Chromatography Forum

Running at 300ul/min Buffer B-100% Acetonitrile. Baseline oscillates with irregular frequency. Agilent 1200 binary pump, UV detection at 280. Anyone see this before

"Anyone seen this before" ? *Why yes. Everyone that has ever operated an HPLC system. The questions to ask are, (1) what is your method and settings and (2) what are you doing to cause it?

As you have not provided any routine details, my first suggestion would be to contact someone with practical experience at your school or company for help. Local training and assistance is what you need when first learning the basics of HPLC (which takes many years to acquire). The web is a poor substitute for hands-on learning. No one is expected to know how to solve these problems without first having several years of training.

Once you have someone experienced to work with you, here are a few comments and suggestions that may help you to understand and solve this common issue.

(1) 300 uL/min is a low flow rate to use. Do you have enough back-pressure on the system (> 40 bars) to maintain it?
(2) Is your mobile phase fully degassed ? *It needs to be. Any problems or issues with degassing must be solved before proceeding.
(3) Have you properly primed the pump and HPLC system with fresh, clean mobile phase?
(4) Has the system been maintained and tested (PV) to demonstrate it is fully operational?
(5) Quantify the observed "oscillation" in terms with values and a scale. This includes when and exactly under which conditions it occurs. Please be very specific and include chromatograms, with scales.

(6) A free article which addresses these types of observations: "Common Causes of Baseline Noise in HPLC, UHPLC." https://hplctips.blogspot.com/2014/09/c ... noise.html

Jerk and condescending responses are not helpful.

To the OP: if your flow rate is that low, I assume you're using a column with a smaller particle size on that system. By smaller I mean less than 5um. Sometimes those columns take longer to equilibrate.

Also, I notice your B is 100% ACN. Maybe it has polymerized. This can cause an oscillating baseline. Check the age of your ACN bottle.

I don't think you're having degassing issues. The Agilent 1200 comes with a superb degassing system built into the pump. However, you didn't mention your MP A. I assume it's water or some buffered solution. In that case, you could be creating some small bubbles when the water comes in contact with the ACN in the gradient mixing chamber, which comes after the degasser. I've dealt with this problem by creating a 95/5 aqueous/ACN solution for A and a 95/5 ACN/aqueous solution for B, and then adjusting for it in the gradient. Premixing the solvents like this gets the bubbles out of the way, so that they don't form in the mixing chamber.

Lastly, your flow cell could be dirty. If it's been awhile flush your system with a 10% IPA solution, and then with water.

how big are the oscillations?
Check the system pressure and whether it also has oscillations. They should, if present, be very small (1bar or less). If they're not, then it is likely that you have an air-bubble in your pump (purge a lot to get rid of it) - this will cause erratic flow, which can also cause oscillations in UV chromatograms. If purging won't get rid of oscillations then you may have a pump problem (difficulty with check-valves, pistons etc.)
If you don't have bad pressure oscillations then the pump is probably OK. Are the oscillations in the UV chromatogram enormous? If they're very small (< 0.1mAU) it may be that you're looking for peaks that are really pushing the system to its limit, and there isn't a "problem" to fix as such, it's more a matter that you're going to struggle to see a peak that tiny on the baseline. You might be able to improve the situation by subtracting a background wavelength (preferably during data-processing rather than data acquisition) - the oscillations may happen at a wide range of wavelengths, while your target compound absorbs over a narrower range.
Yes, check your solvent is clean, as bad solvent certainly won't help. Also check that your mixer is the right size for the job. Some solvents and additives are harder to mix than others (TFA...). I think the 1290 has a mixer that can be reversed to change its volume, I can't remember off-hand.
Back pressure is more important than actual flow-rate; the 1290 can definitely handle 300uL/min easily (it's a typical rate for a lot of MS methods). But pure ACN won't give as much back-pressure as a more aqueous method. If your column is short, or has large particles, you may benefit from a longer column, a higher flow-rate, or a back-pressure regulator placed upstream of the column. If the oscillations are very slow (over many minutes) then it's probably nothing to do with pump or solvent.

Since the OP did not provide any of the conventional details about their method, it is best to start with the obvious and then if they contribute more info, we can evaluate further.

Good general advice is to not reply to posts with advice when you do not have the experience or training to do so. Example: "I don't think you're having degassing issues. The Agilent 1200 comes with a superb degassing system built into the pump." - THE Agilent 1200 binary pump was never equipped with a built-in degasser. The newer 1260 and 1290 pumps do. The 1200, like the 1100-series must use a secondary stand-alone degasser so your assumption is incorrect. The OP's degasser may not be present or may be defective (very common as they only last about 5 years before requiring service). So, yes, a lack of degassing is a very common issue and may indeed be the issue here. Even if the pump HAD an integrated degasser, that would in no way mean it was working properly (how would you judge that with no information at all to go on?).

"

if your flow rate is that low, I assume you're using a column with a smaller particle size on that system. By smaller I mean less than 5um. Sometimes those columns take longer to equilibrate

." ??

For the purpose of this post, particle size is not related to flow rate. Particle size would relate to back=pressure on the column. If the OP is running at 300 ul/min it suggests they may be using a 2.1 mm ID column (column, not particle size), not 3.9 or 4.6 mm ID. Sticking with a linear velocity of 300 uL/min on a 2.1 mm ID column would be the same as using 1.5 mL/min on a 4.6 mm ID column. Flow usually relates to column dimensions. Equilibration should not change in a significant way when using a 5 u or 2 u particle. Now, yes it is certainly true that they could be using a column of any reasonable dimension with small particles inside, but we have no details regarding their method so a column of ID 2.1 or a particle size of 2u just makes no difference at this point in time. Certainly not in any way that would effect noise or baseline disturbances as reported here. IOW: Useful, but not relevant.

ACN polymerization is far rarer in real life than the number of times it appears in web discussion forums like this one. It is also extremely rare to be observed causing a problem in an Agilent 1100 or 1200 Binary or Quat pump with standard AIV installed. Those are extremely rugged pumps and typical problems that all new users have, with any HPLC system, are keeping their HPLC pump properly primed and flushed before starting an analysis. Degassing and proper priming are key to getting things running smoothly. The lower flow rate of 300 ul/min is easily within the limits of a std 1200 binary pump, as long as sufficient back pressure is maintained (> 40 bars).

My earlier post included a wealth of practical, detailed information and article links to help educate this new user and allow them to begin the troubleshooting process. These types of problems are best sorted out at the end-user's site, by professionals with training to both teach and assist new users like yourself and the end-user OP to begin to acquire the skills needed to insure their system is running properly.