Bad Baseline in High Speed

[syx]

We have Hitachi LaChrom Elite HPLC System in our laboratory. We found a problem when run with flow rate 2 mL/min or faster: the baseline will be very wavy. What should we do to repair this problem?



Any suggestion will be appreciated.

Regards,
Siswanto

[Mark Tracy]

That is pump noise being detected. It might be a mixing problem, but more likely a pressure/flow problem. First make sure the pressure ripple is within factory specifications; if not, fix the pump. It could be a bad checkvalve or seal. Some detector cells are very sensitive to variations in flow rate, usually caused by thermal mis-match between the mobile phase and the flow cell. Using a heat exchange device on the flow cell is the cure. If you have disconnected it, put it back. If there never was one, see if your Hitachi rep has an upgraded cell.

[DR]

You may also benefir from a post detector back pressure regulator (or a length of .005 ID tubing to generate a little back pressure post-detector

[rhaefe]

Question: are you using the L7100 quaternary pump and is the static mixer (~0.9mL volume, filled with glass beads) still attached?


The LaChrom L7400 UV detector usually comes with a heat exchanger already set up. You have to be carefull when adding tubing to the detector cell outlet: the standard anaytical cell on the L7400 has a maximum pressure rating of 150psi (if I remember correctly). I have destroyed more than one of these flow cells in my time....



cheers,

[syx]

It is L-2130. The pressure is normal, there is no fluctuation. The wavy baseline won't be appeared when we use slower flow rate than 2 mL/min. I try to ask the same question to the rep.

[rhaefe]

syx,

are you running isocratic or a gradient and what are your %A and %B? I remember vaguely experiencing a very similar problem with a Hitachi L7100 pump. It turned out that the pump pulses only showed up at certain %A and %B settings and ONLY at higher flow rates. As far as I recall a new static mixer solved the problem as did premixing of the mobile phase.

cheers,

[JM]

Check pulse dampner , can creat such base line problems if not working .

JM

[syx]

Raeffe,
It is isocratic run: 45%A and 55%B. What kind of static mixer that you recommend for our system?

[rhaefe]

syx,

Try premixing the mobile phase. If you already do that than it is not a mixing problem. As far as I remember I added a static inline mixer from ASI. They work pretty good. Don't recall the volume I used.
The old L7100 did not have a pulse dampener, don't know about the L2130.

cheers,

[ym3142]

I am not sure but it seems that some bubble can make similar impact.

[danko]

Hi syx,

My bet is, that one of your MP (A or B – doesn’t matter) absorbs much more energy (light) than the other.
Probably not intentionally, but just in this instance (some contamination in one of the reservoirs).
I would find out which one it is by changing from 100 % A to 100 % B whilst monitoring the baseline. Once identified I would prepare a fresh portion of the "trouble-maker"

Another probable cause could be a new column or a column used previously in another separation procedure. It seems to me it is "bleeding" quite a lot (no stable/horizontal baseline - it is isocratic elution right?) and the stuff coming out absorbs a lot of light.
So you're just seeing the pump pulsation, but only because something is absorbing energy in large quantities.
Just allow much more washing – a couple of hors equilibration or more.

Best Regards

[koen_shimadzu]

I agree to first check the mixing. So pre mix the solvent.

If this does not solve the problem, use the premixed solvent, bypass the auto-sampler and use a manual injector.

I would personally not search in chemical problems like MP or the column, because its a frequent profile.

The damper, if you have one, this can be a problem maker. But weird that you don’t find that back in the pressure profile.

Another problem can be that there is solvent leaking somewhere when you increase the flow. The pressure on the pre-column connections is twice as high, which can cause a tiny leak.
one, this can be a problem maker.

[ym3142]

syx, did you solve your problem?
if did, how ?
Thanks,

[John Lim]

Dear Siswanto (Syx),

This is a mixing issue when performing in-line eluent mixing...the standard glass bead static mixer offered by this vendor is spec'd out around 1.5 mL/min (confirm with the pump manual specs) and the degree of baseline oscillation is also dependent on the observed wavelength (the higher the WL, the lesser the effect since it will be moving away from the solvent UV cutoff...if you change to >260 nm, for example, you may see this problem disappear on the baseline if you are using acetonitrile or methanol as your main strong solvent (your observed WL @ 220 nm reinforces this), but this may not be an acceptable change for your method).

For 'fast LC' conditions, mixing efficiency becomes important at higher flow rates...especially if you are using monolithic columns (don't know what type of column you are using here). This type of issue is not limited to this vendor's LC, of course, and is dependent on the mixing efficiency and design of the specific plumbing characteristics of the system wholistically in any LC vendor's system, but the greatest contribution will be from the mixer.

Recommended solutions (from most economical to $$):

1. Pre-mix your solvents as suggested by the other posters...if the oscillations disappear then you know with greater certainty that this is the source of the problem and you can explore options #2 and #3 below...

2. Add another OEM mixer in series (will add to your delay vol), or substitute with a single larger volume static mixer (3rd party vendors can make optimum recommendations based on flow rate target)...the mixer suggested by rhaefe is a good one (theirs uses a convection design instead of glass beads to effect the mixing)...you might also consider adding in series just the semi-micro static mixer version that your vendor offers which should be able to handle the added mixing requirements @ 2 mL/min without greatly adding to the delay volume

3. Bypass the OEM mixer and add a low-volume dynamic mixer (my preference...you will need to source this as a 3rd party item but is an expensive option); this way one can access higher flow rates while minimizing delay volume

[syx]

syx, did you solve your problem?
if did, how ?
Thanks,

not yet... I have no idea to solve it, but I will try premixing as John Lim said. this time I use other system...