Jul 01, 2024
BAF_S01_DIONEX Ultimate 3000 HPLC
- 1University of Virginia Biomolecular Analysis Facility Core
Protocol Citation: Nicholas Sherman 2024. BAF_S01_DIONEX Ultimate 3000 HPLC. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvok98xl4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 12, 2024
Last Modified: July 01, 2024
Protocol Integer ID: 101688
Abstract
Description of HPLC operation and a standard step-by-step for reversed-phase chromatography of peptide mixtures on a 5 um C-18 column 300A, 150 x 2 mm.
Materials
ACN - Fisher chemical A955-4, Acetonitrile, optima LC/MS
Water - Fisher Chemical, W6-4, Optima LC/MS
TFA - Pierce‱, Trifluoroacetic Acid, part number: 28903.
MeOH - Fisher Chemical, Methanol, Optima‱ LC/MS Grade, part number: A456-4.
Pipette tips - Fisher Brand, yellow, part number: 02-681-151
Glass Amber vials - Thermo, 6PK1655
Before start
Buffers:
A - 0.1% TFA H20
B - 0.1% TFA 95% ACN
C (and syringe) - 10% MeOH
Sonicate the buffers for 20 min.
*These are all prepared in the bottles provided that sit on top of the equipment.
Start up the HPLC:
Start up the HPLC:
Prepare 10% methanol for seal wash and injector wash
Check solvents, prepare new if needed and see that lines are not likely to come out of solutions
Check that the waste bottle is not full.
Check if all components are turned on
Open the CHROMELEON 7.0 application. Check if all components are connected.
Once the application opens, click on the PUMP tab
This tab will display the module status, information on the left and right pumps and the pump pressure
If pump seal wash does not run, turn it on under commands
After the pump seal wash has finished, open the cover of the loading pump and twist open the purge valve
Once the purge valve is open, turn on the motor and purge by clicking the empty box to the left of the words. This box will then turn green showing that it is indeed on
Purge solvents. Allowing a long purge may reduce the period of pressure fluctuations.
For autosampler, prime syringe, and wash syringe.
Once the purge finished, close purge valves.
Start a low flow rate with 5% B, at 0.100 ml/min, and let it run for 10 min. Increase the flow rate by 0.50 ml/min until reaching 0.250 mL/min. Let isocratic flow running for 20 min.
TIPS:
When lines from solvent bottles are dry, it helps to use a syringe with a fine tip to suck liquid through.
Attaching a syringe with a tapered tip to the end of the waste line works to suck air bubbles through the lines.
If pumps have sat for long in acetonitrile, check valves may be sticky. Storing pumps in propanol may help, but pressures are high. A pump purge is probably needed. Washing out propanol takes a long time.
Preparing samples:
Preparing samples:
Prepare a new amber glass vial of a fresh blank containing 1.5 mL of 0.1% TFA and put it in the first spot on the autosampler rack you will be loading samples.
Dilute the peptide mixture down into 0.1% TFA to a max of 30 ug of peptide mixture or 3 ug/peptide/injection. Centrifuge sample at top speed for 5 min. Collect supernatant to a new amber glass vial.
Running samples
Running samples
Make sure the module is connected. this is the section under MODULE STATUS on the PUMP page
Turn on the UV - it will take 10-20 min to warmup.
Prepare your samples by diluting them to the correct dilution and putting them in labeled amber vials.
Load blank and samples into the autosampler rack on a known order.
In the software you will need to create a sequence to run all samples and blanks automatically.
Creating a sequence run
Creating a sequence run
In the main window, click CREATE then SEQUENCE
In the pop-up, click UVA_U3000, then click NEXT
In this new pop-up, make sure the injection volume is set to 100 uL and that the start position is correct. click NEXT
In our example, the start position is RA1 (Red row A spot 1) for blank but this could be green or blue also. depending on which rack you load
Make sure the correct method is loaded, click NEXT
Make sure AUTOMATICALLY is selected, click NEXT
Make sure the GENERAL SEQUENCE SETTINGS match what you need and then click FINISH
A small pop-up window will then appear asking the project name. If adding to an existing project, make sure to select that correct folder.If creating a new project, type in the appropriate project name in OBJECT and click SAVE
The DATA window will then open for your project. This is where you will create your sequence
In the first sample spot, name it BLANK 1 and change the TYPE from the drop down arrow to BLANK and make sure the position is correct: RA1
Create a second sample line, name this appropriately for your first sample (sample 1), and change the TYPE from the drop-down arrow to UNKNOWN. Make sure the position is correct here as well, in our example, sample 01 is on RA2 position.
Continue creating your sequence in the 'blank, sample, blank, sample...' pattern until all of your samples are added. Make sure all of your positions are correct (RA1, RA2, RA1, RA3, RA1..) as well as sample type is correct as well (blank = blank, sample = unknown).
You will always start with a blank AND end with a blank. This will prevent carry over among samples.
When your sequence is complete, click START at the top of the window
Allow the sequence to run to completion. The HPLC will automatically stop when sequence is complete.
Saving data
Saving data
Double click the first section on the sequence, UV_VIS_1, this will enlarge the chromatography specific to that sample and open the DATA PROCESSING page
When DATA PROCESSING is open, click REPORT DESIGNER in the bottom left corner
In REPORT DESIGNER, you can customize your chromatogram as much as needed: axis limits, peaks labels, retention time, peaks limits and so on.
Once you get your preferred design, click the green LIZARD head in the top left corner and then click EXPORT
This will open a new, small, pop-up window. in this window make sure CURRENT INJECTION is selected under the APPLY TO section
Make sure the parent folder matches your lab/where you need the data saved to and make sure export format = PDF
Finally, click SAVE and repeat for all remaining samples and blanks