Chromatography Forum

Hello,

I'm working with pretty complex samples of urine and blood serum. Currently I'm running the samples with 1D reversed phase LC, but the complexity of these samples demands an extra separation dimension. Therefore I'm looking for some advice for selection of column for second dimension. I don't have a need for certain selectivity, just want to enhance the resolution.

Our current method that I plan to keep as a first dimension:
Column: Poroshell 180 EC-C18 4.6 x 150 2.6um;
Buffer: 50mM Formic acid, pH 4.25;
Gradient: MeOH 1-80%in 40min; Flow rate 0.6ml/min;

Current ideas for the second dimension:
Column: Polaris Amide C18 ?size?
Buffer: >50mM? Ammonium bicarbonate, pH ~7.5;?

System is Dionex U3000 with Bruker micrOTOF-Q MS/MS (connected thourh flow splitter).

There's a plan to run the 2D-LC analysis in online mode, all fractions of 1D through the 2D. Therefore I have some worries about the stability of pH in 2nd dim. Logically thinking, if the buffer concentration of 2nd dim should be x times higher of the 1st one, the pH chance should be quite marginal, right? The column of 2nd dim should be as orthogonal as possible and I found another column from Agilent there - Polaris Amide C18. My budget for the column shopping is limited and I have just one shot for a buy, therefore I'm not sure if Polaris would be the best hit...

If you haven't already done so, look at the PQRI column selectivity database on the USP web site (http://www.usp.org/app/USPNF/columns.html ; the PQRI database is about halfway down; it has more columns than the USP database at the top of the page; aside from that, either one will work). Pick your first column and search for the 10 most different columns. That will give you candidates with the best shot at orthogonality.

Orthogonality is a statistical concept; you;re looking at the probability of selectivity differences. That means there's no way to know *for sure* which two columns are the most different for your sample; you have to play the odds.

Depending on your sample, you may not need two different columns. Dealing with ionizable compounds, that big pH change will confer a degree of orthogonality. Or, to look at it another way, if you have a high degree of orthogonality in the columns, you might not need the different pHs. If money is tight, you might want to look at running the same column at low pH and then re-running your fractions on the same column at high pH. If nothing else, that may give you some insight into what you will need to do i the way of buffer concentrations.

Since you are (at least according the the mighty GOOGLE) working with uremic toxins most of the stuff you will be looking at is pretty hydrophillic. The best separation mode for hydrophilic compounds is HILIC.
There are a few ways that RP and HILIC has been utilized to gain complementary information in metabolomics.

Poor mans 2D: C-18 SPE is used to divide the sample into a hydrophilic (not bound to SPE) and a hydrophobic part (bound to SPE) the two parts are then analysed by RP for the hydrophobic and HILIC for the hydrophilic part.
REF.
Metabolic Fingerprinting of Rat Urine by LC/MS: Part 1. Analysis by Hydrophilic Interaction Liquid Chromatography-Electrospray Ionization Mass Spectrometry
H. Idborg, L. Zamani, P.-O. Edlund, I. Schuppe-Koistinen, S. P. Jacobsson
J. Chromatogr. B, 828 (2005) 9-13

Four injections of same sample
The sample is analysed by
RP-MS positive
RP-MS negative
HILIC-MS positive
HILIC-MS negative
REF
Hydrophilic Interaction Chromatography for Mass Spectrometric Metabonomic Studies of Urine
S. Cubbon, T. Bradbury, J. Wilson, J. Thomas-Oates
Anal. Chem., 79 (2007) 8911-8918

Full 2D
I do not have any good ref but for proteomics these papares have been published.
Evaluation and Optimization of ZIC-HILIC-RP as an Alternative MudPIT Strategy
P. J. Boersema, N. Divecha, A. J. R. Heck, S. Mohammed
J. Proteome Res., 6 (2007) 937-946
and
Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC and ZIC-cHILIC) provide high resolution separation and increase sensitivity in proteome analysis
S. Di Palma, P.J. Boersema,A.J.R. Heck,S. Mohammed
Analytical Chemistry 83, (2011), 3440-3447

Creative man 2D - mixed-mode and column/guard combination:
http://www.sielc.com/Technology_2D_Properties.html

and the approach for your separation;
http://www.sielc.com/upload/file/pdf/SI ... r_2004.pdf

contact me if you would like me to help you designing the procedure.

Thank you for your advices, I appreciate it a lot

If you haven't already done so, look at the PQRI column selectivity database on the USP web site (http://www.usp.org/app/USPNF/columns.html ; the PQRI database is about halfway down; it has more columns than the USP database at the top of the page; aside from that, either one will work). Pick your first column and search for the 10 most different columns. That will give you candidates with the best shot at orthogonality.

Yes, the PQRI website helped me to find out the Amide C18 column, so this should have the most orthogonal separation.

