Drift Help

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

9 posts Page 1 of 1
Hi everybody - First time post. I'll try to as brief as possible.

Instrument: Shimadzu Prominence-i LC-2030C Plus (UV detector)
Other detectors: Shimadzu RID-20A
Column: Phenomenex Kinetex 2.6 µm C18 100Å, LC Column 150 x 4.6 mm, Core-shell silica, R Phase (00F-4252-E0)
Guard: Phenomenex SecurityGuard UHPLC C18 4.6mm AJ0-8768 with AJ0-0000 Holder.
Mobile Phase: H20:MeOH:Formic acid (80:20:0.16) Analytical grade chemicals and HPLC grade filtered H20.
UV Signal: 265 nm
Oven Temp: 45 C
RID Temp: 45 C
Flow Rate: Started at 0.5 mL/min and slowly increased to 1.0 mL/min over 24 hours. Trying to wash off/out the column, UV cell, and everything.
Pressure: 25.6 MPa at 1.0 mL/min

I'm an chromatography novice. I did it 25 years ago with glass plates, silver stain, sampling with capillary tubes from packed silca gel column samples only.

Before I installed the column, I washed the system with HPLC filtered water for about 32 hours. Then I washed with the H20:MeOH:FA for about a day. I installed the column with the guard end first and let the column purge before I connected it inline. Attached the column in the oven. And slowly increased the flow rate of the mobile phase as described above.

I finally have everything looking good in the LabSolutions v5.97 software. During the mobile phase/washing the UV signal and RID signal were very jumpy. The HPLC has not been used in months or possibly a year or so. I let the mobile phase just run at 1.0 mL/min and 25.6 MPa. This morning the noise levels of the baseline are very low, but both signals (UV and RID) have been dropping at about -0.1 every 15 minutes. The flow mode is isocratic.

I would appreciate any thoughts. The temperatures in the log seem to be steady. The mobile phase seems to be mixed well.
You may try to increase the percentage of methanol as a washing step.

It seems some junk coming out , and the percentage of methanol in your mobile phase is not adequate , to wash it out completely.
Thanks, uzman. I made a 70:30 H20:MeOH mobile phase. I'll load it on in the morning and see if I can clean it out more. I'll start with low pressure again and slow increase the flow rate.

My only question is, the column and guard column appear to be as old as the HPLC. I have no idea if the C18 column was stored properly either. Would you advise the C18 column be removed during the MeOH wash or do you think it would be ok to wash them all together?
You can wash all together , also increase the methanol percentage up to 90%
I washed the instrument for about 48 hours with a 30:70 H20:MeOH mixture at a flow rate of 1.0 mL/min. A lot of junk came off. Pressure stayed around 23.7 MPa. The UV signal would periodically spike and be all over the place during the 48-hours. The drain fluid was tan colored at times. It's clear now. The RID signal was fairly calm.

I slowed down the pump and stopped it. I switched it back to my H20:MeOH:FA 80:20:0.16 mobile phase this morning. I increased the pump slowly to 0.5 mL/min. Purged the RID. Purged and rinsed the auto sampler. The mobile phase has been running for about 4 hours from this morning. The RID signal is pretty solid, but the UV signal is still decreasing. About -0.1 mV every 50 minutes.

Should I try to MeOH wash again at higher concentration? What flow rate should I wash at? How much time should I give it this time? Or do you think there is another issue that I've not thought about yet?
You may want to have a look at this document from Waters about general considerations and tips about cleaning and keeping system

Controlling Contamination in UltraPerformance LC®/MS and HPLC/MS Systems; 715001307:
https://www.waters.com/webassets/cms/su ... l_cntm.pdf

For your actual case I would first clean the system with the "magic-mix" H2O/MeOH/ACN/IPA/HFor 25/25/25/25/0.2-0.5
- disconnect the column; replace by union
- place all solvent lines into the same bottle with the mix; probably replace the bottle sinkers or clean them separately with ultrasound with this solvent mixture before reconnecting
- flush the system (make sure all lines are used, maybe also do some injections of this mix) with about 50 ml of the mix (set the flow rate to what the system can handle in means of pressure. But maybe not more than 1 or 2 ml/min, to get a reasonable flush time)
- then I wouldn't hesitate to also flush the column with this mixture, but maybe not connected to the detectors. Flush with about 30-50 column volumes (e.g 50-90 ml), adapt the flowrate according to the backpressure
- prime all lines with pure methanol and keep them primed with methanol if not used
- optional: flush the column with about 10-20 column volumes of methanol or with acetonitrile for storage.

Now you should be ready to go with your desired method.
Depending on the RIDs sensitivity, equlibration may take a while but I guess overnight at low flow should be more than sufficient.
For column equilibration, normaly again about 20 column volumes are sufficient (think in column volumes not time. The time will result with the flowrate).
Also generally no need to ramp your flowrate over that long period. Start low and then go up every 1-2 minutes in about 0.1 ml/min steps.

Maybe you first want to inject some performance test to check the efficency of the column. You may use some caffeine in mobile phase (e.g. 25 mg/L ?) at UV273 nm; your setup with MeOH 20% should work well, also with the RID.

And maybe check the drift specifications of your detector. Some drift maybe normal and if it's not excessive or a problem for your analysis: don't chase shadows
Great tips, Hallow!!

I like the "magic mix". Given that I have no information on what was ran in the instrument before me, the "magic mix" maybe enough to solubilize whatever is still stuck in there. I did find one file with terpenoid info. Looking at most terpenoid solubility on PubChem, these may be very sparingly soluble in MeOH which would explain my result. But no info on if that's what has been in the system or not.

I will make the mix and load it to run overnight. I'm hopeful I have a better result to report in the next days!
Hey everybody - I wanted to thank everyone who commented in this post. The Magic-Mix did the trick. I made 5 liters of Magic-Mix. I started seeing so much baseline bouncing. But then I just let it run, walked away, and stopped watching it. It ran for a bit over 4 days. I had to get a larger waste container.

But today, I loaded some known samples and made my first standard curve. The baseline is perfect. You can zoom in on the chromatogram and the baseline is flat as flat can be! Whatever was in the UV cell and the RID was nasty, but it's all clean now.

Again, thanks for the help.
I'm glad it helped.

And thank you to update us with the outcome.
9 posts Page 1 of 1

Who is online

In total there is 1 user online :: 0 registered, 0 hidden and 1 guest (based on users active over the past 5 minutes)
Most users ever online was 1117 on Mon Jan 31, 2022 2:50 pm

Users browsing this forum: No registered users and 1 guest

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food & Beverage, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry