RP-HPLC Baseline drift

[wayne]

I am running RP-HPLC with 65% Actonitrile in water. I did not use any gradient. The baseline increased stedily for about ten minutes and then suddenly decreased, this cycle repeated. The total trend is increase.

I use A: 100% Actonitrile,B: 100% water.

Somebody can help? Thanks.

[Chris Pohl]

Wayne,

Although you haven't supplied crucial information (such as the absolute magnitude of the drift), a likely cause for this problem is temperature based oscillation. To test whether or not this is a cause, try one of the following:

1). Turn off the heating/air-conditioning in your lab temporarily to see if the oscillation goes away.

2). Temporarily cover any exposed fluid lines with insulation. Styrofoam, polyurethane foam or even a blanket can be used to cover the portion of the instrument exposed to temperature fluctuations. Again, look for reductions in the magnitude of the oscillation.

In either case, improvement is a strong indicator of a temperature induced problem. If so, look to see if your instrument is situated under an air vent in your lab. You might get better results if you move your instrument to a more environmentally isolated location. Otherwise you may need to minimize exposure to the environment through judicious application of insulation, minimization of exposed tubing lengths or consider buying a column oven.

[HW Mueller]

If you are using a UV detector (or similar) and "suddenly decreased" means virtually straight down it could be the air problem I mentioned in the "baseline drift" chain. Dissolved gas can outgas and collect in the flowcell, steadily increasing the bubble ((basline updrift) until it is swept out (baseline drops). The average increase in baseline could mean that your column (or whatever) is on the way to be saturated with gas/air and is not outgassing totally. (We call this a sawtooth baseline here).

[Ghost Writer]

I believe HW is correct on the air problem. Degas and purge.

[wayne]

Thany you all. I will try.

[BenNC]

I'm fighting a similar problem (see post under Baseline Wandering), but I don't have the sharp drop-off. However, when speaking with the servive rep from Waters he noted that the cycle for the inline degasser was about 10 - 13 minutes and resulted in a sharp drop when it came on. Even if you don't use a Waters system, if you use an in-line degasser it may have a similar pattern.

[wayne]

I used Helium for degassing. How long would be the proper time?

I degassed and primed the system again. I still see the sawtooth baseline. Any suggestions on purging the system efficiently?

[HW Mueller]

Almost all sawtooth baselines in this lab where caused by leaks in the plumbing which drew air (Venturi) into the system, rather than bleeding mobile phase. Tightening the connections erased the problem.

[wayne]

I still have the problem.
A chromatograph is shown in this link:
https://netfiles.uiuc.edu/wwang11/hplc.bmp

I ran pure water without column. 1 ml/min. Pump presure was 68-78 psi.

I tighened the conection, washed the detector cell, replaced the water source, degassed the solvent, primed the pump, but I still have the problem. Temperature may not be a issue, because other system works fine.

Any more suggestions?

[Einar Ponten]

Assuming you are using a UV detector I would check the time the lamp has been in use. The drift and the sharp shifts in baseline level indicate an electrical or a lamp problem. A UV lamp may work nice for 2000 hours or more, but the lifetime is normally specified for a shorter period and problems can occur already after 200-500 hours.

If it is a lamp problem it usually persist even if you turn the flow off. Check also the short-term noise, if increased it indicates an old lamp.

An other aspect;
I would suggest that you try to use a pre-made eluent or mix 90% ACN in water with 10% ACN in water. Mixing heat may release air into the mobile phase and mixing two pure solvents require a highly efficient mixer.

[HW Mueller]

ACN-H2O mixing is endothermic.

[Einar Ponten]

Yes, I am sorry. I mixed up with alcohols. Still, mixing pure solvents may cause problems.

[Chris Pohl]

Wayne,

One possibility that occurs to me is that perhaps you have outgassing problems in your flowcell due to inadequate backpressure. How much backpressure do you have on your flowcell (if you don't know this, what the internal diameter and length of tubing do you have on the cell wasteline)? Generally you should have at least 50 pounds per square inch backpressure on your flowcell.

[Kostas Petritis]

Hi Wayne,

From what I saw from your chromatogram, I would probably blame the UV detector. What Chris suggested is a possibility, although I think it is unlike as you should see spiked peaks in your chromatogram with this kind of a problem.

Can you please take another UV and connect it with your HPLC and see if the problem persists? Also, what is the wavelength that you see that problem? Do you have the same problem in all the different wavelengths?

Let us know...

[HW Mueller]

Kostas,
Einar already gave a good suggestion on what to do to prove/disprove it´s the detector : Stop the flow (one can still have a drift, though! But no sudden drop).
Now, as I have mentioned several times, one can get a sawtooth baseline due to gases. This is caused by slow outgassing in the cell. Spikes are usually produced when gas bubbles pass the cell (bubbles are formed ahead of the cell, or maybe?? suddenly in the cell).