I was wondering if anyone who is running 2D chromotography can give some rough estimates on how long it takes them to run one fraction. This would inlcude sample loading times, buffer loading times, washing, and Elution off RP columns.

Prof Phillip Marriot has succeeded in GC x GC and is venturing into multidimensional LC methods of HPLCxHPLC. The researcjh is in collaboration with AsProf Mike Guilhaus; Waters Corporation.

Try Phil on: pjm@rmit.edu.au

CV = http://www.across.utas.edu.au/per-rmit.html

Luke Roenneburg,
are we thinking about the same thing, namely, running a sample through a column, collecting a desired portion of the eluate and running this through a second column, etc.? If so, there is nothing different, per column, than doing one column (one dimension, or one step) chromatography. One can sometimes interpose whatīs going on on the different columns to save time. The real problem that one faces is that the eluent of one column must be compatible with the chromatography of the next column.

nic,
sounds like something is new here? What? Twodimensional (I prefer to call it two-step) GC (Deanīs switching...) was old hat when I started with fatty acid analysis in 1977.
Multi-step HPLC is monographically presented in Frei, Zech, J Chrom Library, vol. 39A, 1988, especially the article by Huber and Zech on p. 81. This article inspired multi step HPLC in this laboratory shortly afterward.
Gilson had mashines on the market by the 1980īs for doing this automatically, even including an ultrafiltration step.
Now if the world is really being reinvented I am with it.

I agree with H.W. Mueller that nothing has been lately invented, I would rather say that most of them are revisited due to current biological analytical needs. In the case of "multidimensional" chromatography the extended analytical needs in the proteomic field is the main reason (I would say that capillary chromatography have been bousted from that as well).

As a result, several terms that were not very common became very popular like "peak capacity" and "orthogonal separations". Adequate credits are given though to the pioneers (like Giddings) and nobody claims that presents something new while reinvented the wheel.

Now lets come back to the initial question which has not been yet answered which was how long it takes to run a fraction in 2D chromatography...

I'll answer that it depends, and people have made 2-D systems with an analysis per fraction time from 4 min. (six SEC columns, 2 RP columns / Opitec et al. Anal. Chem. 69, 1997, 2283)to at least 1.5 hours (speaking always for on-line systems).

However proteome complexity is so high, that even these approaches can not handle very well and people have start speaking about 3D chromatography and so on. Main problems is the dymanic range of the expressed proteins, the huge number of peptides yielded after tryptic hydrolysis etc. The real chalenge will be of course to deal with comparative proteomics where you should inject your sample at least 3 times (which will rapidly eliminate the proposed 2D methods with an overall cycle time (analysis of one sample) of a day or so.

Hope the above helps,

Kostas

PS: If you need more references you may contact me directly.

Mr. Mueller,

The basic types of applications I am refering to our using ion exhange separation followed by Capillary RP columns all online. There have been a large number of papers on this all of which seem to me to be time consuming and complicated plumbing schemes. I have been working with new methods to shorten the run times and provide much less plumbing. Basically to make this process more efficient and easier to work with.

I have spoke with some people and seen various methods for achieving the above concept. Some methods take longer than others but basically the ion exchange column is (sometimes) larger 1mm or 300 um ID than the RP (usually 75 um) and in most cases a 3rd cartridge column is incorporated for pre-concentration and de-salting prior to RP. To perform all these steps can take a significant amount of time. Some people I have spoke with are spending almost an hour just to prepare the fraction. This is in addition to the RP elution time which can also be close to an hour to achieve good separation.

What is a fraction here?
The series chroms we did (one published) were performed with conventional RP columns and/or ultrafiltration in three steps (columns in line, manually switched). Though I have no experience with the described systems, it seems self evident that one should strive for an optimum separation of each column, for the purpose on hand. That is what determines the time. If you cut corners you may need an additional step (column).

I have not yet seen any well optimized 2-D HPLC to date. If one thinks the problem through, one will discover that the real limitation is the slowness of the detectors that are available for this task. Of course, you can beat it with sufficient money and put multiple MS systems downstream....