I am intending to quantify insulin, a large peptide using LC-MS/MS system and am wondering whether it is possible to combine two different columns for one separation. For example, a size exclusion column combined with a reverse phase LC?
No. Most SEC columns use straight water as a mobile phase while a C18 column (reverse phase) is non-polar and cannot tolerate conditions of 100% water. It uses a mobile phase containing an organic solvent and water.
Why don't you perform your SEC offline using a SPE (solid phase extraction) cartridge and analyse the eluate by C18 reverse phase LCMS?
Yes you can IF you use an advanced 2D Multi-dimensional chromatography to do so (See Link below to a commercial LC x LC system that we produce for this application), but just connecting two unrelated HPLC columns together will generally not work at all. *Different column types will require different mobile phases, different equilibration conditions and different wash systems. These are not compatible.
An advanced 2D Multi-dimensional LC x LC system provides you with a way to: maintain each column in the correct mobile phase for separation and elution; provide a means to wash and even back-flush the columns offline; Transfer peaks from one column to another; concentrate the sample on each column; Do all of these things with just one HPLC pump. Much skill in method development is required to use these tools, but they have been used to solve many technically advanced separations over the past decades.
http://www.hplctools.com/switching.htm
You don't need to use two columns to analyze insulin. Agilent has a single column solution published on their Web site. I have used it; it works quite well.
Thanks for the much informative replies. To be honest, I am not a chemist per say and a rather new researcher looking at utilizing LC-MS/MS for quantification of insulin. There are quite a few published methods out there but mostly using rather expensive immunocaptured methods for sample preparations. This is purely a theoretical question :)
The question arises when I was thinking in theory by using different types of columns with different selection criteria, I would be able to specifically select for elution of insulin. However, as pointed out, with different columns, they come with different mobile phases and other parameters.
SPE is my ideal choice of sample preparation but due to lack of the specific equipment, I will not be able to use SPE and hence have to resort to other "SPE" related methods. If by using LC, the run will be automated and will require less sample preparations which will limit human errors and variability. I have came across a few very interesting columns by Agilent and will be figuring out whether any of them will be compatible with the reverse phase that I am planning to use.
I am hoping with multiple columns, the separation will be better with less signal to noise ratio and higher abundance being detected.
Thanks again
You should be able to use this article.
https://www.ncbi.nlm.nih.gov/pubmed/11216004
Insulin is a smaller protein compared to many other proteins and on a large pore C18 column should have no problem giving a nice peak. Given that, you haven't mentioned what matrix it is in and that can be challenging. You could try the molecular weight centrifugation filters and that can either capture your protein and everything greater than the molecular weight, or let it pass (for example MW cut off of 20,000). The latter lets all the small molecules through as well. Your sample should technically be about 50% cleaner at this point. This is a cheap alternative. A pack of these centrifuge filters is about $100. These things always seem so easy to the reader but can be challenging when you're the one who has to do it. Good luck.
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