[Analytical2015]

Hello all,
I am running a related substances method on a Waters Aquity Binary UPLC with high sensitivity flow cell, and am having trouble trying to eliminate noise /ghost peaks in the baseline which appear characteristically, when the gradient changes to higher organic. The wavelength values I am working with are 225nm onwards and it’s at 225nm where the problem arises. At higher wavelengths, the peaks are not as noticeable; however, the method requires several impurities to be integrated at 225nm.
I think I have narrowed down the source of the contamination to the water in the aqueous buffer i.e. dissolved TOC residue that builds up at the head of the column,  then elutes with high organic. However, once on the column, they cannot be removed with our wash down procedure.
The reason I say that is because I wash out ALL glassware thoroughly with organic solvent before use, I have sonicated the filters, on all lines (A1, A2, B1, B2, both injector washes, seal washes) but I am open to suggestions as to what it could be.
As for the chromatographic conditions, I am using a Phenyl Hexyl CSH Column. Mobile Phase is: Line A - Acetonitrile 100%: Line B - 20mM Sodium Dihydrogen orthophosphate dihydrate, Gradient starts at 20:80 (Organic: Aqueous) increasing to 80:20.
Here is an example of the types of peaks and noise that appear on the baseline. Can anyone help with this?

Baseline

[Arkcon]

We could certainly all benefit for the image, so go ahead and attach it to your next post.

As a general rule, at shorter wavelengths, you can expect greater refractive index effects as the gradient changes, you'll have to accept it.  Even the salts will refract light at shorter wavelengths.

Here's something to try:  At this juncture of your separation, you're going from 80:20 buffer:ACN to 20:80 buffer:ACN, and you're using pure buffer and solvent.  Instead, mix up the starting and ending proportions and run a linear gradient between the two bottles.  Then, you'll have treated each bottle exactly the same, and you may get a better baseline.

Now, I know you can't always do this, because you want to be able to alter a gradient dynamically, but this will let you know what may be happening.

[How Dont]

Hi,

Your post is of great interest to me as I also find myself in a similar position. Like you, I also suspect the aqueous mobile phase is the culprit. I have tried different suppliers of water as well as our own high quality Milli-Q, but the result is always the same.

I have done much research, and I suspect impurities in the water are concentrating at the head of the column during equilibration (high aqueous/Low organic). When the gradient changes to higher organic, these impurities are set free and retained on the column. I have shown that the peak areas of the ghosts increase some-what proportionally with equilibration times.

I have read much about this subject, but very little as regards solutions.

My next step is going to be having a look at in-line mobile phase clean-up columns, such as 'Ghost Guard LC' or 'Ghost-trap DS'. This sounds like an interesting thing to try. If the trap is set say after the pump or mixing chamber, maybe the aqueous impurities will concentrate here rather than on the column?

Does anyone have any experience using these? If so I would be interested to hear how you got on.

I will order some and let you know how it goes.

How Don't.

[Arkcon]

Those scavenging columns can be useful in pinning down the problem.  I've never used them, but I do wonder:  When are they used up?  And what effect do they have on complex eluents, do they say, absorb ion pair reagents or do other things that change the composition of the mobile phase before it reaches the column?

Dirty water can be a problem.  Neither of you has yet mentioned what water you use:  in -house deionized?  Type I or Type-II from an in-house generator? (and how is that PM'ed) Or bottled HPLC Grade water (And how fresh is the bottle?)  The O.P. has given us a little bit of their eluent bottle cleaning, but we may have to try to figure out if these are the source of contamination.

The buildup of things at the head of the column may be traceable to the analyate.  A simple blank run between analytical runs may help.  Or it may not, and the column may need a longer flush, or a gradient with more solvent, or a different solvent, methanol as an example can flush contaminants off a column the acetonitrile leaves behind.   

To track this sort of thing down, we'll have to see what happens when we inject more blanks, or equilibate at initial conditions for an extended period of time.  Which of those events produces a worse result?  If neither, then its a refractive index problem, as I described first.

[How Dont]

Thanks for the input Arkcon.

