· Qualitative Method (Thin-Layer Chromatographic Detection Of Acesulfame Saccharin And Cyclamate):

Apparatus:

· UV Lamp (360 Nm);

· Ion-Exchange Resin: Amberlite LA-2.

Reagents:

· Polyamide

· 2,7-Dichlorof Luorosein

· Bromine

· Formic Acid

· Ammonia 5%

· Xylol

· Propanol

· Methanol

Procedure:

· Extract The Sweetener From Acidified Food Product With Water Or Take Acidified Aqueous Extract And Pass Through The Ion-Exchanger And Wash With Water. Elute The Sweeteners With Dilute Ammonia Solution. Evaporate The Ammonical Solution Under Vacuum To Dryness And Take Up The Residue In 1 Ml Of 50% Methanol (Alternatively Extract These Sweeteners From Acidified Sample, Ph 0.6, With Ethyl Acetate And Use Concentrated Ethyl Acetate For TLC).

· Apply 2-10 µl Of Sample Solution Along With Standards On TLC Plates Coated With Polyamide. Develop The Plate To About 15 Cm Height With A Developing Solvent Consisting Of Xylol: N- Propanol: Formic Acid (5:5:1). Dry The Plates In A Current Of Air And Spry With 0.2% Solution Of Dichlorofluoresein And After Being Dried, Examine Under UV Light. To Identify The Spots In Day Light, Place The Plate In Chamber Containing Bromine And Then Expose To Ammonia Vapour. Spots Appear On A Reddish Background.

· Quantitative Method: (Analysis Of Acesulfame By High Pressure Liquid Chromatography)

Apparatus

· Beaker, Pipette, Flasks,

· HPLC Instrument Of Any Suitable Make With A UV Detector At 227 Nm, Column; Lichrosorb-RP 18 (10 µm).

Reagents

· Mobile Phase: Methanol : Water ( 10 : 90) : Adjust This Mixture To 01M Using Tetrabutylammonium Sulphate,

· Standard Solution Of Acesulfame: 1 Mg/Ml In Distilled Water.

Operating Conditions:

· Pressure: 160 Bar

· Flow Rate: 40 Ml/Hr

· Temperature (Ambient)

· Sample Volume: 10-20 µl

Preparation Of Sample:

· Liquid Samples Such As Juices: Filter Through 0.45 µm Filter (Millipore Inc.) And Inject 10-20 µl.

· Solid Samples: Stir 10 Gm Of Sample Vigorously With 100 Ml Distilled Water For 30 Minutes And Centrifuge. Pass An Aliquot Of This Solution Through 0.45 µm Filter, Discard The First Few Drops Of Filtrate And Collect The Filtrate And

Procedure:

· Inject Standard Solution Ranging From 5-20 µl And Record The Peaks.

· Calculate The Peak Area And Draw A Calibration Graph Using µg Of Substance Vs Peak Area. Inject Samples Solution Ranging From 10-20 µl And Record The Peak Area For Sample. Calculate The Acesulfame Content Of The Sample From Its Peak Area And The Calibration Graph.

· Determination Of Acesulphame – K, Aspartame And Saccharin By High Performance Liquid Chromatography

· Principle :Extraction Of Sample With Water Or Eluent, If Necessary, Clarification On Solid Phase Extraction Column Or With Carrez Reagent, Chromatography At An HPLC Reversed Phase Column And Spectrophotometrically Determination At A Wavelength Of 220 Nm

Reagents

· Acetonitrile For HPLC

· Methanol For HPLC

· POT. Dihydrogen Phosphate

· Dipotassium Hydrogen Phosphate

· Tetra Butyl Ammonium Hydrogen Sulphate

· Phosphoric Acid 85% (W/W)

· Phosphoric Acid 5% (W/W). Carefully Pipette 6 Ml Of Phosphoric Acid At (6) Above Into A 100 Ml Volumetric Flask Which Already Contains 80 Ml Water. Dilute To Mark With Water.

