Assay Calculation on Anhydrous basis

                   Assay Calculation on Anhydrous basis

 

What happens if my sample solvent is stronger than my mobile phase?

We do not recommend injecting in a stronger solvent because it usually results in peak distortion, broadening, poor sensitivity, and shortening of retention times.

This happens because some analytes will tend to travel too quickly through the column, instead of eluting in a symmetrical band.

If you absolutely must do this, keep the volume as small as possible and make sure the solvents are miscible.

 

 

Ideal injection volume vs column dimension

Optimal injection volumes are directly related to the cylinder volume of your  column and are, therefore, dependent on the cross sectional area (A=π r2) and length (L) of your column.

Therefore you can estimate any adjustment from an existing method for injection volume. If you are converting to a different size ID (with the same packing material and length), just multiply your current volume by the ratio of the radius squared to determine the correct volume for your new method. Injection volumes, as well as optimal flow rates, are limited by the size of the column. Ideally, your sample volumes should be, for the different column id:

Column ID	Volume (µL)
2.1 mm (30 -100 mm length)	1-3
3.0-3.2 mm (50-150 mm length)	2-12
4.6 mm (50-250 mm length)	8-40
10 mm (50-250 mm length)	40-100
21.2 mm (50-250 mm length)	150-300
30 mm (50-250 mm length)	300-700
50 mm (50-250 mm length)	1000-2000

Larger volumes may be acceptable if peaks are still symmetrical. Earlier eluting peaks will exhibit broadening first if the volume is getting too large.

If the solvent in your sample is stronger than the mobile phase (starting ratio if using a gradient), you will need to use a smaller volume.

Likewise, if your sample solvent is weaker than the mobile phase, you may be able to use a larger volume.