protein isocratic HPLC..preparatin of tris buffer+n-propanol

[alicee!]

hi all.. my protein isocratic HPLC.. involves a preparatin of tris buffer + n-propanol mix as a mobile phase.. due to variations in buffer preparation.. i m getting variations in Retention time..

how does one make such a buffer mix??
i have done volume measurement in a measuring cylinder calibrated well..

let me know!!

thanks!

[danko]

In my humble opinion, the procedure you’re describing is OK.
Could the variation originate from the TRIS buffer itself?
Have you tried to mix several (2 – 3) portions of the same (same preparation) buffer with n-propanol and if so; did you se any variation in the retention time?
It could be the buffer pH (differences in pH from preparation to preparation).
How do you mix these to liquids – more precisely? Do you fill the cylinder with the first liquid to a certain volume and then fill it up to a final volume with the second liquid? Or du you measure both liquids separately and then mix them? The latter is the procedure of my choice. So, it might be that different coworkers use different procedures.

Best Regards

[sassman]

This might help you:

http://www.analyticalchem.com/?q=buffercalc

[Uwe Neue]

What type of chromatography are you doing? Ion-exchange? Reversed-phase?

[alicee!]

In my humble opinion, the procedure you’re describing is OK.
Could the variation originate from the TRIS buffer itself?
Have you tried to mix several (2 – 3) portions of the same (same preparation) buffer with n-propanol and if so; did you se any variation in the retention time?
It could be the buffer pH (differences in pH from preparation to preparation).
How do you mix these to liquids – more precisely? Do you fill the cylinder with the first liquid to a certain volume and then fill it up to a final volume with the second liquid? Or du you measure both liquids separately and then mix them? The latter is the procedure of my choice. So, it might be that different coworkers use different procedures.

Best Regards

i'm doing an isocratic reverse phase chromatography..
with different proportions of 1-propanol.. i see variations in retention time.. and resolution..

i consider the latter.. any ideas on how else i can reduce the variation in the preparation..
i see that if i get a low retention time.. and then i add a li'l propanol the existing buffer.. i see a high retention time and better resolution.. i need consistency!!

[danko]

As I understand/realize you’ve been filling up to final volume in the same cylinder. And I believe that’s the source of the variation you’re experiencing.
So, if you follow the second (in my mind most appropriate) procedure i.e. measuring both liquids separately and then mixing them, I would expect a much better reproducibility.

Best Regards

[alicee!]

As I understand/realize you’ve been filling up to final volume in the same cylinder. And I believe that’s the source of the variation you’re experiencing.
So, if you follow the second (in my mind most appropriate) procedure i.e. measuring both liquids separately and then mixing them, I would expect a much better reproducibility.

Best Regards

okay i shal try this multiple times and see..
but why would the variation come if i made up the volume? why?

thanks!

[alicee!]

As I understand/realize you’ve been filling up to final volume in the same cylinder. And I believe that’s the source of the variation you’re experiencing.
So, if you follow the second (in my mind most appropriate) procedure i.e. measuring both liquids separately and then mixing them, I would expect a much better reproducibility.

Best Regards

okay i shal try this multiple times and see..
but why would the variation come if i made up the volume? why?

thanks!

[danko]

but why would the variation come if i made up the volume? why?

The keywords here are mixing and volume change upon adding organic solvent to the aqueous liquid. There is even a temperature consideration involved in this case.
The first: complete mixing is not possible in a cylinder, unless there is some sort of mixer involved in the procedure and I don’t believe that is what you’re doing. So you’re filling up to virtually unknown final volume.
The second: When mixing organic and aqueous liquids the end volume is not the sum of the two volumes. The final volume will most probably be less than the sum. So, when adding the one on the top of the other (without an active mixing) there will be some mixing going on in the interface between the two liquids and you are not in control of the process. It is dependant on the diameter of the cylinder, how fast the second liquid is added etc.
The third: Depending on the liquids, there will most often be temperature changes in the mixture. In some cases worming up while in other cases there will be cooling down. In turn the temperature changes will introduce volume variation, which you do not control, for the same reason as mentioned above.
All in all partial mixing introduce volume variations, thus inconsistent final volume, resulting in inconsistent mixtures.

Best Regards

[alicee!]

but why would the variation come if i made up the volume? why?

