Changing between mobile phases

[mezman129]

Hi Guys,

I'm pretty new to HPLC so this might be a very easy question to answer!

We currently use our HPLC system for analysing Thiamine and Nicotinamide, using a 25:75 acetonitrile:water (plus 0.5% sodium phosphate) mobile phase.

We want to start analysing vitamin D2, and the method uses a 99:1 n-hexane:propan-2-ol mobile phase.

Can somebody advise me the best method (and solvents) to use when flushing the system to change between the two methods?

Any help will be greatly appreciated!

[kubowicz.tomasz]

Hello

I'd do as below:

1.Flush out mobile phase/buffer with 25:75 ACN-Water
2.Flush system with 2-propanol (it is the best solvent to flush LC between normal and reverse phases, miscible with water and also with hexane)
3.Flush with your mobile phase - Hexane-2-propanol

Note: don't forget to flush injection system (loop/needle/syringe - depends on LC system you've got)

Regards

Tomasz Kubowicz

[Gerhard Kratz]

And please don't flush your column. Please disconnect the column from the system if it is a reversed phase column.
Good luck. Check also the sealing's if they are compatible with your new mobile phase.

[Fernando]

Hi mezman129

Why don't you use a reverse phase method for vitamin D? You could resolve the three of them.

Only a suggestion.

Best regards

Fernando

[mezman129]

Tomasz/Gerhard

Thank you very much for your very swift replies, really appreciated! That makes perfect sense! I have got a manual injection system so I will remember to flush that too!

When I finish analysing for thiamine and nicotinamide I flush the system and column (syncronis C18) with 60:40 ACN:H2O (to remove the phosphate buffer).

I will remove the syncronis C18 column before flushing the system with 2-propanol.

The column I will be using for the vitamin D2 analysis is a syncronis amino column (stored in 100% ethanol). As ethanol is miscible with n-hexane and 2-propanol, am I correct in thinking that flushing with my mobile phase will equilibrate the column prior to use?

Also, once used, how would use suggest storing the amino column (i.e. which solvents)?

Thanks again guys!

[mezman129]

Hi Fernando,

Unfortunately I am running the two methods for two different customers, each of whom insist we follow their specific method!

:-/

But the customer is always right! Lol!