Has anyone ever injected their sample material into a system with no column, but an equivalent volume of tubing (if that's possible), and compared area counts to a "column" injection.

If so, can someone explain the results to me. I am curious as to what information is obtained from such a procedure. Is it a valid test of the "assay"? What kind of results should I expect?

We have SEC method (TosoHaas 3000xl / Waters LCM-1) and I am suppose to perform this procedure. I have not heard a good explanation from anyone here as to what they hope to accomplish by doing this test. Any information is greatly appreciated.

tia

irie
otny
Tony Diem
PDL Inc.
Plymouth, MN
tdiem@pdl.com

This isn't something that I've seen done very often (anyone else want to comment?), but what is should do is to confirm that you're getting 100% recovery of your analyte. The important thing is have enought mixing volume in the system that you don't go outside the linear range of your detector (mostly about 0.5 AU or so). If that's the case (and if flow is the same in both experiments!) then you should get the same area both times. If you get a smaller area in the chromatogram, that indicates that some of your sample was lost.

This is *a* valid test of the assay, but is not, in and of itself, *the* valid test of the assay (i.e., this only indicates recovery, not necessarily accuracy, precision, specificiy, etc.)

-- Tom Jupille / LC Resources Inc.

We occasionally use this technique (removing the column) to see if we have gross losses of compound on the column. For example, we recently suspected we weren't getting all of an oligonucleotide off the column, so we injected directly to the detector and found a signal about twice as large, so this helped us track down on-column loss. Because there are so many variables involved, we take the results with a grain of salt (+/- 10-20%).

Comparing areas is going to be very difficult. From HP's "Applications of narrow-bore columns in HPLC" monograph, p17,1991.

"Although the peak height depends only on the maximum instantaneous concentration of the component, the peak area depends on both the instantaneous concentration and the velocity at which the component band moves through the flow cell."

This means that the peaks with and without the column must be the same shape. That will not be easy to do because most of the dispersion takes place in the column, which has been removed.

Just changing gradients significantly will cause significant peak area differences, in my experience. Yes, interpret results carefully.

Good advice about intepreting results carefully! However, so long as the flow rate is the same in both systems, the band *will* be moving through the flow cell at the same same velocity. If the linear range of the detector is not exceeded, this means that the area of the peak will represent the product of concentration (proportional to the y-axis) and volume (proportional to the x-axis). This should be true regardless of peak shape.

There are a lot of "so long as . . ." statements in here; in practice, YMMV.

-- Tom Jupille / LC Resources Inc.