Depending on your sample, you may not need two different columns. Dealing with ionizable compounds, that big pH change will confer a degree of orthogonality. Or, to look at it another way, if you have a high degree of orthogonality in the columns, you might not need the different pHs. If money is tight, you might want to look at running the same column at low pH and then re-running your fractions on the same column at high pH. If nothing else, that may give you some insight into what you will need to do i the way of buffer concentrations.

Currently I can play a bit and find the most suitable way for future routine work, so I will test both ways - keeping the column same and changing the pH; and chancing the column and keeping the pH; and just for fun - changing both the column and pH.

Since you are (at least according the the mighty GOOGLE) working with uremic toxins most of the stuff you will be looking at is pretty hydrophillic. The best separation mode for hydrophilic compounds is HILIC.
There are a few ways that RP and HILIC has been utilized to gain complementary information in metabolomics.

HILIC has been in my sight for a while but there are few disadvantages that do not fit with our work. We're running routinely hundreds of samples for creating the statistical database of metabolites/uremic toxins and this makes the running costs quite critical. HPLC grade AcN is unfortunately too expensive (starting 130€/L). Also large amounts of waste AcN does not fit well with the terms of "green chromatography" (meaning extra costs for the waste disposal)... So I had to find alternatives for HILIC.

here is the way to address ACN issue
http://www.sielc.com/upload/file/pdf/SI ... l_2009.pdf

We wrote this newsletter when ACN was 4 times more expensive than now. Mixed-mode is 2D and it is alternative to RP and HILIC.

Thank you Vlad for interesting suggestions.

Creative man 2D - mixed-mode and column/guard combination:
http://www.sielc.com/Technology_2D_Properties.html

This 2D chromatogram is probably done with a software solution. I did not find any information about software products on your website - is there any commercial product also for this "2D-software-LC" solution? I had an idea of similar software 2D-LC, however with our complex samples it turned out to be quite difficult.

and the approach for your separation;
http://www.sielc.com/upload/file/pdf/SI ... r_2004.pdf

It is an interesting idea, will test it as well.

To jurgen.arund.

Somehow this one fits in the context: “If it ain’t broke don’t fix it”.
Your quest - as you put it - is: I don't have a need for certain selectivity, just want to enhance the resolution.
2d dimension implies that you need to selectively separate something that is not separated under the 1th dimension run. But it’s not what you need – is it?

And now to your particular need: There are different ways of resolution enhancement. Longer column for one. Temperature optimization (2) flow rate optimization (3) (the latter is not optimal in your case and should be increased by 40 – 50 %, ion strength (4) etc.
So, do it right and you’ll save money and time – as well as potential trouble.

Best Regards

Somehow this one fits in the context: “If it ain’t broke don’t fix it”.
Your quest - as you put it - is: I don't have a need for certain selectivity, just want to enhance the resolution.
2d dimension implies that you need to selectively separate something that is not separated under the 1th dimension run. But it’s not what you need – is it?

Currently our 1D-LC method does not offer sufficient resolution for using our chromatography results in our large scale statistical analysis. For large scale database creation for statistical analysis we need automatic chromatography data processing. Current chromatography data processing software (I'm using Chromeleon 7.1) do not offer enough reliability for peak recognition and identification of tens of larger peaks and couple of hundreds of smaller peaks, where too many poorly resolved peaks interfere the efficiency of current commercial chromatography data processing algorithms. With the 2ndD-LC for all of the time fragments from 1stD-LC we hope that the conclusive resolution is sufficient enough to apply automatic data processing algorithms and to get reliable data output (I need to do some software development for that). Therefore I would say our current 1D-LC-based method is still "broken" and method as whole needs some enhancement.

And now to your particular need: There are different ways of resolution enhancement. Longer column for one. Temperature optimization (2) flow rate optimization (3) (the latter is not optimal in your case and should be increased by 40 – 50 %, ion strength (4) etc.

With the first dimension we have done the large scale fine tuning: temperature optimization, flow rate optimization, pH fine tuning. I haven't done the tuning for ion strength yet. I've done some column testings with various C-18 columns, latest ones were with solid core particles from Phenomex (Kinetex 2.6um particles) and Agilent (Poroshell 120 2.7um particles), both 4.6mm x 150mm, which is the largest available. Poroshell 120 turned out to have the best resolution of all of the columns we tested. I had an idea to get a column with smaller inner diameter, but then we should have limited the amount of injected sample, which would have decreased the signal intensity of MS too low for the compounds of interest with very low concentration. Therefore this idea stayed as an idea. The 1stD-LC had to be as universal as possible for quantitative analyze of wide chemical spectrum of uremic solutes; for using large scale routine work; as well for using with ESI-MS.

Kind regards

it is not software, just demonstration how mixed-mode provide you with a 2D on a single column. If you compounds are ionic and even slightly hydrophobic mixed-mode will give you the biggest resolution possible in most of the cases, since it is exploring small differences in properties. This differences are enhanced due to synergy of at least two mechanisms.