Qualitative Methods:

Preparation Of The Test Sample:

·       Non-Alcoholic Beverages: Add 3 Ml Hcl To About 25 Ml Of The Sample In A Separator. If Vanillin Is Present Remove It By Extraction With Several Portions Of Petroleum Ether. Discard Petroleum Ether. Extract With 50, 25 And 25 Ml Portions Of Diethyl Ether + Petroleum Ether (1+1) And Wash Combined Extracts With 5 Ml Water And Remove The Solvent By Evaporation.

·       Semi-Solid Preparations: Transfer 25 Gms Of Sample To 100 Ml Volumetric Flask With Small Amount Of Water And Add Enough Boiling Water To Make About 75 Ml, Let Mixture Stand One Hour Shaking Occasionally. Then Add 3 Ml Acetic Acid, Mix Thoroughly, Add Slight Excess (5 Ml) Of 20% Neutral Lead Acetate Solution, Dilute To Volume, Mix With Cold Water And Let It Stand For 20 Min And Filter. Transfer 50 Ml Filtrate To Separator And Proceed As In (A).

Detection:

·       By Conversion To Salicylic Acid (Applicable In The Absence Of Salicylic Acid): Dissolve Residue In About 10 Ml Hot Water, Add 2 Ml H2SO4 (1+3). Heat To Boiling, Add A Slight Excess Of 5% Kmno4 Solution Dropwise And Partly Cool The Dissolve About 1 Gm Of Naoh In It And Filter Mixture Into Silver Crucible (Silver Crucible \With Lids Are Available). Evaporate To Dryness And Heat For 20 Minutes At 210- 215ºc. Dissolve The Residue In Hot Water, Acidify With HCI And Add A Few Drops Of Neutral Feci3 Solution (0.5%). Violet Colour Indicates Salicylic Acid Which Is Formed From Saccharin. By This Method All So Called False Saccharin And Any Salicylic Acid Naturally Present ( Also Added Salicylic Acid When Not Present In Too Large Amount) Are Destroyed And 5 Mg / Litre Saccharin Is Detected.

·       Phenolsulphuric Acid Test:  To The Residue Obtained After Removing Solvent, Add 5 Ml Of Phenol H2SO4 Reagent (Pure Colourless Crystals Dissolved In Equal Weight Of H2SO4) And Heat For 2 Hrs At 135-140ºc. Dissolve In Small Amount Of Hot Water And Make It Alkaline With 10% Naoh. A Magenta Or Reddish-Purple Colour Develops If Saccharin Is Present.

·       Resorcinol Sulphuric Acid Test: To The Residue Add 5 Drops Of Resorcinol-Sulphuric Acid (1:1) And Heat On A Low Flame Until The Product Turns Red. Dissolve In 10 Ml Of Water And Make It Alkaline Using 10% Naoh Solution And Add Few Drops Of Iodine Solution. A Green Fluorescence Is Developed If Saccharin Is Present.

Quantitative Methods:

General Method For Non-Alcoholic Beverages:

·       Principle: Saccharin Is Extracted From A Known Quantity Of Acidified Sample With Diethyl Ether. The Solvent Is Removed And The Residue Is Digested With Hcl And Made To A Known Volume. An Aliquot Is Treated With Nessler’s Reagent And The Absorbance Of The Coloured Product Is Measured At 425 Nm.

Reagents:

·       Hydrochloric Acid

·       Diethyl Ether

·       Nessler’s Reagent: Dissolve 100 Gms Of Hgi2 And 70 Gms Of KI In Small Amount Of Water, Add This Solution Slowly, With Constant Stirring To The Cooled Solution Of 160 Gms Of Naoh, In 500 Ml Water And Dilute To 1

·       Distilled Water (Ammonia Free)

·       Standard Solution: Dissolve 0.2921 Gm Of NH4Cl In 1 Litre Of Ammonia Free Distilled Water (Equivalent To 1 Gm Of Saccharin In 1 Litre Water). Dilute To Get 200 µg/Ml Of Saccharin Equivalent.

Procedure

·       Add 2 Ml Of HCI To 50 Gm Of Accurately Weighed Sample In Separator Funnel Nd Mix. Extract With 3 × 50 Ml Of Diethyl Ether. Filter The Ether Extract Through Cotton Into A Clean 250 Ml Conical Flask And Evaporate The Solvent.

·       Add 6 Ml HCI And 5 Ml Ammonia Free Water And Evaporate On A Hot Water Bath To About 1 Ml. Again Add 6 Ml Hcl And 5 Ml Water And Evaporate To 1 Ml. Dilute The Solution To 50 Rnl With Ammonia Free Water. To 2 Ml Of This Solution In A 25 Ml Volumetric Flask Add 1 Ml Of Nessler’s Reagent And Make Up To Volume. Similarly Take 0.5, 1, 2, 3 And 4 Ml Portions Of Standard Solutions (200 µg/Ml) Into 25 Ml Volumetric Flasks And Develop The Colour With Nessler’s Reagent. Read The Absorbance Of The Product At 425 Nm Against Reagent Blank Similarly Prepared. Compute The Saccharin Content Of The Sample From The Calibration Graph.

