid – 10% By

·       Sodium Nitrite – 2% Freshly

·       Sodium Sulphate – Anhydrous.

·       Solvent Ether

·       Procedure : Add 5 Ml Of 10% H2SO4 To 10 Gm Of Sample And Grind With Sodium Sulphate In A Mortar Until The Sample Is Dry. Add About As Much Sodium Sulphate Again. Grind The Sample With Small Successive Quantities Of Ether And Decant The Ether. Filter The Ether Extract, Evaporate At A Low Temperature And Dissolve The Residue In Methanol (1 Ml). Spot 20 µl Along With Standard And About The Same Amount Of The 2 % Standards On The TLC Plate And Develop The Chromatogram With Developing Solvent. View The Plate Under UV (254 Nm). Para-Hydroxy Benzoates Show Black Spots. Interfering Substances May Be Present, So Caution Should Be Used In Interpreting The Results Mark Any Quenched Area Lightly With A Pin And Spray Lightly With Denige’s Reagent. P- Hydroxy Benzoate Gives A White Spot, Visible By Its Different Reflectivity From The Background. Heat At 100ºc For 5 Minutes And Spray Lightly With 2% Sodium Nitrite Solution. Appearance Of Red Spots Indicate The Presence Of P-Hydroxy Benzoic Acid Esters. 

·       Interpretation :If The Object Of The Test Is To Confirm That The Amounts Of Any P – Hydroxy Benzoates Present Are Below The Prescribed Limit, The Quantities Of The Standard Spotted Can Be Chosen To Correspond To That Maximum. Sample Spotsof Lower Intensity

Qualitative Test For Para -Hydroxy Benzoic Acid:

·       The Test Is Applied On Neutral Ammonium Salt Of Para – Hydroxy Benzoic Acid. Extract 4-Hydroxy (Para) Benzoic Acid From The Acidified Food With Ether And Remove The Solvent. Dissolve Residue In Few Drops Of Dilute Ammonium Hydroxide Solution In A Test Tube. Add A Few Drops Of Millon’s Reagent (Dissolve 3 Ml Mercury In 27 Ml Cold Fuming Nitric Acid And Dilute With An Equal Volume Of Water). Presence Of 4-Hydroxy Benzoic Acid Is Revealed By Rose-Red Colour. Many Aromatic Substances With A Hydroxyl Group Attached To The Benzene Ring Give Red Colour. (Eg. Salicylic Acid Gives Orange Red Colour With Millon’s Reagents). The Test Cannot Be Considered Specific For 4 – Hydroxyl Benzoic Acid. Salicylic Acid Can However Be Distinguished By Intense Violet Colour Given With Ferric Chloride

Quantitative Method:

Principle:

·       The 4-Hydroxy Benzoic Acid Esters Present In The Sample Are Hydrolysed Using Alkali And 4-Hydroxy Benzoic Acid Is 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 518 Nm.

Reagents :

·       Dilute Sulphuric Acid: Dilute 100 Ml H2so4 To 300 Ml With Water.

·       Denige’s Reagent: Dissolve 5 Gm Of Mercuric Oxide In 20 Ml Of H2so4 And Dilute To 100 Ml With Water.

·       Potassium Ferrocyanide (15%): Prepare In

·       Zinc Sulphate (30%): Prepare In

·       Sodium Hydroxide (5%) : Prepare In Water.

·       Diethyl

·       Sodium Nitrite (2%): Freshly Prepared In

Procedure :

·       To 2 Gm Of The Sample Add 60 Ml Water At 50ºc And Adjust The Ph To 7.5 With Naoh (5% Solution). Heat At 50ºc For 30 Minutes With Occasional Stirring. Add 2 Ml Of Potassium Ferrocyanide And Mix Carefully. Add 2 Ml Zinc Sulphate, Mix And Dilute To 100 Ml And Set Aside For 30 Minutes. Filter, Take 50 Ml Filterate And Add 1 Ml Of Dilute H2so4. Extract With 3 X 50 Ml Portions Of Diethyl Ether. Wash The Combine Ether Extracts With Water ( 3 X 5 Ml/30 Sec), Add A Drop Of Phenolphthalein And Shake With 3 Ml Of 0.25m Naoh Solution. Wash With 3 Ml Of Water And Combine The Alkaline Extracts, Remove Any Traces Of Ether On Hot Water Bath And Make Upto Volume (10 Ml). Take 5 Ml Of Solution And Add 5 Ml Of Denige’s Reagent. Heat In A Boiling Water Bath For 5 Min. Cool, Add 5 Drops Of 2% Aqueous Nano2 Solution And Allow To Stand For 45 Min. Measure The Absorbance Of Pink Colour At 518mm. Dissolve 50, 100, 200, 400 And 600 Mg Of Ester In 3 Ml Quantities Of 0.25n Naoh, Make Upto 5 Ml And Carry Out The Above Method Starting From Addition Of 5 Ml Denige’s Reagent To Prepare A Calibration Graph And Determine Concentration

Estimation Of Benzoic Acid, Sorbic Acid And Parabens From Food Samples:

HPLC-UV Method:

·       Principle: Benzoic Acid, Sorbic Acid And Parabens Are Separated From A Known Quantity Of The Sample By Saturating With Nacl And Then Acidifying With Dilute Hcl And Extracting With Chloroform. The Chloroform Layer Is Evaporated To And The Residue Is Dissolved In Neutral Alcohol And The Amount Of Benzoic Acid Is Determined By HPLC–UV Method.

