Determination of void volume

1. 38. Connect the outlet of the pump to the column via an injection valve having a 500 μl capacity loop (Figure 22.4).

39. Allow one bed volume to run through the column at the flow rate used for column packing.

40. Meanwhile, prepare a 1 mg/ml ‘blue dextran 2000' solution in the buffer and filter it through 0.22 μm filter.

41. Bring the injector valve into the load mode.



Figure 22.4: Injector valve (A); the diagram showing tube connectivities in load (panel B) and inject (panel C) mode

2. 42. Inject blue dextran solution (≤2% of the bed volume) in the injection loop and change the injection valve into the injection mode.
3. Take out the injection syringe.

43. Start collecting 1 ml fractions until the blue dextran is completely eluted. The migration of blue dextran can easily be tracked within the column. Stop collecting fractions once the blue coloured dye is completely eluted.

Results and analysis:

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

45. Set the instrument to absorbance mode and wavelength to 620 nm.

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

47. Measure absorbance of all the fractions (washing of cuvette in-between is not required for any of the fractions).

48. Record the absorbance in an observation table (Table 22.2)



Table 22.2: Table for recording absorbance of collected fractions



49. Plot A₆₂₀ against elution volume to obtain the chromatogram of blue dextran 2000. A typical chromatogram is shown in figure 22.2.



Figure 22.5: A chromatogram illustrating the void volume of a gel filtration column

50. From the chromatogram, it can be deduced that the void volume of the column is V₀ .