·       Caramel Samples Produced By Ammonia And Ammonium Sulphite Processes Are Found To Contain A Process Contaminant Namely 4-Methyl Imidazole. The Compound Has Been Subjected To Toxicological Evaluation By Bureau Of Indian Standards.

Determination Of 4-Methyl Imidazole In Caramel Samples:

·       Principle: Caramel Colour Is Added To A Basic Celite Column And Eluted With A Mixture Of Chloroform And Alcohol. The Eluant Is Extracted With Dilute H2SO4 And The Aqueous Extract Is Made To A Known Volume. Taking Aliquot Of The Extract, The Colour Is Developed With Diazotised Slulphanilic Acid In Alkaline Medium. The Amount Of 4- Methyl Imidazole Present Is Calculated From The Standard Curve.

·       Apparatus: Volumetric Flasks, 100 Ml Capacity And 25 Ml Capacity; Separatory Funnel, 250 Ml Capacity; Beaker, 250, 100 Ml Capacity; Conical Flask, 250 Ml Capacity; Pipettes, 2 Ml And 5 Ml Capacity; Spectrophotometer Chromatographic Column With Stop Cock, 25.0×2.5 Cm.

Reagents

·       Naoh, 2N

·       Na2CO3, 20% M/V And 5% M/V

·       Elution Solvent: Chcl3: Alcohol

·       H2SO4: 05/0.1N

·       Chromogenic Reagent:

·       Dissolve 1 Gm Of Sulphanilic Acid In 9 Ml Of Hcl And Make Upto A Known Volume And Cool To 4 ºc.

·       Prepare By Dissolving 1 Mg Of Nano2 In 100 Ml Water And Cool To 4ºc. Prepare Freshly By Mixing 25 Ml Of (A) And 25 Ml Of (B).

·       Celite

Procedure

·       Prepare A Basic Column Packing Material By Mixing Well, Celite 545 + 2N Naoh In The Proportion Of 2 Ml Of 2N Naoh To 3 Gm Of Celite. Place Fine Glass Wool Plug At The Base Of The Chromatographic Column Followed By 5 Gm Of Basic Column Packing. Tag The Packing Firmly To A Uniform Mass. Take 10 Gm Of The Caramel Sample In A 100 Ml Beaker And Add 6 Gm Of 20% Na2CO3 Solution And Pack It On The Prepared Column. Place A Plug Of Glass Wool At The Top Of The Column. Elute With Elution Mix, Until 150 Ml Of Eluate Is Collected Into A 250 Ml Beaker. Transfer The Eluate To A 250 Ml Separatory Funnel And Extract With Three 20ml Portions Of 0.05N H2SO4 {This Extract Should Be Strongly Acidic (Ph 3), Test This By Test Paper}. Concentrate The Combined Aqueous Extract Using Flash Evaporator At A Temperature Below 50ºc. Make Up The Residue To Known Volume.

Preparation Of Standard Curve:

·       Into A Series Of 25 Ml Volumetric Flasks Containing 0.0, 1.0, 2.0, 3.0 And 5.0 Of Working Standard Solution, Add 1.0 Ml Each Of Diazotized Sulphanilic Acid (Chromogenic Reagent) And 2.0 Ml Of Na2CO3 (5%) Solution. Make Up To Volume And Read The Absorbance At 505 Nm And Plot The Standard Graph. Take 2.0 To 5.0 Ml Solution Of The Aqueous Extract And Develop The Colour. Read The Absorbance Of The Sample Solution And Calculate The Quantity Of 4- Methyl Imidazole Present In The Sample From The Standard Curve.