Reagents :

·       Anhydrous Ethanol Ar Grade

·       Methanol Hplc Grade

·       Glacial Acetic Acid – Hplc Grade

·       Deionized Water (Di) – ≥ 10 MΩ

·       Ammonium Acetate – Hplc Grade

·       Chloroform – Hplc Grade

·       Acetonitrile – Hplc Grade

·       Sodium Chloride – Ar Grade

·       Hydrochloric Acid – Ar Grade

·       Anhydrous Sodium Sulfate – Ar Grade

Preparation Of Samples:

·       Beverages And Liquid Products: Mix The Sample Thoroughly And Transfer 100 Gm Of The Sample Into A 250 Ml Volumetric Flask, Using Saturated Nacl Solution. Make Alkaline To Litmus Paper With 10% Naoh Solution And Make Upto Volume With Saturated Nacl Solution. Shake Thoroughly And Let It Stand For 2 Hours. Filter The Sample And Use The Filtrate For Determination.

·       Sauces And Ketchups: Add 15 Gm Salt To 150 Gm Of Weighed Sample And Transfer Into Volumetric Flask. Rinse With Saturated Nacl Solution, Add 15 Gm Pulverized Nacl And Then Add 10 Ml Of 10% Naoh Solution And Make Upto Volume With Nacl Solution. Let It Stand For 2 Hrs. With Occasional Shaking. Filter And Use The Filtrate For Determination.

·       Jams, Jellies, Preservatives And Marmalades: Mix 150 Gm Of Sample With 300 Ml Saturated Nacl Solution. Add 15 Gm Pulverised Nacl. Add 10 Ml Of 10% Naoh Solution. Transfer To 500 Ml Volumetric Flask And Dilute To Volume With Saturated Nacl Solution. Let It Stand For 2 Hrs With Frequent Shaking, Filter And Use The Filtrate For Determination.

Methodology:

·       Preparation Of Stock Solutions: Stock Standards (4.0 Mg/Ml Benzoic Acid, Sorbic Acid, Methyl, Ethyl, Propyl, And Butyl Parabens): Weigh 400.0 Mg Each Of Benzoic Acid, Sorbic Acid, Methyl, Ethyl, Propyl, And Butyl Parabens Into A 100 Ml Volumetric Flask. Add Approximately 50 Ml 70% Ethanol To Dissolve, And Dilute To Volume With 70% Ethanol. Dilute The Stock Stock Solutions To 0, 10, 20, 40, 60, 80 And 100 µg/Ml In 70% Ethanol For Preparation Of Calibration Curves.

·       Determination: Pipette 100 Ml To 200 Ml Of The Filtrate Into A 250 Ml Separatory Funnel. Neutralize To Litmus Paper Using HCI (1+3) And Add 5 Ml Excess. Extract Carefully With 40, 30, 30 And 20 Ml Portions Of Chloroform. Avoid Formation Of Emulsion By Shaking Gently With Rotatory Motion. If Emulsion Forms, Break It By Stirring Chcl₃ Solution With A Glass Rod After Each Extraction, But Do Not Drain Any Of The Emulsion With Chloroform Layer. Transfer The Combined Chloroform Extract In To A Separatory Funnel And Wash It Free From Mineral Acid By Shaking Gently And Rinsing With Water. Drain Off The Water Phase. Dry The Chloroform Layer Over Anhydrous Sodium Sulphate And Distil Off The Solvent. Remove The Last Traces Of The Solvent Under A Current Of Nitrogen At Room Temperature. Dissolve Residue In 100 Ml Of Alcohol.

HPLC – UV Method:

·       Column: Column – 15cm X 4.6mm ID, C-18, 5 Μm Particle Size

·       Mobile Phase A: 1.5% Acetic Acid + 1.5% Ammonium Acetate In DI Water

·       Mobile Phase B: 100% Methanol

·       Gradient: (Flow Rate: 1.0 Ml/Min)

·       Detections – 254 Nm UV (By Using UV/PDA Detectors)

·       Injection – 10/20 µl Of Sample

Calculations ;

·       Calculate Concentration Of Each Preservative In Sample As Follows: Using Peak Areas Or Peak Heights And Concentrations Of Standards, Construct Linear Standard Curve For Each Compound Based On Formula Y = Mx + C, Where ‘X’ Is Concentration (Ppm), Y Is Peak Area Or Height, M Is Slope, And C Is The Intercept. Calculate Recovery Of Fortified Sample And Sample Results.

Reference :

·       Ali, M. Sher. J. Assoc. Off. Anal. Chem., 1985, 68. 488-492.

·       Us Iso 22855:2008 – Fruit And Vegetable Products — Determination Of Benzoic Acid And Sorbic Acid Concentrations — High-Performance Liquid Chromatography Method First Edition 2009-Mm-Dd