Chromatography Forum

i saw some SEC column has pore size down to similar pore size of C18 column, I just do not understand. so C18 column also can behave as sec column? I thought we need to have big pore size in SEC column to differentiate big molecules. it seem like it is not case at all.

if pore is very small, then we need much higher plate count to resovle different molecules since difference between vi and vt will be very small.

i saw some SEC column has pore size down to similar pore size of C18 column, I just do not understand. so C18 column also can behave as sec column? I thought we need to have big pore size in SEC column to differentiate big molecules. it seem like it is not case at all.

The SEC separation depends on three independently controllable properties: the pore size and the specific pore volume of the packing, and the surface of the packing, which needs to show no interaction with the analyte.

Since the pore volume is the only space where separations are happening, manufacturers will maximize the pore volume of SEC materials. Thus a packing that covers the molecular weight range from 100 to 5000 has the same pore volume as one that covers the MW range from 5000 to 600 000. Packed inside the column, the pore volume ranges from somewhere around 35% to maybe up to 80% of the column volume. This is your separation space, and you can enlarge it by putting columns in series.

The MW separation range of a packing is determined by the size of the pores. Packing materials range from very small pore sizes to extra-ordinarily large pore sizes. With the smallest pore size, you can separate closely related molecules, such as a styrene monomer from its dimer, trimer etc... An example is the Styragel HR 0.5 packing. The largest packings have pores that are comparable in size with the spaces between the particles. Again, an example is the Styragel HR 6 packing, whose separation range vastly exceeds MW 10 000 000 for polystyrene standards.

In SEC, you do not want any interaction of the analyte with the surface. Therefore you can find packings that are rather hydrophobic and are used in non-aqueous solvents, as the Styragel family mentioned above, or packings that are very polar and designed for the size-exclusion chromatography of proteins. We just introduced a UPLC version of a packing for the SEC separation of IgG dimers and higher from the monomer.

Hope this clarifies things. As always, you have the option to read Chapter 8 of my book on "HPLC Columns" for more information...

To answer your question if a C18 packing can work for SEC: in principle yes, but it was not designed for this purpose and may have many disadvantages compared to a real SEC packing, mostly a smaller pore volume and a limited MW range.

To your second question: the pore volume does not vary with the pore size of the packing. These are two independent parameters, as explained above.

thanks a lot. the following is my understanding. please correct me if anything wrong.
based on your instruction, pore volume determine vt-vi
pore size determine separation range,
so particle size determine plate count,
with pore volume and particle size, you can determine peak capacity.

should pore size, pore volume and particle size have some relationship when you design the SEC column?
for example, when particle size go down to 3 or 5 micron, will this small particle impose some limitation on your pore volumn or pore size?

The SEC separation depends on three independently controllable properties: the pore size and the specific pore volume of the packing, and the surface of the packing, which needs to show no interaction with the analyte.

Since the pore volume is the only space where separations are happening, manufacturers will maximize the pore volume of SEC materials. Thus a packing that covers the molecular weight range from 100 to 5000 has the same pore volume as one that covers the MW range from 5000 to 600 000. Packed inside the column, the pore volume ranges from somewhere around 35% to maybe up to 80% of the column volume. This is your separation space, and you can enlarge it by putting columns in series.

The MW separation range of a packing is determined by the size of the pores. Packing materials range from very small pore sizes to extra-ordinarily large pore sizes. With the smallest pore size, you can separate closely related molecules, such as a styrene monomer from its dimer, trimer etc... An example is the Styragel HR 0.5 packing. The largest packings have pores that are comparable in size with the spaces between the particles. Again, an example is the Styragel HR 6 packing, whose separation range vastly exceeds MW 10 000 000 for polystyrene standards.

In SEC, you do not want any interaction of the analyte with the surface. Therefore you can find packings that are rather hydrophobic and are used in non-aqueous solvents, as the Styragel family mentioned above, or packings that are very polar and designed for the size-exclusion chromatography of proteins. We just introduced a UPLC version of a packing for the SEC separation of IgG dimers and higher from the monomer.

Hope this clarifies things. As always, you have the option to read Chapter 8 of my book on "HPLC Columns" for more information...

To answer your question if a C18 packing can work for SEC: in principle yes, but it was not designed for this purpose and may have many disadvantages compared to a real SEC packing, mostly a smaller pore volume and a limited MW range.

To your second question: the pore volume does not vary with the pore size of the packing. These are two independent parameters, as explained above.

You will get into particle size limitations only for the largest pore packings, where the size of the pores in the packing approaches the particle size. A 30 nm pore packing has pores that are 100 times smaller than a 3 micron particle, so from the standpoint of the specific pore volume, the particle size does not make any difference.
You will get better plate counts for smaller particle columns, and better separations in a shorter time.

So in theroy, I can narrow my pore size a lot to separation analytes MW smaller than 1000 based on size. it should be very very small, down to single digit nm diameter.
why I did not see any product that works well in this range?

You will get into particle size limitations only for the largest pore packings, where the size of the pores in the packing approaches the particle size. A 30 nm pore packing has pores that are 100 times smaller than a 3 micron particle, so from the standpoint of the specific pore volume, the particle size does not make any difference.
You will get better plate counts for smaller particle columns, and better separations in a shorter time.

Because you did not look...

Look here: http://www.waters.com/webassets/cms/lib ... 0205en.pdf

for HR 0.5, how large MW difference they can separate. can it separate from 100 to 200.
what kind of typical application of this product?

Because you did not look...

Look here: http://www.waters.com/webassets/cms/lib ... 0205en.pdf

The typical application for Styragel packigns is the molecular weight analysis of polymers. This specific application can be run with THF as a solvent or DMF as a solvent, or closely related solvents.

For an analysis from water, you can use the equivalent packings for aqueous analysis, which is Ultrahydrogel 120.

And yes, you can separate MW 100 from MW 200 on either one of these columns. You can see this from the calibration curves, which can be found in the catalogue or on-line.