Do you have any advice as I scale up?

Discussions about gel permeation chromatography / gel filtration chromatography / size exclusion chromatography

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I use a Waters Delta 600 system with 717plus A/S to do size exclusion chromatography.

I have been working on a purely analytical scale so far. I work with environmental samples of dissolved organic matter, which are extremely complex mixtures. Since molarities can't be defined for these mixtures the concentrations are in mgC/L. My samples are currently about 40 mg/L. I certainly spread the material out according the to molecular size of the solution components. My initial work with small injections and columns has been successful and we now are seeking to collect fractions as they elute off of the SEC column for hopeful analysis by other analytical techniques. We want to study some properties (like fluorescence) of the fractions as a function of molecular weight. At this time I get barely enough material coming off for any analyses. You could say I want to repeat the experiments on larger, preparative scale so I can get fractions containing more workable amounts of materials.

I will simultaneously move towards 1) injecting greater quantities of sample and and 2) increasing the amount of stationary phase in the flow path.

In the area of issue 1) I have two options.. I can either use more concentrated samples (up to 700 mgC/L) and continue to use the same configuration of my autosampler (100 microliter loop) or I can go to a 2.5 mL loop (with corresponding larger syringe) and keep my sample concentration at about 40 mgC/L.

Hence, my question to the chromatography community is this: Which manner of injecting more sample is chromatographically preferable? Small injections of highly concentrated sample, or larger injections of relatively lower concentration sample?

I am aware of one potential pitfall to increasing my loop size. I was told by Waters tech support that if I went to the larger loop/syringe that the relative variability on injection volumes would increase for small injections.

Also what do you think I should keep in mind as I explore larger columns? Similarly, I see two ways to increase the amount of stationary phase: Increase column inner diameter while keeping length the same or increasing column length while keeping ID constant. Any ideas about pros/cons of these two approaches?

with all things left the same except for the concentration of the sample or the injection volume you will get the same major effect
the "resolution"/ separation between your peaks will worsen. by how much depends on the chromatography you have
Because size exclusion has no "retention" mechanism per se, some of the rules of thumb you could otherwise apply are invalid. You *can* make the following generalizations:

1. If everything is working OK on the analytical scale, you can increase the loop volume in proportion to the column volume (i.e, double the column diameter => 4 times the volume; double the column length => double the volume). You *may* be able to push it a bit further, but how far you can go would have to be determined empirically (similar to item 4, below).

2. Increasing column volume by increasing length or by increasing diameter is pretty much a wash, since you *will* be overloading in any case and peak width will be affected far more by the overload than by the column's analytical-scale plate number.

3. Don't forget to scale the flow rate to the column cross sectional area (diameter squared).

4. The mass loading has to be determined empirically. Do a series of injections keeping the volume constant but doubling the concentration each time and see how far you can push it before the resolution deteriorates unacceptably.
-- Tom Jupille
LC Resources / Separation Science Associates
+ 1 (925) 297-5374
Thanks Tom,

Do you have any reference to point me to in regards to your point #3? I wasn't thinking that there would be any need to change the flow rate. Thanks, Dave
dkreller wrote:
Thanks Tom,

Do you have any reference to point me to in regards to your point #3? I wasn't thinking that there would be any need to change the flow rate. Thanks, Dave

Uwe Neue, HPLC Columns: Theory, Technology, and Practice pg 291 - "All scaling of all parameters of the separation should be in proportion to the column volume."
Time flies like an arrow. Fruit flies like a banana.
Could you give us some more information about the chromatography you're doing? For example, what column, or packing material are you using? What is the size of the column that you're currently using? What is the size range of the analytes you're separating? Are they proteins?

Thanks for your interest. As I mentioned in my first message: 'I work with environmental samples of dissolved organic matter, which are extremely complex mixtures.' Polydisperse materials with average molecular weight around 1000-2000 Da typically.

I typically use a 100mm x 3.2 mm ID column.

You say you're doing size exclusion chromatography in the initial post. I haven't seen many applications of size exclusion with molecules that small with such a narrow range of sizes. What type of stationary phase are you using? Have you considered that other factors besides size may be influencing the separation you're seeing? If so it may be advantageous to exploit those factors instead when scaling up.
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