-
- admin
- Site Admin
-
- Posts: 647
- Joined: Wed Aug 11, 2004 12:19 am
By VZM on Monday, June 7, 2004 - 08:14 am:
Hi
I am new to HPLC. I am trying to purify a 14kD protein on a C4, rp column. When I really slow down the acetonitrile gradient (1%/3min at 1mL/min), the peak getts very very broad (20 min). I was expecting an on/off effect. What should I try to improve resolution?
Thanks for your help!
ps. Also, is it safe to mix 50%MeOH50%H2O with 5%Acetonitrile95%H2O0.1%TFA on a C4 column, HPLC system?
-------------------------------------------------------------------------------------------------------
By Anonymous on Monday, June 7, 2004 - 03:08 pm:
There is no on/off effect; peaks get wider when you run slow gradients.
-------------------------------------------------------------------------------------------------------
By Anonymous on Monday, June 7, 2004 - 09:39 pm:
Dont keep more of aqueous phase in your C4 column because as u go from c18 to c8 to c6 to c4 the column becomes more and more suitable for normal phase chromatography. Change ur column to C18 if u want to use more of aqueous phase
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, June 8, 2004 - 02:53 am:
Get nano hplc
-------------------------------------------------------------------------------------------------------
By MG on Tuesday, June 8, 2004 - 07:03 am:
The original poster is trying to purify a protein. I'm not sure why nano-LC would help, since a much smaller amount would be loaded onto the column.
VZM, as you go to very shallow gradients, you begin to approach isocratic conditions. In isocratic methods, peaks get broader the longer they stay on the column. If you cannot get the resolution you need by increasing retention, you can go to a longer column, smaller particle size, different mobile phase modifiers (either changing pH or changing organic modifier), or changing the stationary phase type. I see no danger in mixing those solvents you ask about.
What did you mean by on/off effect?
-------------------------------------------------------------------------------------------------------
By VZM on Tuesday, June 8, 2004 - 07:40 am:
Hi again,
Thank You very much for your responses!
Could someone suggest a resource that outlines examples of different stationary phases, mobile phases, and modifiers that can be tried?
Right now I am using an acetonitrile gradient with 0.1%TFA.
Also, could someone tell me how long a column should be equilibrated for before and after loading the sample?
Thank You again,
VZM
-------------------------------------------------------------------------------------------------------
By MG on Tuesday, June 8, 2004 - 08:41 am:
Disclaimer: I usually handle small molecules, not proteins, so my suggestions should be considered from the standpoint of general chromatography. I have heard that proteins can behave quite differently, and there are others here with more experience with proteins who can hopefully add to or correct these suggestions.
Stationary phases: In addition to C4, there's C8, C18, CN, "phenyl" and others for reverse phase. All of them can have different selectivity, either giving you better or worse resolution under equivalent conditions. For proteins, you'll need a large pore size (like 300 angstrom), else your compound will be excluded from the pores. C4 seems like a popular choice for proteins, and there may be good reasons for this. E.g. I understand that proteins can be very strongly retained on C18 colums.
Mobile phases: It is easy to try methanol in place of acetonitrile as the organic modifier, and at least with small molecules, this will often cause a significant change in selectivity (either for better or for worse). I have developed methods where mobile phase "B" was a mixture of methanol and acetonitrile, for optimum selectivity. You can also add isopropanol in some proportion to your acetonitrile; selectivity will be similar to methanol but isopropanol is stronger in terms of elution strength. Both methanol and isopropanol will raise your backpressure, and with high isopropanol concentrations it may become intolerable.
pH modifiers: TFA seems to be the popular one for proteins. There have been discussions on this forum as to whether TFA functions as an ion pair reagent. Alternatives are acetic and formic acids, and these can be mixed with their ammonium salts to adjust the pH. For non-MS work, there may be other alternatives I have neglected.
column equilibration: The time depends on your flow rate and the dimensions of your column. In terms of volume, the rule of thumb I use is 5 to 10 column-volumes at initial conditions before injecting the sample and starting the next gradient.
-------------------------------------------------------------------------------------------------------
By VZM on Tuesday, June 8, 2004 - 09:05 am:
Hi,
Thank You for the suggestions. I have couple of more questions.
