Experiment-20 Microscale Spectrophotometric Measurement of Iron in Foods by Standard Addition BY SIBY FRANCIS

Reagents 2.0M HCl: 15mL/student Hydroquinone: 4 mL/student Trisodium citrate dehydrate:6g/student O-phenanthroline: 4mL/student St.Fe (40 µg Fe/mL):4 mL/student 6M HCl for cleaning purpose

Procedure WEAR YOUR SAFETY GOGGLES.

Procedure Fill the clean porcelain crucible with 6M HCl and allow to stay overnight. Rinse the crucible with distilled water and dry it in the oven. Dry the crucible until the weight agrees to 0.001g. Get the unknown from the stockroom and add 5-6 g of food particles in the crucible.

Procedure Cont… Weigh the crucible again to get the exact mass of the food particles. Heat the crucible for 3 hours. First, at low flame to dry the food then increase the flame temp to char the sample. After charring,use the hottest flame possible flame to ignite the black solid. If the sample bursts into fire use the lid to smother the flames.

Procedure Cont… Burn until we get white ash. Cool the crucible to room temp and add 10mL 2.0M HCl to the sample. Swirl the crucible gently Filter the mixture through a small filter and collect the solution in a small vial. Weigh 0.71g of trisodium citrate to four 10-mL volumetric flasks. Using the 2mL Vol. pipet add 2.00mL of the ash solution to each of the flasks.

Procedure Cont.. Add 4mL of distilled water and swirl to dissolve the citrate. (pH 3.6) Using micropipet add 0.20 mL of hydroquinone solution and 0.30 mL of phenanthroline solution to each flask. Label the vol. Flask 0-3 and add 0.250mL, 0.500mL,0.750mL St.Fe to flask 1-3 respectively. The flask marked Zero have 0µg/mL of St. Fe.

Procedure (Blank prep) Blank is prepared by mixing 0.71 g of trisodium citrate dehydrate,2.00 mL of 2.0M HCl, 0.20 ml of hydroquinone solution, 0.30 mL of phenanthroline solution, and diluting to 10mL.

Using a spectrophotometer. Measure the absorbance of each solution at 512nm in a 1-cm cell with dist. water in the reference. With the help of a pasteur pipet wash the cuvet and start measuring from the blank solution in the order of increasing concentration. Wash the cuvet with dist.water and rinse it with the solution to be used before measuring any absorbance.

O-Phenanthroline Trisodium citrate Hydroquinone WHY WE ADD?? O-Phenanthroline Trisodium citrate Hydroquinone

O-Phenanthroline o-Phenanthroline is a colorimetric reagent for iron detection. It is also used as a reagent for the solvent extraction of anions and iron chelator.

O-Phenanthroline Fe 2+ ions are colorless because it has molar absorptivity of zero for visible light. Thus, to determine the concentration of Fe2+ ions in solution using a visible wavelength of light, one must react the ions quantitatively with something that will form colored species. In this experiment Fe 2+ ions (Lewis acids) react with o-phenanthroline molecules, C12H8N2 , (Lewis bases) to form orange-red [Fe(o-Phen)3]2+ ions

Trisodium citrate There is another Lewis acid in the solution -- the H+ ion. These ions compete with Fe 2+ ions for the o-phenanthroline molecules. Citrate ions, C6H5O73-, are added to react with the hydrogen ions to form citric acid molecules and, thus, to remove them from competition.

Hydroquinone Another complication is that Fe2+ ions are easily oxidized to Fe3+ ions with air. Hydroquinone, HOC6H4OH, is added to reduce any Fe3+ back to Fe2+ forming quinone, OC6H4O.