Shimadzu HPLC issue

Hi there,

I am having some peak shape issues, with the Shimadzu HPLC system SPD-10A VP UV
detector.
S.E. and R.E. values (mv) are keep changing and blinking on the system monitor.
The D2 lamp used time is 1192 hours.

Any suggestion regarding this issue will be appreciated.
Subi

Dissolved gasses in the solvents will degas and form bubbles when they enter
the low-pressure region after the column.  The bubbles will disturb the light
in the flow cell of the detector and cause the output to be erratic.  This is
especially problematic on high pressure mixing HPLC systems which typically do
not have in-line degassers or helium sparging of the solvents.  You should do
two things, degas your solvents by vacuum filtration, or vacuum and sonication.
Second you can add a smaller inner diameter restrictor line on the outlet side
of the detector to maintain a higher pressure on the solvent all the way
through the detector.  Be careful not to use too small a restrictor for your
flow rate and solvent viscosity which could generate too much back pressure
causing the flow cell to leak or break.  For example, using a length of tubing
on the detector outlet with inner diameter of 0.005 inches will generate
several hundred PSI depending on flow rate and prevent degassing in the flow
cell.  The Waters detectors I've used can take up to 1000psi.

You also mention that the sample energy and reference energy readouts are
fluctuating.  If you do have solvent flow going through both the sample and
reference cells, then the above is the possible problem.  I typically never
used the reference cell so if both energy readouts are fluctuating with no flow
going through the reference cell, then it's likely your lamp is going bad and
not much light is getting through the flow cell to the photodiode resulting in
lots of noise.  The noise will become greater when there is absorbing material
flowing through the flow cell.  For example, even with a new lamp, if your peak
contains a large amount of absorbing material that causes the detector response
to go off scale, that can cause erratic output during the center of the peak
elution because the analyte is absorbing nearly all the light and little energy
reaches the photodiode resulting in erratic output by the detector.  If you
combine this situation with a weak/bad lamp this will exacerbate the problem.

All of this troubleshooting assumes your pumps are working properly.

Best regards,
Brian

________________________________
From: ABRF Discussion Forum <email obscured>> on behalf of Anderson, David
Eric (NIH/NIDDK) [E] <email obscured>>
Sent: Thursday, January 14, 2021 5:01 AM
To: <email obscured> <email obscured>>
Subject: Re: [ABRF Discussion Forum] Shimadzu HPLC issue

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I am not familiar with this unit but you have 3 different things to isolate.

You may have bubbles in the detector. refreshing the cell or line with water
than lots of methanol then water (all degassed/sparged) would treat that.

It doesn't sound like an actual peak shape problem from fluid connections but a
bad connection can help put in bubbles etc.

Finally you can check electrical connections including the outlet connections
if you use those.

This sounds like bubbles or noise.

Hopefully someone with knowledge of this actual unit will respond later in the
day.

All the best,


Eric

D. Eric Anderson
Mass Spectrometry Facility, NIDDK
NIH


Building 8 Room B2A19, 9000 Rockville Pike, Bethesda MD, 20892 USA
(301)-496-7546



________________________________________
From: OKARVI, SUBHANI MOHAMMAD <email obscured>>
Sent: 14 January 2021 02:28
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] Shimadzu HPLC issue

Hi there,

I am having some peak shape issues, with the Shimadzu HPLC system SPD-10A VP UV
detector.
S.E. and R.E. values (mv) are keep changing and blinking on the system monitor.
The D2 lamp used time is 1192 hours.

Any suggestion regarding this issue will be appreciated.
Subi

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