Regarding the water, I have the Milli-Q Direct purification system producing Type I water.  However I also use 'gradient grade' water from various suppliers.

I also thoroughly wash all storage vessels with water/organic/water. I have run several system blank runs (No injection) and the Ghosts are observed at low wavelength, and increase in area with increase in equilibration time.

Until I install the trap - and perform the analytical exorcism, thus removing these demonic ghost peaks - I wont know what knock on effects it will have on the composition of the mobile phase.   

Early stages, but its just an option for the OP and indeed myself.

How Don't.

[Analytical2015]

@ Arkon, thanks for the reply. The baseline image is attached to this post. I use in-house Milli-Q-Water for the mobile phase and washes. This system is calibrated regularly to ensure it stays at the optimum of 18.2 MegaOhms. As a trial, I tried using HPLC Grade Gradient Grade Water to make up the mobile phase and the washes but it made no difference to these stubborn peaks. Its interesting that you mentioned methanol as a solvent to wash any peaks which may come off from the Acetonitrile; my current post-run washdown incorporates firstly a high aqueous/high temp flow to remove buffer, followed by 0.5mL flow of 100% Acetonitrile, then no flow but high column temp in order to evaporate these dissolved ions I believe are in the water. Would it be worth changing to Methanol for washing down the system after the runs?

You also mention the salts will even absorb or precipitate at such low wavelengths. One of the actions I am considering is using both a higher quality grade of Buffer salt and I have also ordered a case of the highest purity HPLC Grade Water on the market (very low ppb dissolved ions) to see if this will clean up the baseline. The flow cell is new so shouldnt have that much residue on it plus these peaks appear on a "Condition Column" injection with just mobile flowing through the system which narrows the source of these peaks down to system - mobile phase, column or innards like detector or flow cell. The machine was recently PMd giving it a thorough cleaning and replacement of worn parts from the BSM to the PDA Detector. We have used brand new columns- still there! Any suggestions are very welcome. 

@ How Dont, that's crazy how similar our issue is. I have tried Chromatography literature, articles, learned fellow colleagues and other forums for help with this issue so Im at my wits end now. I have a slight ulterior motive for getting this method running perfectly though; Im in line for a promotion soon if I can prove myself to my superiors on Analytical Method Validation and this is the sweetener needed to nab that (well paid!) higher rung on the ladder. As you can imagine, Im tearing my hair out and havent been sleeping properly worrying about this method, so Ill try anything at this stage. Maybe you can pick up some tips from this thread! 

[How Dont]

Hi analytical 2015,

I'm sure we are not the only ones experiencing this problem. Those are some fine ghosts by the way!

Do any have a similar UV profile to your compounds of interest. In my case they don't.

At this stage all I see is something like this:



I can relate to your sleepless nights thinking about it, have you ever woken up to find fully integrated peaks scratched into the bedroom walls, well I haven't - just yet.

Getting back to to the issue in hand, Arkcon's suggestion of methanol (and as you say possibly as part of the wash-down procedure) is certainly worth looking at.

And best of luck with that promotion - hopefully a suggestion here will aid you up on to that ivory tower and you wont have to worry any longer! But you wont have any fun up there, like you do now.

I will certainly keep you informed if I make any progress.

How don't.

[MOTOBALL]

Has anybody run blanks with Water/MeCN, NO SALTS, yet ?

[How Dont]

Hi Motoball,

I have. I substituted my aqueous buffer solution with 100% water and I get the same effect.

That's what is leading me to the water. I have also tried various suppliers of high purity acetonitrile.

In my case the ghosts don't seem to interfere with quantitation, but I just have a worry this may happen at some stage down the line.

How don't.

[Arkcon]

There's lots to work with here, lets try a few points.

You seem to have access to the cleanest solvents.  That's a good first start.  Like MOTOBALL: suggested, try to run a few runs of the gradient, with just water and acetonitrile, in your cleanest glassware, and look to see those peaks again.  Do they go away after time, do they get smaller, things like that.  Changing to the best source of salts may be useful too.

