by
penxx » Sat Jun 19, 2021 6:08 pm
TylerSmith123 wrote:
Hi Penxx,
I was actually in your exact shoes about a year ago, with the exact same system and software! Thankfully, through online resources like this one, my knowledge and love for the instrument has grown parabolically.
When it comes to column conditioning, I would recommend a few things. At the start of the day, or whenever you plan on starting the instrument, you should wash your columns and lines with specific method files that can both clean and equilibrate your column. On my instrument, this is all compiled into a single method (binary pumps) that can run through a wash sequence and, depending on the column and flowrate, execute an equilibration for (my preference) ten column volumes. If it is asking you to inject a sample, this setting can actually be turned off or you could just manually click start on the window.
There is also a setting (for example if you run your HPLC every day, for the same amount of time) called start-up (as well as shut-down), that ensures your machine is started (which you can link a method to) before you even enter the lab (requires the instrument to be on). I'm a little confused regarding the latter half of your question however. Are you executing all of these from an autosampler? If so, simply keep a "wash" vial that has amenable/miscible liquids that can serve as a placeholder for the wash run in your first autosampler vial port.
As for your method/gradient, that seems like a fine way to write it out. Albeit, a little redundant if you already stated that your mobile phase is buffered. Typically, when writing our gradient profiles, A is assumedly your aqueous phase, while B is your organic. However, as long as it's easy to read and well understood, I'm sure your way of writing it will not cause any issues.
As for column flushing, I would wash the column for as long, if not a little longer, than you would for equillibrating the column at the start of the day. Essentially, you want to ensure that whatever liquid that is on your column is exactly what you need for analysis (or for this case, shut-down). So a flush of roughly ten column volumes should leave the column clean-enough and buffer free. Remember to flush any/all lines that use the buffer, including the column and all the detector! If not, you have a chance for your salts to precipitate onto the column which will lower it's lifespan over time, and this effect could happen realistically anywhere along the way if the right conditions are met. This includes having a high% organic portion of a gradient or wash sequence and you should always make sure that your buffer works at all of your gradient's concentrations. For example (and I don't expect this to be the case currently), when your method dips to a lower buffer percentage (~33% at 13min), you should assure that the salts are still soluble in the ACN/buffer mixture.
I hope some of this helped you!
Tyler
Hi Tyler,
You have no idea how touched I am at seeing your reply to my post T_T Thank you so much for your valuable inputs!!
I would like to clarify on a few more points:
If I were to incorporate a wash sequence at the end of the sample run and executed an "auto shut-down" of system after this and I come back the next day to start on another run (using the same analysis method), I have to purge the system first before starting right? My understanding from a colleague was that purging has to be done at the start of every analysis...
Regarding the method written in my previous post, I wanted to check if it is the correct way to write the time program for my method if I want to command the system to use only mobiles B and C through the whole analysis? Since other users in my lab mostly use water as their mobile phase A, I want to save them the trouble of switching the phases if i were to replace that with my buffer (i prefer to dedicate a flow line for buffer MP
) However, I do see that by default, users would assign A as aq solvent and B as organic solvent so in the time program, the command would state only the B conc.
Well noted on your point on the flushing! After flushing, I will store my column in 100% ACN (as recommended in manual) if not in use for several days or longer. So for my next use, will I have to condition my column first before starting to equilibrate with my mobile phases (buffer/ACN)? If so, do I condition with the same set of gradient ratios used in my analysis but without buffer (i.e. replace buffer with water) since there is risk of buffer precipitating out if I deliver it through the column (that contains 100% ACN) straightaway?
Thank you very much once again for your advices! God bless you!