Chromatography Forum

Tom: But presumably you answer if you are confronted with a back-inquiry, unless you don´t want to have your car fixed?

Thanks
We are doing quantitation of some metabolite.After doing protein precpitation .I want to do dry my sample & recon ..in minimum amount .During that period sample are degrading(actually it's light & temp.sensitive) & some time I can't see metabolite.It's in very trace amount.To avoid that thing we are inecting supernatent in HPLC but it's diluted now & It's binds with protein .So I am using acid to overcome this problem.

But presumably you answer if you are confronted with a back-inquiry, unless you don´t want to have your car fixed?

You're right, of course. But I might get very frustrated and give up if the exchange had to happen via a discussion group board

We are doing quantitation of some metabolite. After doing protein precpitation .I want to do dry my sample & recon ..in minimum amount .During that period sample are degrading(actually it's light & temp.sensitive) & some time I can't see metabolite.It's in very trace amount.To avoid that thing we are inecting supernatent in HPLC but it's diluted now & It's binds with protein .So I am using acid to overcome this problem.

OK, there are many others on the forum who are more expert in sample preparation, and I'm sure you'll get better answers from them.

Can we obtain an indication of how quickly the pressure rises with injections, and whether a subsequent flush with solvent reduces the
pressure?.

Presumably you have some method of ascertaining that degradation is occuring during evaporation and reconstitution, regardless of how careful you are during preparation. Knowing that heat and light are potential problesm, it should be possible to greatly reduce their effect, but also watch for other possible degradation paths - such as pH.

The problem is presumably high pressure caused by either precipitation of some part of the sample when it contacts the mobile phase or guard column, or some particulate material already in your sample.

The insolubility can fixed by ensuring all of your sample remains completely miscible. You have to eliminate the problem material from your sample, or at least make it soluble in the mobile phase. To do that you need to understand the problem and/or perform some experiments.

I'd be inclined to try and premix your sample with some of the mobile phase and then either filter and/or centrifuge the solution. Choose filters and microcentrifuge tube materials that don't bind your sample - contact filter suppliers like Millipore and ask for advice. If such experiments reduce the rate of pressure increase/sample, it's worth pursuing optimising the larger injection volume of sample diluted with mobile phase.

I suspect there are better solutions that others are aware of.

Bruce Hamilton

OK, the problem is typical for samples containing proteins. You can´t precipitate all of them. Not yet mentioned (?) additional cleanup methods are size exclusion and ultrafiltration. None of them will do you any good if you don´t get the instability under control. If you don´t have a standard whith which you can play you may have a life´s project at hand.

Anupama,

did you try to precipitate proteins with acetonitrile? You can obtain very clean samples with this method.....

So Tom, if I understand your words right: There is a gap in the HPLC market to start a garage for HPLC users

[quote="bert"]Anupama,

Yes I tried Acetonitrile,Methanol & ethanol too.

[quote="Bruce Hamilton"][quote="anupama"]


After 20-30 injection I was playing with pH too. Right now I am using almost similar procedure to inject my sample, as you mention here.

After 20-30 injection I was playing with pH too. Right now I am using almost similar procedure to inject my sample, as you mention here.

OK, can we please have a little more detail. Does flushing the column ( with suitable solvent ) reduce the pressue, or do you throw away the guard after 20 - 30 samples?. Have you tried backflusing the guard?.

If centrifuging, what is the sample, solvent and RCF ( or speed and diameter ), if filtering what filters are used?. Did diluting with mobile phase have any effect, as compared to diluting with water?.

For the 20-30 runs, is the data acceptable, or does the accumulation of gunk also compromise the chromatography?.

Bruce Hamilton

If the guard column gets clogged, it is doing its job. You need to decide if you want to do more work in sample preparation to save some money on the guard column.
The simplest way to do a solid-phase extraction is to load your entire sample onto a reversed-phase SPE cartridge after adjustment of pH to eliminate the protein - analyte interaction. (This has been worked out for an Oasis SPE cartridge.) Then a wash with water at the same pH, followed by elution of the analyte with methanol, evaporation to dryness and reconstitution in mobile phase.
You can do more sophisticated SPE methods, which give better results, i.e. cleaner samples. This is up to you. I can advise, if you so choose to.

Errr.....it's a guard column a "sacrificial" column? If so, I wonder if an appropriate in-line or particle filter might be a cheaper option.

Sorry, I mean't in the above post: Isn't a guard column.......

There is gunk that adsorbs strongly on the packing at the column inlet. This is especially true for samples of biological origin that have undergone only a limited cleanup. You can not filter that stuff out.

Therefore I have always been a strong advocate of guard columns. I realize that there are trade-offs. You can work out a sample preparation protocol that give you a cleaner sample. This costs time and money. Or you can inject the only partially cleaned sample on a column. The column will die quickly, which costs money. Or you can buy guard columns, which die and cost money.

Yep, thanks Uwe.