Chromatography Forum

Dear LC-Forum experts,

I need use 2-dimentional chromatography to be eliminate interference
from some blood-serum samples of end-stage renal disease patients.
First column is silica HILIC/Trifluoroacetic acid/MeCN/H2O and the other
is ReversedPhaseModePrimesep200/Trifluoroacetic acid/MeCN/H2O.

Sample, after deproteinization with HCLO4 and diluted with MeCN, is well
separated on the HILIC column with peak retention time of 7.1 min.
Interference size is ~ 0-10% of usual peak size, and not visible.

Instrument is Ultimate 3000 microHPLC with dual 3-gradient pumps and
autosampler. Theare are both 2-swithable 10-port Rheodyne valves
controlled by Chromeleon. Left is not in usage. Right valve is connected
to autosampler and 2 columns, by serviceman as 1-2 and 10-1 modes.
This may not to be change.

I have some examples of computer Programs on Chromeleon.

My problem is about how to made connections to columns and outputs
to be make 2-dimentional chromatography using above free left valve?

sincerely

You need the brochure that comes with the Rheodyne (or maybe a catalog) to see how the ports are related to the slits on the rotor. With the help of plumbing diagrams (which you should find in the mentioned material) you can figure out how to connect the columns.
Are you sure that your mobile phases are at an optimum?

I think you told about rheodyne 7610 valve.
Best way to take manual http://www.rheodyne.com/pdfs/2320662a.pdf

Actually, we have a free LCi Solutions CD that should hold all the information you are looking for.
I'll get one to you.

Dear Dr Mueller, Elsico & Wulffniedner,

Thank you for suggestions. I try to construct Flow Diagram for 2-dimentional HPLC.

http://s3.tinypic.com/1zqaik8.jpg

Flow diagram for 2-dimentional LC, using a Model 7610 Rheodyne
2-Position, 10-Port Left-Valve and Model 7610 Rheodyne 2-Position,
10-Port Right-Valve, connected to autosampler in 1-2 Mode.

Using Position B Autosampler inject 25 ml sample into HILIC column.
Flow is acc. to black line (ports 1,10,7, 6). Reversed phase is pumped
from Pump B across into 3,2,5,4 into PrimeSep200 column until to
detector acc. to red line. Connection 2-5 is Rheodyne sample loop.

Using Position A at time between 6.9-7.2 min., peak from sample is filled out the loop 2-5

Using Position B, again, after 7.4 min sample in 2-5 loop is directed to
the PS200 analytical column and to HPLC detector.

sincerely

To check whether your switching is correct you can inject a standard directly into the PrimeSep, then do an identical injection into your 2-step system. The chromatograms should be identical after any corrections due to time delays caused by "transporting" the sample onto the second column.

I'm just received attractive article from Prof. Pawel Jandera J Sep. Sci.
31, 1421-37, 2008. Figure 3 and Figure 4 are display 10-port valve for
2-dimensional LC using trapping pre-columns or without trapping columns,
respectively.

I need to solve 2 problems:

1) Interest peak fraction from 1-st dimension takes 1 minute * 0.7 ml/min.
= 700 microliters capacity for this trapped fraction. I could change
column diameter from 4.7 mm to 2.1 mm to be obtaining smaller
elution volume.
2) I need use HILIC as first before ODS because any extraction of serum
sample is not practical and only HILIC column is resistant to lipid
inpurities. But PrimeSep200 or 100 column serves more separation
mechanisms.

I am a bit confused, so here is just a very general hint: One chooses a 1st column to separate out the group of analytes you want to do in such a way that the mobile phase does not cause chromatography on the second column (you want the "heart cut" from the 1st column to get stuck at the beginning of the 2nd column). This way you practically don´t need to limit the sample volume coming off the first column. The chromatography on the 2nd column is then done with an optimum mobile phase for that job.