Chromatography Forum

Goodday,

I just bought a new Chromolith RP-18 endcapped LC column and used it for separation of steroids and steroid conjugates. The column is fairly new (3 month) but over the last month peaks were getting more tailed and now I have shoulders on them. I regualarly washed the buffer away with water and stored it in 60/40 ACN/water. However, it seems that even though I'm using inline filter and a guard column the column head is somwhow blocked as I have an unusual backpressure of about 410 psi (with 100 % ACN at flow rate 2.0 ml/min). It should be about 190. I already run the complete washing procedure with water-ACN-n-propanol-ACN-water.
The shouldering is getting better after having about 1 L of water passed over the column but has not yet dissappeared. I just wonder were the contaminats came from since I use all prodection and washing. Anyone with a bright idea pls answer!!

Kind regards

Frank

What are you injecting, exactly?

During the last three months I ran two types of samples:
1. CaCl2 (5mM) soil extracts, those are centrifuged before injection,
containing estrogens

2. Estrogens reconstituted in 20% Methanol in water resp. 20 % ACN in
water.

Any sugestions, I'm looking forward to.

Frank

Soil is another highly complex material. My experience with it is limited to helping somone with radiotracer detergent studies. There is only the remembrance that even with radiotracers the workup was not effortless.
Nevertheless, can you and did you wash the columns backwards? Filter the samples? Do you perform any other workup besides extraction (of soil samples) prior to injection?

Maybe someone more knowledgeable can jump in here.

Some natural polymers in soil, such as humic acid, can be trapped by reverse phase column. They are not easy to remove.

Hey there,

Thanks for the hints.
Well, in order to satisfy the first question: My only treatment is centrifugation prior to the injection. I don't employ filtration as different glass fibre and syringe filters adsorb my compounds.

The humic and probably fulvic acids I think are not the reason as I can see a big peak after the void volume and since those compounds are charged I'm pretty sure that this peaks is due to those humic and fulvic acids.

I cleaned the column in reversed flush modus already. In the reversed modeus the backpressure is more or less all right. Therefore I think there's an particle accummulation on the column head. However, where should it result from I was always running the column with dubble protection (inline filter and guard column).

Regards

Frank

I've been recently running prep HPLC of some steroid derivatives on a C12 column ( Phenomenex Synergi Max RP ). The peak shape deteriorated suddenly to develop a large shoulder. As the pressure didn't noticably increase, it probably is not the same problem as yours.

I discovered that inadvertently flushing with a small volume of mobile phase with high water content caused the problem. Pumping 100% acetonitrile for 10 mins restored the column and peak shape. Could be worth a try if you get desperate, but my samples are much cleaner than yours..

I use PTFE membranes in PP bodied syringe filters, and haven't seen any significant steroid losses. I would definitely want to filter your samples, even if there are some losses, just to determine if the pressure stays constan or increases at a different rate than before.

Bruce Hamilton

Did the reverse washing lower forward flow resistance? If yes you had something break through the pre-filter + guard, either by overloading the guard or via having larger protecing frit pores (or more "reactive" ones?) than those on the column. If the forward flow was not any better after your backwards washing you may have something acting like a backup walve (I have seen this after coinjecting some proteins, my only explanation: Some proteins attached themselves at one end to the column inlet frit. In forward flow they were pressed into the pores, closing some. In reverse they were pushed out of these pores, but not washed away....) If you are not careful you may just wash "blockers" back and forth in your system, though.