· Hydrochloric Acid 25 % (W/W)

· Carrez Solution 1 – Dissolve 15 Gm Potassium Hexacynoferrate ( K4[Fe (CN)6] – 3 H2O) In Water And Dilute To 100 Ml

· Carrez Solution 2 – Dissolve 30 Gm Zinc Sulphate (Znso4 7H2O) In Water And Dilute To 100 Ml

· Phosphate Buffer Solution II – ( KH2PO4 0125 Mol / Litre, Ph = 3.5. Dissolve 1.70 Gm Potassium Dihydrogen Phosphate In 800 Ml Of Water In A 1000 Ml Beaker. Adjust To Ph 3.5 With Phosphoric Acid. Transfer The Solutions To A 1000 Ml Vol. Flask And Dilute To Mark With Water.

· Phosphate Buffer Solution III – Ph 5 Dissolve 5.46 Gm Of Potassium Dihydrogen Phosphate In 500 Ml Water In A 1000 Ml Beaker. Adjust To Ph 6.5 With Dry Dipotassium Hydrogen Phosphate. Add To The Solution 3.4 Gm Of Tetra Butyl Ammonium Hydrogen Sulphate And Stir To Dissolve. Adjust The Ph To 6.5 By Addition Of More Dipotassium Hydrogen Phosphate. Add 250 Ml Of Methanol And Adjust The Ph To 4.0 By Drop Wise Addition Of Hcl (8).Transfer This Solution Into A 1000 Volumetric Flask And Dilute To The Mark With Water.

· Mobile Phase – Phosphate Buffer And Either Acetnitrile Or Methanol. Filter The Phosphate Buffer Used For The Mobile Phase And Either Acetonitrile Or Methanol Separately Through Suitable Membrane Filters, Of Pore Size 45 µm And De Gas For 5 Minutes In An Ultrasonic Bath. Add Carefully Measured The Required Amounts Of Phosphate Buffer And Acetonitrile As Given In A,5 And Mix. Prepare The Mobile Phase Freshly On The Day Of Use

· Control Solution – Containing Acesulphame – K ,Sodium Saccharin And Aspartame (And Optionally Diketopiperazine, Aspartylphenyl Alanine, Phenylalanine,Caffeine, Benzoic Acid, Theobromine, Hydroxyl Methyl Furfural,And Vanillin)

· Standard Solution 1 – Pipette 10 Ml Of The Stock Solution (15) Into A 100 Ml Vol. Flask And Dilute To Mark With Water.

· Standard Solution 2 – Pipette 5 Ml Of The Stock Solution Into A 100 Ml Vol Flask And Dilute To Mark With Water.

· Standard Solution 3 – Pipette 1 Ml Of The Stock Solution Into A 100 Ml Vol Flask And Dilute To Mark With Water.

Apparatus

· Analytical Balance

· High Speed Blender Or Homogenizer

· Volumetric Flasks – 100, 250, 500 And 1000 Ml

· Beaker 1000 Ml

· Pipettes – 1, 5, 10 , 20, 25, 100 Ml

· Micropipette 1000 Ul

· Graduated Cylinder – 1000 Ml

· Funnel

· Fluted Filter Papers, Medium Fast Qualitative

· Water Bath

· Ultrasonic Bath

· Centrifuge

· Degassing System

· Membrane Filters – Pore Size 45 Um Or Smaller With Filter Holders And Suitable Syringe.

· Solid Phase Extraction Column

· High Performance Liquid Chromatograph – Equipped With UV Detector (Capable Of Operating At A Wavelength Of 220 Nm, Preferably A Diode Array Detector) And Equipped With Recorder Or Integrator Which Allows Measurement Of Peak Heights And Peak Areas)

· Column , Reverse Phase – A RP C 18 Stationary Phase Of 5 Um, A Length Of 250mm, Internal Dia 4 Mm, A Guard Column, RP C 18 ( Optional But Strongly Recommended For All Solid Sample

Procedure

· Clear Liquid Products (Lemonades, Cola, Beverages) Dilute 20 Ml Of The Liquid In A 100 Ml Volumetric Flask With Water. Filter The Solution Through A Membrane Filter Of Pore Size 0.2 Um Before Injection

· Cloudy Liquid Samples ( Juices , Flavoured Milk Drimks) Dilute 20 Ml Sample With 50 Ml Water In A 100 Ml Volumetric Flask. Add 2 Ml Carrez Solution 1 , Mix And 2 Ml Of Carrez Solution 2 , Dilute To Mark With Water And Filter Through A Fluted Filter Pass The Filterate Through A Membrane Filter Of Pore Size0.45um Before Injection.

