Proteomics @ Liverpool
An internal website for the Protein Function Group and Collaborators
An important part of maintaining reproducible and reliable chromatography, and to maintain a good spray at the emitter, is a high level of care in buffer preparation. Ways to compromise the chromatography include: NOT following these instructions carefully, using bottles other than those set aside for the nanoAcquity systems, using anything other than ultrapure reagents and ignoring the changeover frequency

We have decided to change all nanoAquity buffers weekly. This is intended to be a shared responsibility, and there must be no sense of deliberately avoiding Monday morning sessions to avoid the buffer change. In fact, you could argue that being the first person to use the system after a buffer change ensures the highest chance of success.

Lastly, it is essential that the buffer changes be logged, in a single forum entry per instrument, with a one line update

Step 1: Solutions

Buffer A
HPLC water, 0.1% formic acid
200 ml HPLC grade water
Add 200 µl formic acid
(prepare in cylinder, mix on transfer to Duran)

Buffer B (also ‘strong wash’)
HPLC acetonitrile, 0.1% formic acid
200 ml HPLC grade acetonitrile
Add 200 µl formic acid

Weak wash
0.1% trifluoroacetic acid

500 ml HPLC grade water
Add 0.5 ml trifluoroacetic acid

Seal wash
10% acetonitrile

450ml HPLC grade water
Add 50 ml acetonitrile

MDA formic acid]
MDA trifluoroacetic acid]
MDA acetonitrile]

[PDF version]

Created: Amy Claydon, 16/04/2011
Revisions: Amy, 21/04/2011 (changes to volumes, and to procedures)

Step 4: Change and purge wash solutions

Throw away old buffer and completely replace with new.

Sample manager
Prime syringes
Sample & wash syringe

Run for 5 cycles

Step 5: Log the buffer change on the group forum

"Buffer change by Xxxx on xx/xx/xxxx. All pressures OK."

Step 2: Change and purge Buffer A/B

Sonicate buffers for 10 min to de-gas

Change Nano-Acquity flow from 0.3 µl/min to 0 µl/min (in 0.1uL/min steps) and wait for pressure to drop

Replace previous buffer bottle with new (keep old buffer in its Duran bottle) and be careful with the frits

Binary solvent manager-
Prime A/B solvents (A1, B1)

Run for 4 min at 4 µl/min (automatically set at 4 ml/min per solvent, so 8 ml/min)

Also need to “Prime syringes” (see Step 4) as Buffer B is also the strong wash)

Step 3: Auto zero flow transducers Buffer A/B

Change nanoAcquity flow to 0.3 µl/min - 50% Buffer A

Binary solvent manager
Auto zero flow transducers

Pump A/start/results

Check previous and current readings are very similar.

Binary solvent manager
Auto zero flow transducers

Pump B/start/results

Check previous and current readings are very similar.

If the readings are not in good agreement, purge buffers again and repeat from Step 2.