Fortunately, you have a PDA.  Do try to extract the spectrum of those peaks.  Do they absorb sharply in the region you're interested in, like How Dont: asked?  That signifies a particular contaminating compound.  Or is it just noise.  Or is it a sharp drop off at the high or low ends of the spectrum, that signifies refractive index change.

I don't like the end of your column cleaning protocol.  Heat with no flow to blow off volatile contaminates seems unlikely.  The sealed column connected by coils of fluid lines inside an instrument isn't going to let anything escape.  Moreover, we generally don't heat a column without flow, that could damage it.  In fact, you may now have to face that these peaks are possibly the silica's bonded phase "bleeding off", both due to regular wear and tear and any thing you may be doing.  You can try with a brand new column, equilibrating it with water and ACN gradients, and see if this region is clean, or if this contaminant starts building up again.  Mass spec grade columns have the bonded phase bonded at multiple points, to prevent this sort of bleed.  So you can look for a similar column, in "low bleed" format to see if they help.

This is why we avoid low wavelengths in HPLC.  You can find a contaminant from everywhere:  The solvent bottles, the water generator, the eluent storage bottles, the fluid lines, contaminants built up inside the HPLC, stuff extracted from seal material.  Then there's your sample vials, the containers used to collect your samples, the samples themselves.  If you've adjusted the pH of the mobile phase by sticking the pH electrode in the buffer, then there are traces of the electrode filling solution in your eluent.  And whatever you used to adjust the pH, which is often not the cleanest acid or base bottle around.

What are you going to do?  I can predict this.  You're going to try everything in sequence, twice, and in the end, the problem may disappear.  But you will not know for sure which single thing was the culprit.  You'll just know that if you have to be at the low levels, at extreme wavelengths, you'll have to be very fastidious with your instrument.

You might want to flush out the system without the column.  One of the best ways to passivate the interior of the HPLC is with a liter or more of 0.1N citric acid, followed by a liter or more of 0.1 N EDTA, delivered slowly, say at 0.25 ml/min over a weekend.  A good boiling MilliQ water rinse before and after can be helpful as well.  These reagents are compatible with and will somewhat clean the detector flow cell.  Harsher acid washes may not be safe for the flow cell parts.

On the subject of the detector, if you're pushing it to extremes like this, maybe you'd like while you're working on this problem, to perform a lamp replace and PM a little bit early, to be sure its at its best.

[How Dont]

For anyone haunted by ghost peaks in their reversed phase chromatography, the results of an interesting study have recently been published on chromatographyonline.com (September 2015)

'Removal of Contaminant Peaks in Reversed-Phase Gradient Liquid Chromatography for Improved Detection of Pharmaceutical Impurities'

http://www.chromatographyonline.com/removal-contaminant-peaks-reversed-phase-gradient-liquid-chromatography-improved-detection-pharmac-0

Its a useful resource for anybody facing into the task of eliminating ghost peaks. Unfortunately, it was published a few months too late for me!

I managed to get a Ghost trap DS (Activated charcoal packed column), and although it helped, it didn't clean up the baseline to the extent I was hoping (In my case anyway). In fact, I never managed to solve the issue entirely, instead have focused on minimising the problem, i.e. trying to ensure I have high quality Type 1 water at all times - changing the filters regularly!

I also installed a small C18 column on the aqueous line between the pump and the mixer - which causes minimal additional dwell volume, cleaning afterwards with an aqueous/organic mix.

If anyone has had any success stories, would be interested to hear how you got on.

[How Dont]

The results from another interesting 'Ghost Peak' study published on chromatographyonline.com (January 2016)

'Ghost Peaks from Nitrile Glove Contamination in Reversed-Phase LC Drug Analysis'
http://www.chromatographyonline.com/ghost-peaks-nitrile-glove-contamination-reversed-phase-lc-drug-analysis?pageID=1

Personally I have found 'high quality aqueous phase and good-to-high quality organic phase' will eliminate ghost peaks in gradient RP-LC.

However, this study may be of use to someone.

Apologies for replying to my own posts, just think its worth adding to the useful info already on this thread.