Inconsistent or lack of volume in the filter plate may occur for several reasons. Below are some suggestions:

1. Water or another solvent was used for making the extraction solvent instead of Methanol. Perform the following steps:
   1. Transfer the extracts back to the filter plate.
   2. Dry the samples under nitrogen.
   3. Dissolve again using pure methanol (volume according to extraction step in user manual).

2. The pressure manifold was not set up correctly.
   1. Check that the pressure manifold settings are correct according to the instructions.

see document “Instructions using pressure manifolds for 96-well plates with biocrates kits”

1. Put the plate back in the manifold and try again.
2. If the problem persists, increase the low flow pressure up to 30 psi

3. Droplets of liquid remaining on the underside of the filter plate. It is normal if a small amount of liquid is on the underside of the filter. Internal standards will account for small volume discrepancies. Take care that there is no cross contamination into other wells when removing the top filter plate. If large droplets remain on the underside of the filter plate:
   1. Put the plate back into the pressure manifold and run at a higher pressure (30 psi).
   2. While the filter and capture plate are still secured together by tape, tap the plate lightly on the table to shake the droplet loose.

4. It is also possible that some amount of volume was lost during the shaking step or when transporting the plate. You can check the wells with lower volume in the capture plate to see if the remaining volume is still in the top filter plate. If the top filter plate is empty, then it’s likely that the volume was spilled at some point during the preparation. Take extra care during shaking and carrying the plate that nothing is spilled when the wells are uncovered!