TITLE: BAF_S01_DIONEX Ultimate 3000 HPLC
AUTHORS: Nicholas Sherman

[DESCRIPTION]
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.


[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. 

[STEPS]
SECTION: Start up the HPLC:
9. 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
SECTION: Start up the HPLC:
10. Purge solvents. Allowing a long purge may reduce the period of pressure fluctuations.
SECTION: Start up the HPLC:
6. Once the application opens, click on the PUMP tab
SECTION: Start up the HPLC:
6.1. This tab will display the module status, information on the left and right pumps and the pump pressure
SECTION: Start up the HPLC:
8. After the pump seal wash has finished, open the cover of the loading pump and twist open the purge valve
SECTION: Start up the HPLC:
13. 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.
SECTION: Start up the HPLC:
12. Once the purge finished, close purge valves.

SECTION: Running samples
17. Make sure the module is connected. this is the section under MODULE STATUS on the PUMP page
SECTION: Running samples
18. Turn on the UV - it will take 10-20 min to warmup.
SECTION: Running samples
19. Prepare your samples by diluting them to the correct dilution and putting them in labeled amber vials. 
SECTION: Running samples
20. Load blank and samples into the autosampler rack on a known order.
SECTION: Running samples
21. In the software you will need to create a sequence to run all samples and blanks automatically.
SECTION: Creating a sequence run
22. In the main window, click CREATE then SEQUENCE
SECTION: Creating a sequence run
23. In the pop-up, click UVA_U3000, then click NEXT 
SECTION: Creating a sequence run
24. In this new pop-up, make sure the injection volume is set to 100 uL and that the start position is correct. click NEXT
SECTION: Creating a sequence run
24.1. 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
SECTION: Creating a sequence run
25. Make sure the correct method is loaded, click NEXT
SECTION: Creating a sequence run
26. Make sure AUTOMATICALLY is selected, click NEXT 
SECTION: Creating a sequence run
27. Make sure the GENERAL SEQUENCE SETTINGS match what you need and then click FINISH
SECTION: Creating a sequence run
28. 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 
SECTION: Creating a sequence run
29. The DATA window will then open for your project. This is where you will create your sequence 
SECTION: Creating a sequence run
30. 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
SECTION: Creating a sequence run
31. 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.
SECTION: Creating a sequence run
32. 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). 
SECTION: Creating a sequence run
32.1. You will always start with a blank AND end with a blank. This will prevent carry over among samples.
SECTION: Creating a sequence run
33. When your sequence is complete, click START at the top of the window 
SECTION: Creating a sequence run
34. Allow the sequence to run to completion. The HPLC will automatically stop when sequence is complete.
SECTION: Saving data
35. 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
SECTION: Saving data
36. When DATA PROCESSING is open, click REPORT DESIGNER in the bottom left corner 
SECTION: Saving data
37. In REPORT DESIGNER, you can customize your chromatogram as much as needed: axis limits, peaks labels, retention time, peaks limits and so on.  
SECTION: Saving data
38. Once you get your preferred design, click the green LIZARD head in the top left corner and then click EXPORT 
SECTION: Saving data
39. This will open a new, small, pop-up window. in this window make sure CURRENT INJECTION is selected under the APPLY TO section 
SECTION: Saving data
40. Make sure the parent folder matches your lab/where you need the data saved to and make sure export format = PDF 
SECTION: Saving data
41. Finally, click SAVE and repeat for all remaining samples and blanks 
SECTION: Start up the HPLC:
14. 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.
SECTION: Start up the HPLC:
1. Prepare 10% methanol for seal wash and injector wash
SECTION: Start up the HPLC:
2. Check solvents, prepare new if needed and see that lines are not likely to come out of solutions
SECTION: Start up the HPLC:
3. Check that the waste bottle is not full. 
SECTION: Start up the HPLC:
5. Open the CHROMELEON 7.0 application. Check if all components are connected.
SECTION: Start up the HPLC:
7. If pump seal wash does not run, turn it on under commands
SECTION: Start up the HPLC:
4. Check if all components are turned on
SECTION: Start up the HPLC:
11. For autosampler, prime syringe, and wash syringe.
SECTION: Preparing samples:
15. 
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.
SECTION: Preparing samples:
16. 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.