· To Make Allowance For The Volume Of Any Precipitate, If The Fat Free Insoluble Matter In The Initial Sample Mass Exceeds Approx 3 Gm, It Is Advisable To Centrifuge The Clarified Solution For 10 Minutes Before Filtering It Quantitatively Into A 100 Ml Volumetric Flask. Wash The Settled Matter Twice With Water And Centrifuge Again, Collect Each Of The Supernatant In The 100 Ml Vol Flask And Then Dilute The Solution To Mark With Water. Before Filtering It Quantitatively Into 100 Ml Vol Flask. Wash With Water And Centrifuge Again As In Case Of Cloudy Liquid Samples

· Jams, Preserves, Marmalade And Related Products. Weigh To The Nearest 1 Mg, 20 Gm Of Homogenized Sample In A 100 Ml Vol. Flask, Add About 60 Ml Water And Place The Flask In An Ultrasonic Bath At 40⁰C For 20 Minutes. The Temperature Should Not Exceed 40⁰ C Since Aspartame Can Get Degraded. Cool To Room Temp. Add 2 Ml Carrez Solution 1 , Mix Followed By 2 Ml Carrez Solution 2 . Shake Vigorously And Allow To Stand For 10 Minutes. Dilute To Mark With Water. Filter The Solution Through A Fluted Filter Paper. Pass The Filterate Through A Membrane Filter Of Pore Size 45 Um Before Injection. To Make Allowance For Any Precipitate, If The Fat Free Insoluble Matter In The Initial Mass Exceeds 3 Gm, It Is Advisable To Centrifuge The Clarified Sample Solution For 10 Minutes At1400 R.P.M

· Semisolid And Solid Products – Weigh 10 – 20 Gm Of Thoroughly Homogenized Sample In A 100 Ml Vol Flask. Add About 50 Ml Water And Place The Vol Flask In An Ultra Sonic Bath At 40 0 C For 20 Minutes. Cool To Room Temperature, Add 2 Ml Carrez Solution 1 , Mix And Add 2 Ml Of Carrez Solution 2, Dilute To Mark With Water And Filter Through A Fluted Filter Paper. In Case Of Very Complex Matrices, Additional Purification Using The Solid Phase Extraction Column May Be Necessary To Protect The Separating Column, Since Colouring, Flavouring And Fat Can Not Be Separated By Carrez Solution. In This Case Add 2 Ml Of Clarified Filterate To The Cartridge, Previously Activated With 3 Ml Of Methanol And 20 Ml Water And Elute With About 20 Ml Of Mobile Phase. Pass The Filtrate Through A Membrane Filter Of Pore Size 0.45 Um Before Injection. To Make Allowance For The Volume Of Any Precipitate Follow Procedure Mentioned Above.

· Custard Powder Weigh 10 Gm Sample In A 500 Volumetric Flask. Add About 400 Ml Of Water And Proceed As Described Above. Add 6 Ml Of Carrez Solution 1 And 2 For Clarification

Identification

· Identify The Intense Sweeteners By Comparing The Retention Times Of The Analyte Concerned In The Sample Solution With That Of The Standard Substance Or By Simultaneous Injection Of The Standard Solution And The Sample Solution.

Determination

· Integrate The Peak Areas Or Determine The Peak Heights And Compare The Results With The Corresponding Values For The Standard Substance With The Nearest Peak Area / Height Or Use A Calibration Graph. Check The Linearity Of The Calibration Graph.

