Results and analysis:

1. 1. Switch ON the UV/Visible spectrophotometer and allow it 30 minutes warm up.

2. Set the instrument to absorbance mode and wavelength to 280 nm (Note 3).

3. Zero the reading taking first fraction as a blank.

4. Measure the absorbance of all the fractions (do not wash the cuvette in-between the readings).

5. Record the absorbance in an observation table (Table 24.2).

Table 24.2: Table for recording absorbance of collected fractions



2. 6. Plot the absorbance values against the fraction number to obtain the chromatogram.

7. The peak corresponding to hen egg white lysozyme can be identified by checking the mass of the collected fractions using mass spectrometry (Note 2).

Notes:

1. 1. The molecules in ion exchange chromatography are also eluted by varying the pH of the buffer. This, however, may not work well for proteins as variation in pH may denature the proteins.
   
   2.  Ion exchange chromatography may not be able to provide 100% pure protein from a complex mixture of proteins. The proteins having same or very similar isoelectric points may not get resolved, thereby eluting in the same fraction. The fraction containing hen egg white lysozyme therefore may have other proteins of very similar isoelectric points.