The keywords here are mixing and volume change upon adding organic solvent to the aqueous liquid. There is even a temperature consideration involved in this case.
The first: complete mixing is not possible in a cylinder, unless there is some sort of mixer involved in the procedure and I don’t believe that is what you’re doing. So you’re filling up to virtually unknown final volume.
The second: When mixing organic and aqueous liquids the end volume is not the sum of the two volumes. The final volume will most probably be less than the sum. So, when adding the one on the top of the other (without an active mixing) there will be some mixing going on in the interface between the two liquids and you are not in control of the process. It is dependant on the diameter of the cylinder, how fast the second liquid is added etc.
The third: Depending on the liquids, there will most often be temperature changes in the mixture. In some cases worming up while in other cases there will be cooling down. In turn the temperature changes will introduce volume variation, which you do not control, for the same reason as mentioned above.
All in all partial mixing introduce volume variations, thus inconsistent final volume, resulting in inconsistent mixtures.

Best Regards

how exactly i prepare the buffer is..
i take 21 ml of n-propanol..
79 ml of aqeous buffer..
in separate cylinders..
pool them.. stir them for 15 minutes.. then sonicate them.. for 5 mins.. and then put it for chromatographyy...
all at room temperature..

[danko]

how exactly i prepare the buffer is..
i take 21 ml of n-propanol..
79 ml of aqeous buffer..
in separate cylinders..
pool them.. stir them for 15 minutes.. then sonicate them.. for 5 mins.. and then put it for chromatographyy...
all at room temperature..

If that is what you’re going to do, then I must say; it looks fine to me.
I would stir for 5 - 10 minutes and sonicate for 15 min. if I were you, just to ensure adequate degassing.

Best regards

[Uwe Neue]

Contrary to other types of chromatography, it is very difficult, close to impossible even to get reproducible isocratic retention of proteins in reversed-phase chromatography. Of course, this depends on the size of the molecule, and a few other things, but the chances are small that you will be successful. On the other hand, you can run reasonably flat gradients. Now, the reproducibility depends on the gradient setup and the quality of the gradient mixing in your instrument.

[alicee!]

yess i m facing a lot of problems on reproducibility..
i have chosen to fix a particular resolution/retention time.. and add propanol or buffer after my first run.. to adjust this. balancing.. its a bad way of doing it.. i konw! that is why i ask for suggestions..

i have even tried weighing the solutions.. based on density calculations.. but even that gives varition..

the reasons for so much variation in isocratic rp?
also.. i see that my protein.. and its reduced and other forms.. do not resolve on an acetonitrile gradient.. they only resolve on the isocratic rp.. why so?
actually i fail to completely understand the principle/reason behind this isocratic rp.. and if something resolves on isocratic.. it shud on a gradient too right? dunno.. any reasons?

and if i may quote
"Reversed-phase high-performance liquid chromatography (RP-HPLC) was utilized for the separation of recombinant human growth hormone (hGH) variants on a C18 silica column at 55°C using an isocratic mobile phase which contained 27% 1-propanol in a 25 mM potassium phosphate buffer, pH 6.5. Three of the obtained peaks were characterized by tryptic mapping and mass spectrometry; two of the peaks were found to contain oxidized hGH (dioxy Met14/Met125 and Met125 sulfoxide) while the third contained a deamidated form (Asn149→Asp149 or Asn152→Asp152). Compared to the European Pharmacopoeia RP-HPLC method of hGH analysis, this new method gives two additional peaks and a 50% reduction in the analysis time"

i see a lot of references on this protein.. all refering to isocratic n not gradient.. WHY?

thanksss!!

[danko]

Alicee wrote:

the reasons for so much variation in isocratic rp?

The isocratic mode setup can not be the source of variation, you’re experiencing.
If you use the same column and mix the eluent components properly, you shouldn’t be witnessing any variations – whether it is god or bad chromatography in terms of separation.
So I’ll suggest to you, to find and eliminate the variation (could be air in the pump, malfunctioning equipment, variation in pH and last but not least column temperature control)
Once you’ve fixed this problem you can begin concentrating on separation, efficiency, or whatever your success criteria are.
Concerning the elution mode, I’ve done some pretty good protein separations in isocratic mode RP and I know that hGH is run in isocratic mode some places, so it’s certainly not an impossible task. But if I were you I’d use in-line mixing of aqueous and organic during the development/testing you’re conducting. Anyway until I find the most promising composition. And even then, I’d premix these two liquids in a non final composition in order for me to have the possibility of adjusting retention time when needed (with a new column etc.)
Finally, as Uwe wrote, you might like to try the gradient option, which in many instances would be the most efficient one.

Best Regards

P.S. If you need some more advices/suggestions, you might like to describe your current situation/setup and maybe provide a chromatogram or two.

[lmh]

How reliably do you think you can measure 21mL in a measuring cylinder?

Are all your measuring cylinders the same? (Really the same, not just looking fairly similar and coming from same manufacturer)

Sorry if this is a double post - I'm getting the dreadful "Service Not Available" error on this forum.