Phenol -H2SO4 Colorimetric Method:

·       Principle: Saccharin Is Extracted From The Acidified Sample With Chloroform And Benzene And The Solvent Evaporated. The Residue Obtained Is Treated With Phenol H2SO4 And Heated At 175ºc For 2 Hrs. After Making Alkaline With Naoh The Absorbance Is Read A 558 Nm.

Reagents

·       Chcl3

·       Ether

·       Chloroform + Benzene (95+5)

·       Methanol

·       Phenol (Colourless Crystals)

·       H2SO4

·       Saccharin

Preparation Of Sample:

·       Soft Drinks (Carbonated Beverages And Low Calorie Drinks):

·       De-Carbonate The Beverage By Repeated Shaking And Pouring From One Beaker To Another. Transfer 10 Ml Sample To 125 Ml Separatory Funnel With Teflon Stopcock. Add 15 Ml Water And 0.5 Ml 1N Naoh. Extract With 50 Ml Chloroform Benzene Mixture Shaking 1 Min. Let Layers Separate And Discard The Solvent Layer (Benzoic Acid And Benzoates Do Not Interfere).

·       Fruit Juices: Transfer 50 Ml Sample To 100 Ml Volumetric Flask. Add Slight Excess Of 5% Neutral Lead Acetate Solution (<10 Ml). Dilute To Volume With Water, Let It Stand For 1 Hour And Filter. Take Aliquot Of Filtrate Containing 1-3 Mg Saccharin And Proceed As In Determination Of Saccharin Beginning “Add 5 Ml Hcl (1+4) And Extract With Chloroform-Benzene Mixture”.

·       Food Sweetener Tablets And Concentrated Liquids: Grind 10-20 Tablets To Uniform Powder. Accurately Weigh 0.5 Gm Powder Or Measure 10 Ml Liquid Concentrate Sample Into 500 Ml Volumetric Flask And Dilute To Volume With Water. Take 10-15 Ml Aliquot For Analysis. If Liquid Concentrate Contains Parabens As Preservatives, Acidify By Adding 5 Ml Hcl (1+4) To Aliquot And Extract With 20 Ml Ccl 4. Discard Ccl 4 And Proceed As In Determination Beginning, “Extract Aqueous Phase By Shaking 1 Min. Each Time”.

·       Jellies And Preserves : Blend Sample And Weigh 25 Gm Into 50 Ml Beaker. Heat On Water Bath To Make Sample Fluid. Transfer To 250 Ml Volumetric Flask Using 25 Ml Hot Water To Rinse Beaker. Dilute To Volume With Methanol And Mix Thoroughly. Let It Stand For 1 Min And Filter. Transfer An Aliquot Containing 1-3 Mg Saccharin To 50 Ml Beaker. Evaporate To 1/2 Volume On Water Bath To Remove Alcohol And Transfer With About 25 Ml Hot Water To 125 Ml Separatory Funnel. Proceed As In Determination Of Saccharin Beginning, “Add 5 Ml HCI (1+4) And Extract With Chloroform-Benzene Mixture”.

·       Low Calorie High Protein Powder, Granules And Liquids: Grind Granules In Mortar To Uniform Powder. Transfer 10-20 Gm Powder Accurately Weighed Into 250 Ml Volumetric Flask With 150 Ml Hot Water. Mix Thoroughly To Dissolve. Add Slight Excess Of 5% Neutral Pb (OAC)2 Solution (30 Ml). Dilute To Volume With Cold Water. Mix And Let It Stand 1 Hr And Filter  For Liquids Use 50 Gms Sample And Proceed As In Determination Beginning “Add 5 Ml HCI (1+4) And Extract With Ether-Benzene”.

Determination

·       Transfer Aliquot As Specified, Of Prepared Sample And Standard (1-3 Mg Saccharin) Solution To The Separating Funnels. Add 5 Ml Hcl (1+4) And Extract By Shaking For One Min. Each Time With 50, 30 And 20 Ml Chloroform-Benzene Solvent Mixture (95+5) Or With Ether: Benzene (95+5) As Specified In Sample Preparation. Filter The Combined Solvent Extract Through Funnel Fitted With Pledget Of Glass Wool And Containing 10 Gm Anhydrous Na2SO4 Into 100 Ml Volumetric Flask. Dilute To Volume With The Same Solvent Mixture Used Above And Mix. Transfer 20 Ml Aliquot To 50 Ml Erlenmeyer Flask. Evaporate The Solvent To Dryness In Shallow Water Bath And Complete Drying In Oven At 100ºc For 20 Min. Pipette 1 To 5 Ml Hot Melted Phenol To Erlenmeyer Flask And Swirl Until The Residue In Dissolved. Add With Caution 1.2 Ml H2SO4 By Pipette And Swirl. Prepare Blank By Pipetting 2.5 Ml Hot Melted Phenol And 1.5 Ml H2SO4 Into 50 Ml Erlenmeyer Flask.

·       Stopper The Flask With Tight Cap Covered With Aluminium Foil And Heat For 2 Hours At 175ºc In An Oven. Cool And Add Approximately 30 Ml Hot Water To The Flask And Mix. Add 10 Ml 20% Naoh Solution And Mix. Transfer Quantitatively To 100 Ml Volumetric Flask And Dilute To Volume With Water.

·       Read The Absorbance Of The Solution In Spectrophotometer At 558 Nm. Determine The Concentration By Comparing With A Calibration Curve.