negative peak used to determine void volume

[alminc]

Hi guys,

In most of the methods I used so far, void volume is determined by injection of NaNO3 solution, and the retention time of NaNO3 (To) is used to calculate k´ of the analyte.


In one method for Omeprazole assay that I started to use recently, a negative peak (at about 1 minute) from injection of dissolving medium (blank) is used to determine k´ of Omeprazole.

I would like to understand what causes that negative peak and why it can be used for determination of To (or void volume Vo)

Mobile phase: 26% acetonitrile, 71% phosphate buffer pH 7.4, 3% of TBAHSO4
Dissolving medium: 20% acetonitrile, 80% phosphate buffer pH 11.0, I =0.01 (that is the blank solution that has the negative peak at RT about 1 minute)
Flow rate: 1 ml/min
Column: Microspher C18, 3um particles, 4,6*100 mm
Detection: UV-vis detector, wavelength 280 nm

What causes that negative peak and why the retention time of that peak can be used as To ?

Almin

[mattmullaney]

Hi Alminc,

I've run more than a few Prilosec samples in my time...nice to see that someone else gets some work that way now!!

Determination of the Void Volume, t0, has been and probably will always be a hotbed of controversy. What does it mean, a "non-retained component"? Sometime that goes through the column, through all of the particles in the column, without interacting with any of them...seems kind of like a "Holy Grail".

In your case--the "negative peak" is likely a bit of air and/or a bit of solvent from the injected sample that has a sufficiently different refractive index that manifests itself in detection oppositie in magnitude than "Real Peaks". Probably as good as anything else you could use as a marker in practice...perhaps less good for rigorous thermodynamic calculations one could do, bringing more theory into it.

Some people use uracil, thiourea, Sodium Nitrate (depending on who's wanting to be "purist," this one gets a rap...too much charge density...gives low estimate of t0 as it may be "excluded" from the pores of the stationary phase...I'm just saying this, I do not say it's "right or wrong"), fructose, acetone, air at the same inj. vol. as the standard or sample...list goes on and on. I think I recall Dorsey at the Florida State Univesity detemining column void volumes using pycnometry...loading columns with Hg metal, weighing the column before and after the filling--lots of ingenious ways to determine t0. Some of the columns in the past 10-15 years for RP are so good at retaining polar substances it's really hard to decide on what to use as a void volume marker...my personal favorite presentation of this is the Waters Atlantis column at the Waters web site.

Ideally...one would want a void marker that would give one the void volume value irrespective of the nature of the stationary phase and/or eluent being used. My personal favorite...depending on my own degree of "anal retentiveness," is borrowing deuterated acetonitrile or methanol from the NMR guy down the hallway (if I am using RI or MS detection) and injecting these, if my organic is ACN or MeOH...works well for these detectors...and comes closest to a substance that would give the same t0 value irrespective of stationary phase (guess the mobile phase, MeOH or ACN, is the trouble here)...though the NMR folks may be a bit unhappy since you've wasted their expensive deuterated solvents!

Enough philosophy from a limited philosopher...Practically, as long as one does the measurement of t0 the same way every time, limits the use of t0 to setting values of k' for analytes and doesn't go any further with it than that, and remembers to say "it's a pseudo-void volume value...just an estimate, honest," when a nit-picker asks, you'll be just fine using that "negative peak" at the beginning of your chromatographic run in your system suitability injection(s). Don't get me started on using mean "t0 values" from the system suitability, or sample injections, or what-not, next.

Have a Good One!