getting the column ready for next injection in gradient HPLC

[mahsary]

hello everyone.
I'm working on "Hydrocortisone acetate" impurity test using the USP35 method which is based on gradient method. and this is the timing for mobile phases:

0-5 min: 90-10%
5-25: 10-90
25-30: 10-90
30-35: 90-10
35-40: 90-10

the only difference between my method and USP is that I'm using a L1 column with 25cm length but USP method is based on using a 15cm column. and i have increased the flow rate from 1 to 1.66 but still the sample gets stuck in the column. and when i wash it with methanol and water i get very large peaks which i know is sum of a few injection that i have done before.
i was thinking may be i don't get the column ready at the first place. and what should i do between my injections? should i get a base line just with 90-10 % mobile phase or what?? plz help
sorry my english is not that good

[mattmullaney]

Hello.

Your English seems good enough to me. I don't know USP 35 at all, but I'll offer that if your column is 4.6 mm x 250 mm and assuming a porosity factor of 0.68, the column-volume is then about 2.8 mL. A general "rule of thumb" that has served me well during the run (even if it's wasteful at first) is to flush the column at high percent organic (say such as your 10:90 A/B at 30 min in your gradient program) for seven column volumes. At a flow rate of 1.66 mL/min, this would take about 12 minutes instead of the five you currently are trying. If you were to try this change, the end of the 10:90 A/B would be at 42 minutes, then I'd use a second "rule of thumb" and at, say, 42.1 minutes, flip the gradient back to 90:10 A/B, allow that to run for 10 column-volumes (at flow of 1.66 mL/min, this would take about 17 minutes), and you should then be fine for the next injection, I'd guess. Total sample run time would be an hour...this is the price of using long columns, though.

You could then perhaps cut back on the length of the high organic flush step and/or the re-equilibration step a bit at a time to shorten things a bit. As I say, this is a conservative approach that is "wasteful" in the early method development that may be cleaned up during the course of development...the waste in solvent and time in my experience is generally compensated for by a continuous opportunity for problem-free series of injections. There are always exceptions, but for me these have been rare.

Please, see what you think...others may have their own experiences to share as well. Best of Luck!

[HPLCaddict]

hello everyone.
the only difference between my method and USP is that I'm using a L1 column with 25cm length but USP method is based on using a 15cm column.

Which "L1 column"? The one that's been described in the USP or any other of the myriad of L1 stationary phases available?

[Gerhard Kratz]

Problem with "L1" column's is, there are so many on the market, from sub-2µm particles up to 10µm (or more?), endcapped and non-endcapped materials, with polar embedded groups, or not, and so on, and so on.
That makes USP methods so challenging!
L1: Octadecyl silane bonded to porous silica or ceramic microparticles, 3 to 10µm in diameter.

Please try to find out more about similar applications to make the right selection of particle size and column dimensions.
Gradient runs often need a long time to get back to the initial gradient conditions. This conditioning step, after a run, you should carry out very carefully.
I would recommend to reduce column length at first. To increase flow rate not always will help.
What is the composition of your buffer and organic modifier?

[Consumer Products Guy]

That makes USP methods so challenging!

Wow. That was VERY "diplomatic" !

I'd make the post-run/equilibration time long, then try to cut back shorter, and add in some leeway, to find the real equilibration time. Remember that it is really column volumes, not time in minutes, that is important, and that guidelines for number of column volumes are just guidelines. You need to determine what works, and what your timeframe is. If throughput is not an issue, allow for extra equilibration time.

[mattmullaney]

Absolutely agree with Gerhard and ConsumerProductsGuy...and also much with HPLCaddict. always giving more details is much better than less details for the troubleshooting!!

Always in (early) MD flush out well with reasonably high %Organic and allow plenty of "column-volumes" for both this high %Organic flush as well as re-equilibration of the column to its original eluent composition (90A:10B in your case). This flushing may be decreased the more you become familiar with any given separation.

When we're talking about "column-volumes" are we/I clear to you in our meaning?--I mean absolutely no offense, but if an explanation would help (due to language differences) I could take some time to explain this idea if that would help you out.

Good Luck!