how important of capacity factor?

[flora0975]

I developped a very simple and consistant method, the retention time of the interest came up at ~ 2 min. The void rention time is ~1.6min. Per the k' < 2 rule, is this method acceptable? the peak shape is very nice and signal is strong.

How to increase the k'? The current mobile phase is

A: B = 90%:10% , isocratic
A: 0.1% H3PO4 in water
B: 0.1% H3PO4 in ACN.
1min/mL

Thanks.

[lcrandall]

Hi
It depends on the goal of your method. Can you give us more information about the purpose/stage of development?

Can you tweak your isocratic ratio slightly to acheive a bit later retention time?

[flora0975]

the method is to analysis the interests concentration, no require to report impurities.

This is early stage of method development. The compound is polar and has high solubility in water (50mg/mL) . I am using C18 column.

I started from 55:45 = 0.1% H3PO4 in water: 0.1% H3PO4 in ACN, isocratic. The peak came up ~1.5 min.

As I decresed the organic%, the peak showed up later. Now I tried 10% organic, the peak came up ~2min.

I like to keep the method as simple as possible, any suggestion based on current setting? thank you!

Hi
It depends on the goal of your method. Can you give us more information about the purpose/stage of development?

Can you tweak your isocratic ratio slightly to acheive a bit later retention time?

[bisnettrj2]

With your retention time you have a k' of 0.25. Depending on what you are eventually going to do with this method you should shoot for at least a k' of 2, or a retention time of about 4.8 minutes. Try 95% H2O, see what you get. You could also try a different reversed phase column, maybe a phenyl or cyano, or a C18 with an embedded polar linkage. Or, switch to HILIC and run 90% ACN : 10% H2O, and see what your k' is then. Just make sure your injection solvent is mostly acetonitrile if you try HILIC.

[DSP007]

Retention factor is just one of the mathematical characteristics of the "sorbent-mobile phase, rather (not) than religious dogma. In exclusion chromatography it ever be less than 1.
If your materials are separated in a satisfactory degree, and additional effects are not expected - you can use this method.

That any advice to increase the retention time, not knowing the composition of the mixture and the target substance - "I wash my hands" (c , Matthew )

[tom jupille]

The US FDA "suggests" that k' should be >2 (as part of system suitability). While that's not a requirement, it *is* good advice for several reasons:
- other things being equal, resolution is proportional to k'/(1+k'). That may not seem to be a problem if you only have one peak, but
- a low k' value implies low chemical interaction with the stationary phase. That compromises specificity
- the act of making an injection upsets the "equilibrium" of the system. It takes a bit of time to "decay" back to it's steady state. During that period, the system is somewhat chaotic and quantitation can suffer.

k'=1 means that your compound elutes 1 column volume after the void. My personal feeling is that is the minimum you would want to see.

Note that, as DSP007 pointed out, the above does not apply to SEC separations, in which all compounds elute at t0 (but different size molecules have different t0 values!).

[lmh]

I appreciate that you've said you don't need to look for impurities, but you have to look at it this way: you have chosen to do chromatography. The whole point of chromatography is to separate things. If the things you wish to separate from your peak of interest elute exclusively after it (rare situation!), then it doesn't matter if the peak is early. If the things you wish to separate from your peak might elute before it, then a very low k' value gives very little space for them to elute without contaminating your peak. If there is nothing in the mix to separate, then why do chromatography anyway?

Also, just psychologically, if you decide to ignore things like FDA suggestions, even if you ignore them for good reasons, you'll have an uphill struggle if you ever need to justify your method to someone else.

[flora0975]

thanks for the suggestions.

I have tried HILIC can get nice peak at 50% organic isocratic, but still ~2 min RT.

I don't want the method to be complex. Can I slow the flow rate to get a decent RT? I know the k' will not be changed.

[bisnettrj2]

If you're trying HILIC, start at 90-95% acetonitrile, then work toward 50% organic. What column are you using for HILIC, and what is your sample diluent?

[flora0975]

I started with 95% ACN, no peak.
Then using 50% ACN isocratic , the peak showed up at ~2 min. I found sample diluents no difference between ACN and 50% ACN. I also tried gradient, from 95% --50% water, the peak is very urgly.

the column I used is Zorbax NH2, 250x4.6

Thanks.

If you're trying HILIC, start at 90-95% acetonitrile, then work toward 50% organic. What column are you using for HILIC, and what is your sample diluent?

[bisnettrj2]

So, no elution at 95%, zero retention at 50%. Did/can you try isocratic at 90% and 75% ACN? Can you post a chromatogram of the 'ugly' gradient peak? Also, what compound are you analyzing?