Determining to for k value

[qrys]

How do I determine to for a new column? I have a home made column, made out of silica particles coated with PEG. Im running phosphate buffer with various salt concentration isocractically. The samples are pure proteins.

Since to is the elution time for unretained compound, that means for different protein samples I should have the same to value, although these samples were individually ran, right? I have read some papers defined to as the elution time when the subject protein is the least retained.

Thank you.

[Uwe Neue]

Yes, the approach of using the protein proper and getting it as unretained as possible is probably the best approach for the correct to of such a sample.

[qrys]

Uwe, what do you mean by using the protein proper?

If I have a column, a phosphate buffer mobile phase with various salt concentration, typically a protein is the least retained at low salt concentration. In my case, I have lysozyme and BSA. I found tr=0.7 min when lysozyme is the least retained, on the other hand, tr=.5 min when BSA is the least retained. So I will have different to for different protein, or I use to=0.5 for all proteins?

Thank you

[Uwe Neue]

You can expect a different t0 for the different proteins due to the fact that they have different sizes and are excluded to a different degree. Nothing wrong with having a special t0 for eahc protein...

[qrys]

I see! Thank you very much Uwe! Btw, I have your HPLC book, it is a very good HPLC book.

[qrys]

Uwe, I'm sorry to go back to this issue again after few months.

You said earlier in the previous posting,

".. the approach of using the protein proper and getting it as unretained as possible is probably the best approach for the correct to of such a sample."

my question is on getting a protein as unretained as possible. Does it mean I should stick to the phospate buffer or I can use other mobile phase for this purpose? For example, for lysozyme, on PEG column I obtained t0=0.7 min with phospate buffer and 0.1 M Na2SO4. On another column that has less PEG content I found that the time the lysozyme gets the least retained is 1.1 min. Both columns roughly have the same height. Does it mean on second column my t0 is 1.1 min? Or should I try other mobile phase to get the condition where lysozyme is the least retained?

My goal here is to compare the S and ko (from plot of log k vs [salt]) across different columns that have different PEG content on their surfaces.


Thank you

[Uwe Neue]

The best approach for the kind of theory that you are planning to do is to get a true unretained signal for the protein whose retention pattern you are investigating on the column that you are studying.

This does not help though in sorting out when the protein is really and completely unretained. The first thing to do is to compare the retention of the lysozyme to that of a small very polar molecule, such as uracil or dihydroxy acetone. If the lyszyme is less retained, you may be in a reasonable spot. If it is more retained, this retention is caused by unsuppressed interaction with the stationary phase. For lysozyme, the first suspect is ion-exchange with silanols. This is usually suppressed with increased salt, but this approach can be problematic because an increased salt concentration will cause the hydrophobic intereaction that you are trying to measure.
The interaction of the lysozyme with the surface silanols can also be suppressed with the addition of an amine to the mobile phase (keeping the pH constant). If you have silanols, it may then be difficult to get the amine out of the column again.

Another approach could be to select a substitute marker for lysozyme, selected on a column that does not show any interactions. An acidic protein, maybe, or a polar polymer, such as a polysaccharide.

Running out of ideas...

[qrys]

What about using t0 of a small molecule that is unretained in the column as the t0 of lysozyme or other proteins that are run through the column? Is this method good? I have seen a paper using sodium nitrate to determine t0 of their column. Thanks.

[Uwe Neue]

Don't use sodium nitrate. It is subject to exclusion effects.

Yes, a small molecule is a second choice, but you can't do proper S and k0 determinations. Such things do rely on a proper measurement of the V0 for analyte.

(Of course, I may be a bit to strict here, I have seen plenty of papers with weird V0s...)

[qrys]

Uwe, pondering on your previous comment,

"Another approach could be to select a substitute marker for lysozyme, selected on a column that does not show any interactions. An acidic protein, maybe, or a polar polymer, such as a polysaccharide."

I thought of 2 questions,

should I get the same t0 for an unretained small molecule and for an unretained protein in the same column? I think I shouldnt. I use porasil particles 125 A 15-20 um size. Lysozyme has a size of 19 x 25 x 43 A^3 (based on literature value) and this is so much bigger than a small molecule like acetone or urasil. And the size of the protein is comparable to the pore of particles. What do you think?

As you mentioned subtitute marker for lysozyme, you are thinking of having molecule of similar size to lysozyme? Which means size matters in determining t0?

Thank you

[Uwe Neue]

No, you are absolutely right: the t0 for a protein and a small moecule will be different and should be different. This is why I suggested up front to get the t0 for your protein, nad not use a small molecule. On the other hand, I understand the difficulty to get such a value.

Size is the important factor. You can try to find a rather inert molecule with the same size as the lysozyme. I suggested a polysaccharide. You now will have to figure out the equivalent size of a dissolved polysaccharide molecule and the lysozyme. You knw the size of the lysozyme, so that is a good start.

[qrys]

Uwe,

Can I use polyethylene glycol in place of polysaccharides to measure t0 as you recommended to me a while ago?

Thank you

[Uwe Neue]

Under conditions in which you are running the PEG column, PEG probes may be retained.

Also, you have to figure out which PEG molecular weight is equivalent to the size of your protein, which is not an easy task.

[qrys]

Uwe,

When I was looking for polysacharides sample to be use for measuring t0, I found out that they are not detected by UV/vis and we only have uv/vis detector in our lab. I was then interested in using small molc i.e. uracil to test in the column. I have problem in dissolving uracil. I have tried water, methanol, acetonitrile and phosphate buffer. Anyway uracil may get retained in the column as it's pKa ~9, so at pH 7 it's protonated and possibly would get attracted to the residual silanol. I then tried TFA 0.01% in water to run in the column. You mentioned before about using acidic protein. For some reason I obtained 2 peaks from the TFA sample. The values below indicates the first peak

mobile phase 100%MeOH: ~0.54 min
mobile phase 100% pure water (pH~5): ~0.54 min
mobile phase 100% phosphate buffer pH 7: ~0.71 min, however I notice a small dip on the chromatogram at ~0.54 min

column size 46 mm x 5.6 cm, flow rate 1 ml/min

Is it safe to conclude that my void volume/t0 is 0.54 min based on small molc measurement? One thing that I dont know is that the possibility of exclusion of TFA in the column.

the minimum retention time for lysozyme, RNAse A, trypsin inhibitor in this column is ~0.64 min.

Thank you

[Uwe Neue]

You would be able to see the polysaccharides in the low UV at 210 nm with acetonitrile as the organic modifier. Even with methanol, you "could" see them maybe at a slightly higher wavelength.

Uracil is NOT charged at acidic pH. It is a very weak acid, and it loses a proton at its pKa, which is around 9.5 if I remember correctly. Under normal LC conditions, it is not charged, and does not interact with silanols.

I just ran a set of data with uracil from 0 to 100 % methanol and acetonitrile. I did not have any solubility problems, nor does anybody else in my neighborhood.

Forget TFA!

A rough (!) estimate for the column dead volume of a C18 column is 66% of the column volume.