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Separation and Analysis of a Mixture Using liquid Chromatography

by Ernie Nicol

PURPOSE: To isolate and compare dyes from two brands of grape soda.

MATERIALS:

  • 3 ml syringe
  • 50 ml distilled water
  • SEP-PAK column
  • 50 ml 30% methanol
  • 6 small test tubes
  • 50 ml 100 % methanol
  • 3 100-ml beakers
  • Spectrophotometer
  • Various brands of grape soda and / or Kool Aid

PROCEDURE
  1. PREPARATION OF THE SEP-PAK (wetting the bed)
      You will use a small syringe as a pump to pass various liquids through the SEP- PAK. When filling the syringe always remove the SEP-PAK. Draw the proper amount of liquid into the syringe and then reattach by inserting the syringe into the shorter of the two tubes extending from the SEP-PAK.
    • Pump 2ml of 100% methanol through the SEP-PAK.
    • Pump 10 ml of distilled water through the SEP-PAK.

  2. ADDING THE SAMPLE
    • Pump 2 ml of grape soda into the SEP-PAK and collect the clear liquid in a small test tube.

  3. ELUTION OF THE DYES
      The dyes that are stuck to the SEP-PAK can now be removed using the proper solvent.

    • Pump 3 ml of 30% methanol through the SEP-PAK and collect the red dye in a small test tube. If some red dye still remains on the column use additional 30% methanol until it has all been removed.

    • Pump 3 ml of 100 % methanol through the SEP-PAK and collect the blue dye in a small test tube. If some blue dye remains flush with 100% methanol until clean. The SEP-PAK can be reused.

  4. SPECTROPHOTOMETRIC ANALYSIS
      If you wish to compare dyes from different brands of soda. Place sample in a spectrophotometer. Record and graph % transmittance at wavelengths from 400 to 760 millimicrons using 20 millimicron intervals.


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