Presence of salt in sample

[yeevoon]

My samples contain salt and I'm worried that it will affect the analysis via HPLC, is there any way that I can minimize the problem as I've tried to remove as much salt as I could but there would still be present.

Besides, I would also like to seek advice on the way to prevent compounds from degrading after injecting them into HPLC columns as I always couldn't reproduce the same chromatogram with my recovery product, and sometimes, I can't get the same chromatogram with the same method which I developed, even on the same day, even though my sample was freshly prepared on that day.

I usually run a gradient system on RP/Silica normal phase column. Please feel free to advise. Thanks alot for the help & have a nice day.

[tom jupille]

More details would be helpful. Like specifically what solvent and column packing you are using (I'm sorry, but "RP/silica normal phase column" doesn't provide a whole lot of information!).

[Consumer Products Guy]

In my business (consumer products, duh !!!), practically of all our samples contain some salt; we make no efforts to remove it. As to the other question, I also don't know what RP silica normal phase column means - doesn't RP usually mean reverse phase?

[yeevoon]

Thanks for the reply & sorry for the confusion, what I meant was, I have a reversed-phase monolithic column, as well as a Si normal phase HPLC column.

Regarding the sample degradation problem, is there a way to overcome besides using volatile buffers?As what I understand, the columns have to be flushed thorougly if buffers were used.

Thanks again!

regards,
yeevoon

[yeevoon]

..continued from above

I usually run a gradient system of water: acetonitrile or water: methanol on a RP column, and hexane:ethyl acetate on a Si normal column. Both of them are monolithic columns.

[AA]

First off, on column degradation (or on column transformation i.e. from one ionic species to another, much more common than degradation) is relativly rare, although it does happen. It usually shows itself as a very distictive peak shape, often described as a shelf or bridge type of peak. What it looks line is a sharp upward rise, sharp down to a point that is much higher than the baseline level and then off this shelp a fairly normal looking peak. So, what you have is the parent on one end, the product on the other, and the molecules that are in transition forming the shelf or bridge. You can often use temperature to drive the reaction one way or the other to confirm if this is what is really going on. If your sample is truly degrading, it is more likely happening during the sample workup, cleanup or what ever other manipulations you do to it befor injection. I dont think I have ever heard of salts in a sample causing this, most salts that are in a sample just elute in the void (in reverse phase). This is why people like to use SPE to de-salt samples. The salts are unretained on the SPE cartridge and go to waste, and the compounds of interest can then be eluted.

[Mattias]

We have had the same problem in the past, mainly with stressed samples that has been dissolved in various buffers from pH 1-10. The pH and ionstrength of the sample solution will most of the times have a huge effect on the retention time and peak shape of the early eluting peaks.

We solved it by column switching: The sample is injected in a stream of low percent organic that goes through a C18 precolumn. The analytes are adsorbed and desalted. Then we start the normal method and the analytes are transferred to the analytical column and separated.

A totally automated solution that works beautifully in most cases!

[yeevoon]

Thank you all for replying.

To AA: I wonder if you could tell me more about the SPE principles as I'm not quite sure of it?Isit the same as the conventional column chromatography? If they're the same, all of my samples were previously fractionated and isolated via conventional column chromatography before subjecting them to HPLC. Does this mean that the salt problem has already been minimized?

To Mattias: I am interested in the column switching method u've mentioned as I'm quite reluctant in using buffer solution, but I wasn't quite sure of this part-"The sample is injected in a stream of low percent organic that goes through a C18 precolumn. Then we start the normal method and the analytes are transferred to the analytical column and separated. "- is this all done in the same analytical column? or u flush the sample through the column & collect them, then inject them again?

Pardon me if I've asked silly question as I'm still a beginner.

Thank you again!

Regards
Yee Voon

[AA]

Yes, SPE is the same as column chromatography. So, asuming your samples have been properly cleaned up, they shouldnt have any salts in them. So that is not likely your problem.

[yeevoon]

Thanks for the reply again, AA.
I guess the only possible reason is due to the acidity of HPLC column, which I can only resolve the problem by using buffer solutions, but I'm quite reluctact to do so, as I've heard that the HPLC columns have to be taken great care if I used any buffer solution in my solvent system.

May I know the precaution which I should aware of?Or is there any preventive maintenance that I can do?

Thanks for the great help again!

Regards,
Yee Voon

[AA]

Yuor comment "that the HPLC columns have to be taken great care if I used any buffer solution in my solvent system. " is way overstated, the column can sit in a bufferd mobile phase (as long as there is some amount of orgnic in there to keep microbial growth from starting. Of bigger concern is leaving the LC system sitting in buffer for days at a time. Depending on the system, this can be overcom easily. If using a quartinary system, one of the unused line can be set up with 90% water 10% organic and used as a shut down method/wash out method at the end of the sample set. If this is not possible, make a shut down method that keeps flow at a very low flow (o.05 mL/min) this will protect both column and LC system (flowing buffered mobile phases is what they are designed to do).

[Mattias]

yeewon: We use a column switch, a schematic picture can be found at:

http://www.rheodyne.com/products/fluidic/manualapps/, under sample enrichement. Igonore the 9 and 10 position of the switch valve and imagine that you inject the sample in the flow of pump A.

Injection performed in valve position B, and elution onto the analytical column in valve position A.

For this technique you need two pumps and a switch valve. We use a six port valve (not 10 port as described in the link).

[pharmason]

Hi all
today is my first day in this forum,
now regarding to the main question, i wanna know
1. degredation in cloumn or degredation during sample preparation?
2. what is your Diluent for sample preparation?
3 what is chemical nature of your Analyte?
as u said you dont get constant result even if you prepare sample at same day ,
let me know above information to understand case