Chromatographic Conditions

· Type – Reversed Phase (RP) Stationary Phase And Column Lengths – Spherical Particles Of 3 Um, For Column Lengths Of 100mm, Upto10 Um For Column Lengths Of 300 Mm Internal Diameter – 4.0 Mm Guard Column –Recommended (Optional) – Bondapak C 18 Or Partisil ODS3, Or Superspher60 RP Select B

· Flow Rate – 0.8 Ml /Min To 1 Ml / Min Injection Volume – 10 µl Upto 20 µl Detection – Photometrical (UV) At A Wavelength Of 217 Nm For Aspartame

Calculation

· Calculate The Mass Fraction W Expressed In Mg / Kg Or Mass Concentration P In Mg / Litre Of The Intense Sweetener As Under

· Liquid Chromatographic Determination Of Caffeine, Benzoate And Saccharin In Soda Beverages:

Principle

· Saccharin, Benzoate And Caffeine Are Simultaneously Quantified In Soda Beverages By Liquid Chromatography On µ-Bondapack-C18 Column Using Acetic Acid (20%) Buffered To Ph 3.0 With Saturated Sodium Acetate And Modified By Adding 0.2% Isopropanol. The Concentration Of The Additives In The Sample Is Determined By Measuring The Peak Heights Using A UV Absorbance Detector Monitored At 254 Nm.

Apparatus

· Beaker, Pipette, Flasks, A Liquid Chromatograph Equipped With A Solvent Delivery System And Injector Or Equivalent, UV Detector At 254 Nm, Chart Recorder/Integrator And U-Bondapak C18 Column 300 X 4 (I.D) Mm, Flow Rate 2 Ml/Min. Detector Sensitivity Adjustable From 0.02– 0.05 AUFS.

Reagents

· Mobile Phase: 20% Acetic Acid (V/V) Buffered To Ph 3.0 With Saturated Sodium Acetate Solution Modify With 0-2% Isopropanol To Obtain Base Line Resolution And Retention Times Of Standards From Mixed Standard Solution In Approximately 10 De-Gas Prior To Use.

· Standard Solutions: Prepare Individual Standard Solutions From Standard Compound To Get Following Concentrations-Sodium Saccharin: 5 Mg/Ml, Caffeine: 0.05 Mg/Ml And Sodium Benzoate: 0.5 Mg/Ml. Use These Solutions To Determine Sensitivity For Detector Response And Retention Times Of Individual Standards.

· Mixed Standard Solution: Prepare Solution Containing 5 Mg/Ml Sodium, Saccharin 0.05 Mg/ Ml Caffeine And 0.5 Mg/Ml Of Sodium Benzoate. Use This Solution To Optimise LC Conditions For Complete Resolution And To Quantify.

Preparation Of Sample:

· Carbonated Beverages: Decarbonate By Agitation Or Ultrasonic If Free Of Particulate Matter, Inject Directly.

· Beverages Containing Particulate Matter: Filter Through Millipore Filter (0.45 µm) Discarding First Few Ml Filtrate. If Large Amount Of Particulate Matter Is Present, Centrifuge Prior To Inject Filtered Solution Directly.

Determination

· Inject Known Volume (10 µl) Of Mixed Standard Solution In Duplicate. Peak Heights Should Agree Within ± 2.5%. Inject Known Volume Of Prepared Sample In Duplicate. Measure Peak Heights Of Standards And Sample Components.

HPLC Method For The Determination Of Caffeine, Benzoate And Saccharin.

· This Test Method Can Be Used For The Determination Of Preservatives And Sweeteners I.E. Benzoic Acid, Caffeine, Aspartame, Acesulfame-K & Sorbate In Soft Drinks By HPLC.

Equipment And Reagents

Equipment

· HPLC With UV Detector

· Calibrated Micro Pipettes- 20 To 200µl And 100 To 1000µl Capacity Ranges

· Ultrasonic Bath

Glass Ware

· 200µl Glass Inserts

· 50 Ml Glass Beakers

· Syringe Filters 22µ Size

· HPLC Vials

· 10 Ml Calibrated Volumetric Flasks

· 5ml Poly Propylene Ria Vials

Chemicals

· Ethanol, HPLC Grade

· Pure Water

· Ammonium Phosphate Mono Basic, GR Grade

· Methanol, HPLC Grade

Reference Standards

· All The Standards Used Have Purity Of ≥ 96%. The Details Of Estimated Analyte Are Given Below

Sl.No

Name Of Standard

Acesulfame

Aspartame

Benzoicacid

Caffeine

Sorbic Acid