column clogged

[anupama]

In RPLC,for bioanalysis, column is getting clogged very fast .

[Bruce Hamilton]

That's a deliberate plot by your column supplier to sell you more columns, so just try to obtain a bulk discount. If you would like some help save your employer money, it would help to provide some details.

In RP, the most obvious candidates particles, wildlife, or crud.
1. check your water quality, ensure it is HPLC grade with final 0.2um filtration ( or 0.45um for older Milli-Q systems). Ensure it is changed regularly are fresh, so no wildlife is growing in your water reservoirs.
2. If you are making up buffers, ensure they remain completely dissolved in any gradient with solvents, and ensure buffers that are friendly to wildlife are also replaced regularly ( eg weekly for phosphate), or stored to prevent wildlife growth. If the mobile phase is the problem, pressure should increase without any injections.

3. Ensure your samples are soluble in the mobile phase, and don't contain particles ( prefilter or centrifuge ). Ensure that your gradient or isocratic system is fully flushing all the sample from the column, if you flush the column with solvents recommended by the manufacturer, you should remove most slightly soluble crud. Consider the column and injector temperatures, and the effect they may have on sample solubility. If the sample is the problem, the pressure will increase according to the number of samples analysed.

Now, to clean the column, reverse it, and don't connect to the detector. then run your solvents through it, gradually increasing the flow until at least twice that of your method. Then apply the column cleaning solvents suggested by the manufacturer, first in the reverse direction, and then in the normal flow direction.

If it all seems to hard, just get you employer to buy columns in bulk.

Bruce Hamilton

[Bryan Evans]

Along the same lines, you can also try using a guard column/cartridge
right before the column - it should help with reducing the amount of particulates reaching the column.

[anupama]

Thanks ,guard column is already attached.

[HW Mueller]

So what got clogged, the column, the guard, or both? What are you injecting?

[Peter Apps]

You need to filter your samples

[anupama]

I can not filter my sample.I will lose my sample & it's degrading at ambient temperature so i can not evaporate up to dryness & it's light sensitive

[Peter Apps]

How will you lose your sample if you filter it ?

Please describe how you are preparing your samples.

[tom jupille]

As has been suggested, you need a better cleanup. If you can't do that (e.g., for sample stability), and alternative is to do the cleanup on-line. You could set up a guard cartridge with a switching valve and a backwash pump. Inject your sample, let the analyte(s) pass through the guard cartridge to your analytical column, the switch the guard column out of line and backwash it. Switch it back into the line when you are ready to inject the next sample.

Check the Rheodyne and VICI websites for applications notes on switching valves.

[Uwe Neue]

I do not agree with Bruce that "that's a delibarate plot by your column supplier to sell you more columns". I hope, Bruce meant this to be a joke...

Columns clog, if you inject junk. For a sample from bioanalysis, you need to do sample cleanup in the sample preparation step. If you want to get solid information how to do such a thing, send me an e-mail and I advise you. Alternatively, you can go to the Waters website and look for the Oasis applications booklet.

My very first attempt to do a "bioanalysis" was an injection of a milk sample onto a reversed-phase column. It immediately resulted in high backpressure, I suppose due to the instantaneous formation of cheese on top of the column.

The better the sample cleanup, the longer your analytical column will live. The worse your sample cleanup, the shorter your column life. You need to decide, where you want to spend your money...

[tom jupille]

I hope, Bruce meant this to be a joke...

I'm sure he was as serious as I am when I tell people about the vast conspiracy by oil companies to suppress information about the high tech carburetor that allows autos to operate on a mix of 10% gasoline and 90% water.

[Bruce Hamilton]

I do not agree with Bruce that "that's a delibarate plot by your column supplier to sell you more columns". I hope, Bruce meant this to be a joke......

It was intended to be a joke. Less supplied information by original poster = less serious responses from me. The fact that it wasn't obviously so, means I should use smilies more often. I'll try to do better.... I have a lot of respect for suppliers.

It's not clear to me that the original poster has confirmed that the sample is the cause. There's no reason why sample prep can't be used on material that is heat and light sensitive, it's done every day with vitamin D assays.

They should continue to work in the dark, and use appropriate techniques such as filtration and freeze-drying. If they want specific help, they need to be more specific.

Completely off-topic. Tom, Honda have/had the Shell ultrathon record, showing that they can make a car that moves a single person around a figure eight course at an average velocity of 15 mph and achieve over 10,000 mpg with gasoline. Our vehicle fuel consumptions that are greater than that must be a combined oil company and car company conspiracy.

Bruce Hamilton

[chromatographer1]

Monday morning, where's my coffee? Oh yeah,

having seen posters place postings with an expectation that this forum is like a magic button which will cure all their chromatography ills miraculously, perhaps a standard reply-email should be sent to these hapless folks instructing them to kindly prepare better questions to the forum if they really seek to gain any information from its members.

Sorry about the grumple, but it is Monday morning, and I only wish to help those who need help and will at least expend enough energy in preparing questions that makes me suspect they think my time is valuable enough to formulate an answer to them.

This is an on-going problem with forums like this. Everyone please forgive me and have a good week.

Now I feel better, even without the coffee.

Rod

[HW Mueller]

Actually, Rod, you stated the problem rather mildly.

[tom jupille]

Hans, Rod, Bruce, et al:

"It comes with the territory". Whether we like it or not, for most users, chromatography is a tool, a chromatogram is a means to an end, and a chromatograph is an appliance. They justifiably expect useful results without requiring deep understanding on their part, just as I justifiably expect my car to start in the morning without requiring me to understand compression ratios, spark timing, fuel injector pulse width, stoichiometric combustion ratios, or exhaust gas temperatures.

When something goes wrong with my car, I often don't know what questions to ask or what information to provide the service tech in order to allow efficient diagnosis. He has to pull the information out of me ("what do you mean by a 'funny noise'?").

Like you, I get frustrated at not being provided the information I need to be helpful, but then I stop and remind myself that extracting that information is a big part of being helpful!

And after that rant, it's obviously time for me to get my cup of coffee!