Regarding getting rid of the acetonitrile TFA after the purification. I have tried flash freezing in liquid N2, followed by O/N vacuum at 60mT. This doesn't seem to gett rid of everything, meaning the product is still acidic. I have read papers that cite rotovaping the sample, prior to lyophilization, but am not sure exactly what is required (time / vacuum) to remove the acetonitile / TFA, or what rotovaping accomplishes over freeze drying.
Also supposing that I try methanol or isopropanol, how could I get rid of that?
Thank You,
VZM
-------------------------------------------------------------------------------------------------------
By RH on Thursday, June 10, 2004 - 10:42 am:
VZM, what is the purpose of your purification? If
you don`t need the protein in its natural
structure, as I think as you use organic
modifiers, how much substance do you load onto
your column (which dimensions, particle size,
pore size?) in what kind of solvent? And can you
give a hint about the protein of interest, mol.
weight, pk, hydrophobicity ,prostehetic groups?
-------------------------------------------------------------------------------------------------------
By VZM on Thursday, June 10, 2004 - 11:09 am:
Dear RH,
It is 14kD protein with a pI ~5.5. It has no modifications. It is not very hydrophobic. It is however, very unstable.
The purpose of the purification is to purify (on a preparatory scale ie 10mg) the protein away from similar degredation products.
IEX, HIC, SEC do not accomplish this, so I decided to try RP HPLC. I have tried a C4 5um particle size, 3.9x150mm column, using an
acetonitrile gradient in 0.1%TFA. I do not get the
resolution I need, meaning that the contaminants are still present and the peak is very broad.
Thank You for your help,
VZM
-------------------------------------------------------------------------------------------------------
By A.Mouse on Thursday, June 10, 2004 - 07:20 pm:
Is this a C4 column recommended for proteins? You need a larger pore size to get narrow peaks for proteins.
-------------------------------------------------------------------------------------------------------
By Anonymous on Saturday, June 12, 2004 - 07:53 pm:
Hello VZM,
I used to purify peptides of about 6kD and I got good results with Phenomenex Phenyl-hexyl column (50 x 250mm, 300A)at 50mL/min, a gradient 0.1% TFA WATER: 0.1% TFA ACN of about 10 to 50% over 120, at 214nm.
ACN is preferd to MeOH because of it's volatility, low cut off and and low viscosity, and TFA fits very well to these conditions.
Good luck
Hi
I am new to HPLC. I am trying to purify a 14kD protein on a C4, rp column. When I really slow down the acetonitrile gradient (1%/3min at 1mL/min), the peak getts very very broad (20 min). I was expecting an on/off effect. What should I try to improve resolution?
Thanks for your help!
ps. Also, is it safe to mix 50%MeOH50%H2O with 5%Acetonitrile95%H2O0.1%TFA on a C4 column, HPLC system?
-------------------------------------------------------------------------------------------------------
By Anonymous on Monday, June 7, 2004 - 03:08 pm:
There is no on/off effect; peaks get wider when you run slow gradients.
-------------------------------------------------------------------------------------------------------
By Anonymous on Monday, June 7, 2004 - 09:39 pm:
Dont keep more of aqueous phase in your C4 column because as u go from c18 to c8 to c6 to c4 the column becomes more and more suitable for normal phase chromatography. Change ur column to C18 if u want to use more of aqueous phase
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, June 8, 2004 - 02:53 am:
Get nano hplc
-------------------------------------------------------------------------------------------------------
By MG on Tuesday, June 8, 2004 - 07:03 am:
The original poster is trying to purify a protein. I'm not sure why nano-LC would help, since a much smaller amount would be loaded onto the column.
VZM, as you go to very shallow gradients, you begin to approach isocratic conditions. In isocratic methods, peaks get broader the longer they stay on the column. If you cannot get the resolution you need by increasing retention, you can go to a longer column, smaller particle size, different mobile phase modifiers (either changing pH or changing organic modifier), or changing the stationary phase type. I see no danger in mixing those solvents you ask about.
What did you mean by on/off effect?
-------------------------------------------------------------------------------------------------------
By VZM on Tuesday, June 8, 2004 - 07:40 am:
Hi again,
Thank You very much for your responses!
Could someone suggest a resource that outlines examples of different stationary phases, mobile phases, and modifiers that can be tried?
Right now I am using an acetonitrile gradient with 0.1%TFA.
Also, could someone tell me how long a column should be equilibrated for before and after loading the sample?
