Chromatography Forum

Hello all,

I have an important question to the community regarding HPLC-MS-MS quantitative analysis using a C18 column. (2.1x100x3micron)

Upon running a sample at 0.5mL/min in 10:90 AQ:ORG with the column in place I Observed a retention time of ~0.5min

While maintaining the flow rate at 0.5mL/min in a 50:50 AQ:ORG with the column in place I observed a new retention time of ~1.7min

The compound is definitely retained, but slightly with lower affinity due to higher organic content. The question is , does a problem arrise if too short a retention time is used, or is this method using the 1.7min RT acceptable?

I am a bit worried due to the small retention, but the calibration curves are consistent and the quantitation is consistent batch to batch.

The elution is isocratic, and an appropriate equilibration and run time is used in every sample.

What is an acceptable short retention time?


Thanks for the help!

I'm contend if k' is about or larger > 2, ie the retention time is about twice the void time. In your examaple the void time should be less tahtn than 1 min, is it so?

Thanks for the reply, is this column void volume or instrument void?

I believe instrument void is 0.2 and column void using the 10:90 was about 0.5min so with the 0:100 should be even less.

What is the easiest way to measure the column void?

Is this a correct calculation
COLUMN DIMENSIONS (I.D. x L(mm)) VOID VOLUME (ml)
2.1 x 100 0.24

Also, when calculating the retention time, we do take into account the instrument void time + the column void time?

Thanks so much

Void time is (pi*r^2*L*0.65)/flow. I add in the 0.65 factor to account for porosity of the column packing. I tend to add in my loop volume if the loop is in-line with the the column, especially at low flow rates and large loop volumes, in addition to any significant tubing volumes between the pumps and the column (except for tubing and loops I leave out the porosity factor, obviously). For your column, without knowing anything of your system, your column void time is approximately 0.45 minutes. So, your injection at 10:90 A:B is probably at t0. The problem with using chromatography at t0 in LCMS is potential ionization suppression from unretained components in your samples that may interfere with quantitation. If you're using a labeled analogue of your analyte as an internal standard, you should be able to compensate for any suppression issues.

So, for your retention time of 1.7 minutes, your k is (1.7-0.45)/0.45 = 2.8, which is marginally acceptable in the normal chromatography world. I would probably want a retention time somewhere near three to five minutes (k=5.7-10), but that's just me.

Thanks for the reply, I found your response most suitable. Having a matrix effects ion suppression study in a method validation is critical to account for any ion enhancement or suppression from nonretained compounds eluting early.

It seems I will stick with my current assay, since I am using a structural analog as an IS and am having consistent quantitative and instrumental response batch to batch but in the future will definitely keep that into account.

I would appreciate any other comments on the topic from any other users.

Kind Regards

I'm thinking that with the specificity of the HPLC-MS-MS detector that you really don't have to obtain that good of separation...

True, but you don't want a method with potential "built-in" problems, and depending on the type of sample being run, you may experience ionization suppression in the early part of a run if you have non-target components in your sample that are unretained, or poorly retained (like the chromatography that shemesh199 is running).

Although I have not performed an ion suppression study, since the column void is around 0.5mL, would most of these unretained components in the sample matrix come out near the void, for example from 0.5-1.0 min, and then at 1.7 min analyte of interest is able to elute?

I think 0.5 minutes is, as has already been said, far too close to the void volume. This means your compound is not being sufficiently being retained to separate it from any other compound in your matrix that is not retained (such as salts) and you are setting yourself up for matrix effect depending on your sample make-up.
If your peak shape is acceptable with the 1.7 minute retention time then this is the way to go, if not at least somewhere between a minute and 1.7 minutes. You might want to give a 65% organic isocratic mobilephase a try. Also note that at at 0.5 mL/min and a 100 mm x 2.1 column you probably have VERY high back pressure. I'd drop down to 0.3 mL/min or so based on my past experience, unless you are running on a high pressure pump (UHPLC). at 90% organic the back pressure will be substantially less (especially fif using acetonitrile).

Thanks guys I really appreciate the input.