Linear Dichroism (LD) measures the difference in UV photo-absorption between horizontally and vertically polarized light of an aligned sample. Alignment can be accomplished by several methods. Some chemical compounds may be aligned in a stretched sheet of polyethylene, but most samples in solution are aligned via a flow method. A Couette flow cell, where the sample is between an outer rotating capillary and an inner stationary rod, both made of UV grade quartz, is well suited for alignment of biological samples. Long molecules, like e.g. DNA are readily aligned in the Couette flow and ligand binding may be followed as the ligand is aligned with the DNA upon binding.
What are the sample requirements?
- A Couette flow cell needs 60-70 uL of sample per load.
- The concentration of the sample should be adjusted to an absorbance of about 1 at 190 nm for the typical pathlength of the Couette flow cell of 0.5 mm. Highly absorbing buffers (e.g. those containing Cl- ions) should be avoided.
What other specific considerations are relevant?
- When a sample is loaded into the cell, first a base line is acquired without rotation after which the LD of the sample is measured at a typical rotation speed of 3000 rpm.
Partners offering this technique
MOSBRI reference partner site for this technique: