Shimadzu PDA SPD-M20A baseline drift

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Hi,

I'm using a Shimadzu SPD-M20A Diode Array Detector. Scan range 215-400 nm. Mainly focused on 270 and 325 nm for compounds of interest. Mobile phase H20 with 0.1% formic acid and MeOH with 0.1% formic acid gradient increase % MeOH throughout run.

For some reason the baseline keeps drifting down. I see no reason why this should be happening. There are no leaks. Cell temperature is same as column temperature (50 C). Not sure what settings to check here. Not using reference wavelength because I don't see why I should have to for the wavelengths of interest. Even if increasing MeOH is the problem the baseline should shift up not down.

Any ideas?
Hi, Justin_ea,

My idea is this, mixing the water and MeOH (both with 0.1% (v/v) formic acid) will result in a volume contraction...the formic acid concentration is changing enough to effect a change in refractive index. Lowering the formic acid content in the MeOH should help with the baseline drift. This phenomenon also is prevalent in water/ACN gradients w/ added TFA.
MattM
Thanks for advice although I do not think that is the issue. When water and methanol mix they create heat so it shouldn't contract doesn't it expand? With water and acetonitrile yea then its the other way around it cools so it would contract. Either way column oven and flow cell are heated to same temperature (50 C). Another HPLC we use almost same method (just different column) type and baseline drifts up as expected as MeOH concentration increases.

I wonder if its the mixer we have. The mixer has 3 volume options 2.6, 1.7, and 0.5mL. Its configured to 2.6 mL which seems high to me. Column particle size is 2.7 micron tubing is like 0.1mm I believe throughout.
Hi Justin_ea,

With regard to the phenomenon of volume contraction upon mixing methanol and water, I respectfully disagree:

http://www.chromatographyonline.com/how ... -degassing

http://laserchrom.com/LaserchromHPLC-HP ... erties.htm

among other references. If you choose, you can easily verify this in the lab by mixing volumes of methanol and water. Changing the temperature of the methanol-water mobile phase is not relevant. The concentration of the formic acid is not likely to be 0.1% (v/v) during the gradient course.

I'll ask these questions. Are these two HPLC instruments both cleaned and maintained in exactly the same way? Is the comparison between the two HPLCs' behavior apt?

In any event, the larger the volume of the mixer, in general, the lesser the noise, for example: (see page 10)

https://mottcorp.com/sites/default/file ... aper_0.pdf

I'm not certain that mixer volume has any impact on baseline drift during execution of gradient chromatography. In any case, the larger the mixer volume, the better the mixing of the mobile phase components.

That TFA concentration affects baseline drift during gradient chromatography is well-known:

http://www.chromatographyonline.com/gra ... &sk=&date=

You're acquiring PDA data below 220 nm. As Dolan notes in the article above; "At wavelengths <220 nm, however, baseline drift caused by differential solvent absorbance can be sufficient to prevent practical use of certain solvents, such as methanol or tetrahydrofuran."

Please, see what you think, and thank you!
MattM
Hello Matt,

Thanks for informed reply. Yes of course you are right when you mix MeOH and H2O volume goes down. Sorry I was a bit confused.

Anyway there are enough differences between method on other HPLC and the HPLC in question. Different instrument model, column, gradient, etc. So its not really comparable.

Either way I've never used an instrument before where baseline is decreasing as MeOH increases when both the aqueous and organic mobile phase contain 0.1% formic acid during gradient run.

Since there is no real reason to have UV spectrum go down to 215nm I'll try increasing it and seeing if that works.

If the formic acid in the MeOH is causing problem how low do you recommend I should try to go to notice a difference? 0.1% is used just because its sufficient to reduce peak tailing for later eluting acidic compounds.
Hi Justin_ea,

In the case of TFA, the TFA content in the organic solvent is lessened, say, to ca. 0.75% (v/v) while the gradient composition is maintained.

I think this may be worth a try...that said, Dolan's article didn't seem to suggest much outside of acquiring data at longer wavelengths. I concur with your idea--keeping above 220 nm may help a bit according to Synder's book. That makes good sense to me.

The old-school solution was to add sodium nitrate to both the water and organic solvents to increase the RI values of both. The nitrate generally will not interfere with the separation. This said it may not be worth the bother. This link:

http://www.chromacademy.com/chromatogra ... Drift.html

covers the use of absorbance matching. You'll need to sign up for a free membership to CHROMacademy to see the entire article.
MattM
Hi Justin_ea,

I am hoping that the experiment went well! I wish for you the best.
MattM
The baseline drifts down even if I am running isocratic.

Increasing wavelength didn't make a difference.
Hi Justin_ea,

Interesting...sorry to hear that you're still experiencing odd behavior. Okay, the isocratic eluent, please, what was its composition (MeOH/water w/some formic acid)? Perhaps an eluent prep w/o the formic acid would be a good idea, one less variable.

Also, did you try to run the isocratic method without a column in place--using a union connector? This way, the column is singled out as a potential problem source.

The second thing to try would be to take a look at the flow cell windows if the flow cell is easily removable--perhaps they need a little cleaning.

The third thing to do is to check the pressure trace against an injection of a blank--say the mobile phase itself.

Please see what you think and thank you!

P.S.: Almost forgot, how is the eluent being degassed prior to use?
MattM
Hi Matt,

So we recently had the instrument serviced and performed a IQ/OQ and all these parameters like baseline stability passed. That was all done with restrictor column. Its only since I've been using the instrument with a column and doing method development that this behavior started to appear. The column is new and is composed of a phase called "Bonus RP" its from Agilent. Its an alkyl amide type phase. This is the major difference between methods I've done in the past. Normally I used C18 columns but have been looking for phases with better resolution of some analylates. Regardless I don't see why this column type would matter too much.

I flowed 50/50% MeOH / H2O through the system (no mixing, isocratic single pump). I ran baseline check under these conditions and the baseline still drifts down. Oven temp and pda cell temp were 40 C so I don't think temperature is the issue.

Yes solvents degassed in sonicator prior to use and all pumps hooked to degassers.

Pressure stable. No leaks no other clues really.

I wonder if any PDA settings are wrong or mistaken? Or zeroing not working. Or something leaking off column ( again I doubt this..).
Hi Justin_ea,

I'm very familiar with Bonus-RP, used it numerous times with no ill effects, good phase. The phase shouldn't matter, agreed, particularly if the column is new out of the box.

Degassing is appropriate, check.

Yet a pre-mixed 50:50 (v/v) MeOH/water eluent in isocratic mode from one solvent line still affords a sloping baseline.

To me, this indicates dirty UV cell windows and/or air bubbles in the UV cell. I'm familiar with Agilent/Waters cell cleaning/LC flushing w/o a column in place, do you have an idea of these protocols for the Shimadzu? This sounds like the next thing to look at. Next would be flushing the instrument as a whole.

Feel free to type in PDA settings...I don't think that's the trouble...of course, the reference wavelength is disabled, yes?
MattM
Hmm. Well I might have figured out the problem. I let system equilibrate for a long time (> 30 min) and the baseline stabilized and passed test.

The column ovens in shimadzu equipment are very large relative to column ovens I'm used to from other vendors. Maybe I've just been too impatient with equillibration? We'll see. I'll post any conclusions.

Reference wavelength is disabled (don't want to use that function during method development).

I doubt flow cell is dirty due to the IQ/OQ all passing (will try to clean as last resort). I can try flushing system for air bubbles (we did have a pump run dry....recently) without column as well.
Sounds good...I'll hope for the best when you run in earnest.
MattM
Interesting development. So this system has 3 pumps (A, B, C).

A = aqueous
B = organic
C = rinsing (50 : 50, water : organic)

I ran each pump isocratic to and performed baseline check. A and C passed. B failed. Pump B seems to have larger pulsation of pressure.

This pump was serviced recently as well and passed all criteria with a restrictor column.

But I did notice the rinsing kit isn't working. I'm not sure why these pumps have or need a rinsing kit (is it for seals? heat?). Its just water. There is a valve broken in the rinsing kit.

This is the part:

hromforum.org/viewforum.php?f=1&sid=0042c7b1c65ed3a95c8d30f25fe2a4b5
Hi Justin_ea,

Link from your last note isn't working on this end...which of the Prominence pumps are you using, LC-20AD or LC-20AT? The Prominence I procured a few jobs ago was for Ion Chromatography (LC-20Ai).

Your system (LC-20AD?) has high-pressure mixing, presumably...and it does seem that the Rinsing Kit is designed to keep the pump seals for each line free of salts. Seems a bit unusual to have 100% water in the Rinsing Kit (should there be some MeOH to keep mold from growing? Wow, I guess not according to the manual. Interesting.).

So A and C lines are working well (flat baseline) with 50:50 MeOH/water, the B line is Not Working Well (large pulsation...and Rinsing Kit on the fritz)?

Interesting also that IQ/OQ "passed" with such trouble evident with the B line afterwards...no further comment on that. I'll note, though, that OQ/PV may not reveal a "dirty UV cell", though maybe this time that isn't the culprit.

Anyhow, pump pulsations can also cause baseline wander...you may have found the "smoking gun" at last.
MattM
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