Chromo - Case study

Whats the scenario?

purify a target protein from a cell mixture. You know the protein has an Isoelectric Point (pI) of 9.0.

what are the steps

1. Evaluation of Method

2. Experimental Procedure (The 5 Stages)

Info for step one

Evaluation of Method

• Choice: Ion Exchange Chromatography is suitable because proteins are charged molecules.

• Phase Selection: You must decide between Anion or Cation exchange.

  • Strategy: You choose a buffer with pH 7.0.

    Application: Since pH (7.0) < pI (9.0), the protein will gain protons and become positively charged (Cation).

  • Conclusion: You must use a Cation Exchanger (which has negatively charged beads) to bind your target protein.

info for step 2

Experimental Procedure (The 5 Stages)

1. Equilibration: Run the pH 7.0 buffer through the cation exchange column to prepare it.

2. Sample Application: Load the mixture. Your target protein (+) binds to the negative beads. Impurities that are negative (anions) or neutral will pass through the column and be discarded.

3. Elution: To recover your protein, you must break the bond.

   • Method: You switch to a mobile phase with High Salt (ions compete for the beads) or High pH (if you raise pH > 9.0, the protein becomes negative and repels the beads). This releases the protein.

4. Collection: Collect the liquid fraction corresponding to the peak on the recorder.

5. Regeneration: Wash the column to remove any remaining stickies so it can be used again.

What case studies can you use for GC?

• Forensic Case Study:

  • Scenario: A fire investigation.

  • Target: Analysis of arson accelerants (volatile organics) or explosives.

  • Why GC? The accelerants are volatile and can be vaporized without decomposing.

• Clinical Case Study:

  • Scenario: Testing a driver or patient.

  • Target: Blood alcohols or drugs.

  • Why GC? Ethanol is highly volatile, making GC the perfect method for precise quantification.

• Environmental Case Study:

  • Scenario: Water quality testing.

  • Target: Trihalomethanes in drinking water or pesticides in soil.

  • Detector: You would likely mention using an ECD (Electron Capture Detector) here, as it is specific for chlorinated compounds like pesticides

Case stufy for HPLC?

• The Scenario: Analysis of a mixture containing 7 different flavonoids (plant compounds often found in citrus).

• The Data: The chromatogram shows distinct peaks for:

  1. Taxifolin

  2. Rutin

  3. Naringin

  4. Hesperidin

  5. Quercitin

  6. Naringenin

  7. Kaempferol

• Application to Exam: You can use this to explain Selectivity and Resolution.

  • Observation: Notice how "Taxifolin" comes out first (shortest retention time) and "Kaempferol" comes out last.

  • Explanation: In a Reverse Phase column (the standard for this), Taxifolin is the most polar (travels fastest), while Kaempferol is the most non-polar (sticks to the column longest)