Waters UV Detector

[qrys]

We recently purchased a HPLC system from waters and Im a beginner student that has been working with this HPLC. I stumbled the following problem. I inject a protein sample through the HPLC with and without column, same concentration, same injection volume (5 ul of 5.5 mg/ml lysozyme in pure water). I then obtained a peak for both settings (ie. for injections with and without column), I integrated the peaks, the area for sample injected without column is always lower than that injected through a column. Can anyone explain me what is happening here? I can understand is the condition is the reversed. Thank you.

[danko]

I'm almost 100% sure that the peak obtained with the zero column setup is very narrow and very high - so high that it reaches 2000000 uAU and there it stops. What I'm trying to say is: You're probably reaching the absorbance scale limit, when injecting without the column on.

Best Regards

[tom jupille]

Danko suggested one possibility. Another (admittedly less probable) is that the flow rate might be somewhat higher with no column to generate back pressure (liquids are somewhat compressible). Since peak area is the product of absorbance and residence time in the flow cell, a higher flow rate = smaller area.

[DR]

Or, since the peak would elute at the void volume, there may be a negative baseline disturbance co-eluting w/ the peak, diminishing its area somewhat.

[qrys]

My goal of injecting sample without column is to quantify recovery of protein through the column. The detector is set to 2 AUFS, and yes, the height of the peak go above 2 (i.e 2.4), but I can still see the complete peak (not like something in the case of column overloading that you dont see the top of the peak). The flow rate of mobile phase is 1 ml/min. At this condition, the integrated area of the peak is ~5.0E6. The area for protein through the column is ~5.6E6.

As Danko mentioned it is above the detector limit, I then carried out injections without column with reduced flow rate, so that I can get the peak to max below 2, and I needed to run at 0.1 ml/min to obtain peak height below 2. The area under the peak in this case is ~5.4E7, I divided it with 10 (due to 0.1 ml/min flow rate) and I get 5.4E6, which is close to the peak area with column (ie. ~5.6E6).

Do you think this is acceptable? Or it is better to adjust the AUFS?

Thank you!

[danko]

I have another (better?) suggestion: Go back to the normal flow (1 ml/min) and inject the half of the volume you're injecting currently. Or just prepare a less concentrated solution (f. ex. the current concentartion divided by 2).

Best Regards