Thank You again,
VZM
-------------------------------------------------------------------------------------------------------
By MG on Tuesday, June 8, 2004 - 08:41 am:
Disclaimer: I usually handle small molecules, not proteins, so my suggestions should be considered from the standpoint of general chromatography. I have heard that proteins can behave quite differently, and there are others here with more experience with proteins who can hopefully add to or correct these suggestions.
Stationary phases: In addition to C4, there's C8, C18, CN, "phenyl" and others for reverse phase. All of them can have different selectivity, either giving you better or worse resolution under equivalent conditions. For proteins, you'll need a large pore size (like 300 angstrom), else your compound will be excluded from the pores. C4 seems like a popular choice for proteins, and there may be good reasons for this. E.g. I understand that proteins can be very strongly retained on C18 colums.
Mobile phases: It is easy to try methanol in place of acetonitrile as the organic modifier, and at least with small molecules, this will often cause a significant change in selectivity (either for better or for worse). I have developed methods where mobile phase "B" was a mixture of methanol and acetonitrile, for optimum selectivity. You can also add isopropanol in some proportion to your acetonitrile; selectivity will be similar to methanol but isopropanol is stronger in terms of elution strength. Both methanol and isopropanol will raise your backpressure, and with high isopropanol concentrations it may become intolerable.
pH modifiers: TFA seems to be the popular one for proteins. There have been discussions on this forum as to whether TFA functions as an ion pair reagent. Alternatives are acetic and formic acids, and these can be mixed with their ammonium salts to adjust the pH. For non-MS work, there may be other alternatives I have neglected.
column equilibration: The time depends on your flow rate and the dimensions of your column. In terms of volume, the rule of thumb I use is 5 to 10 column-volumes at initial conditions before injecting the sample and starting the next gradient.
-------------------------------------------------------------------------------------------------------
By VZM on Tuesday, June 8, 2004 - 09:05 am:
Hi,
Thank You for the suggestions. I have couple of more questions.
Regarding getting rid of the acetonitrile TFA after the purification. I have tried flash freezing in liquid N2, followed by O/N vacuum at 60mT. This doesn't seem to gett rid of everything, meaning the product is still acidic. I have read papers that cite rotovaping the sample, prior to lyophilization, but am not sure exactly what is required (time / vacuum) to remove the acetonitile / TFA, or what rotovaping accomplishes over freeze drying.
Also supposing that I try methanol or isopropanol, how could I get rid of that?
Thank You,
VZM
-------------------------------------------------------------------------------------------------------
By RH on Thursday, June 10, 2004 - 10:42 am:
VZM, what is the purpose of your purification? If
you don`t need the protein in its natural
structure, as I think as you use organic
modifiers, how much substance do you load onto
your column (which dimensions, particle size,
pore size?) in what kind of solvent? And can you
give a hint about the protein of interest, mol.
weight, pk, hydrophobicity ,prostehetic groups?
-------------------------------------------------------------------------------------------------------
By VZM on Thursday, June 10, 2004 - 11:09 am:
Dear RH,
It is 14kD protein with a pI ~5.5. It has no modifications. It is not very hydrophobic. It is however, very unstable.
The purpose of the purification is to purify (on a preparatory scale ie 10mg) the protein away from similar degredation products.
IEX, HIC, SEC do not accomplish this, so I decided to try RP HPLC. I have tried a C4 5um particle size, 3.9x150mm column, using an
acetonitrile gradient in 0.1%TFA. I do not get the
resolution I need, meaning that the contaminants are still present and the peak is very broad.
Thank You for your help,
VZM
-------------------------------------------------------------------------------------------------------
By A.Mouse on Thursday, June 10, 2004 - 07:20 pm:
Is this a C4 column recommended for proteins? You need a larger pore size to get narrow peaks for proteins.
-------------------------------------------------------------------------------------------------------
By Anonymous on Saturday, June 12, 2004 - 07:53 pm:
Hello VZM,
I used to purify peptides of about 6kD and I got good results with Phenomenex Phenyl-hexyl column (50 x 250mm, 300A)at 50mL/min, a gradient 0.1% TFA WATER: 0.1% TFA ACN of about 10 to 50% over 120, at 214nm.
ACN is preferd to MeOH because of it's volatility, low cut off and and low viscosity, and TFA fits very well to these conditions.
Good luck
