Chromatography Forum

I am having an impossible time with stablizing the baseline with my PE 200 series HPLC with RI. The system is a 5 channel degasser, quaternary pump, ALS, column oven, and 200a refractive index detector. MP is premixed 75:25 ACN/H2O, flow 1 ml/min, 150x4.6mm 3um NH2Pr column, det and column temp 30C, ~30psi back pressure. I have purged the reference cell manually and used the unattended mode. I flushed the system many times with 1M nitric acid, briefly with NaOH, strong polar organic, water, warm water,, probably others too and not in that order. I have tried/fixed: check valve, high piston seal, pulse dampener diaphragm, rebuilt injection port, solenoid valve in RID, new lamp, adjusted optics, and adjusted lamp voltage. I tried bypassing the degasser once and noticed it actually got a little better, so then I put some water in an ultrasonic bath to degasser is and the baseline was perfect. I sent the degasser back to the reseller and they said it works fine because the light turns green. They took it apart and noticed a line was a little loose and called again and said they might have found something and fixed it (after first saying it works perfectly). I don't much trust them though because the green light logic, and because I had to replace all of those parts and clean off the salt deposits. The column was replaced by my supplier with no improvement. I also shut down the flow and plugged the in/out on the detector and baseline is beautiful so I think the detector is ok. The system is on a dedicated circuit as well. Is there anything else I can possibly try?

Hi,
I have got similar problems with RIDs (not with the PE 200).
Please try:
1/ Purge flow cell (reference and sample cell) with neat Isopropanol.
2/ Cover the top of the unit with paper board (or anything similar) to insulate
3/ Use HPLC-grade water to make mp.
4/ Install a restrictive capillary (e.g. 0.010" ID) at the waste line of RID to maintain positive back pressure.
5/ Consider to install new check valves. Purge check valves with neat MeOH or IPA after installation.
6/ MP and all solutions (IPA, MeOH, water) must be degassed (or sonication).
7/ Use a voltage stabilizer or power line conditioner for at least the RID.

Good luck
Alfred

30 psi backpressure for a 3 micron 150 x 4.6mm column seems very low , are you sure that your pump delivers 1 ml / min flow ?
You may check it by collecting the waste to a measuring cylinder versus time.

Alfred,
I don't have a degasser right now and I'm not confident on the one I have but I'll certainly try the IPA flush that when it's back. For sonics I can't do that consistently but it helps a little at first with my degas mode on the ultrasonic bath. I'll look into a voltage stabilizer but when I've monitored it, voltage and frequency were very stable. Water is RO passed through a specialty column to 18Mohm deionized, purity is my companies business. The back pressure regulator should be sufficient though I have tried .005" ID post detector.

Uzman,
The flow measures up, 30psi is post detector, precolumn is about 1240-1260psi

Thanks for suggestions anything might help!

Wow, you did already full maintenance to your HPLC system, excellent.

If flow rate is ok please disconnect your column from the system.
Maybe your baseline is than stable. Please check for extra column effects and check the capillary connection to your column.
With your MP it should be ok to do ultrasonic before the run and disconnect your degasser.
Temperature is also constant for column and detector?
Good luck.

Gerhard, is there a way to fix my issue if it is in fact the column causing my issue?

If it is at the inlet of the column YES.
Make a new connection. If you use stainless steel capillaries with ferrule and nut make sure that the capillary is going into the endfitting before you fix the ferrule. If you use finger tide fittings do it the same proper way.
If your endfitting is damaged, how ever that happened, order a new endfitting.
You can change that by your own. If you do it replace the frit at the same time.
Good luck.