HPLC for peptides

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rohit2008
rohit2008's picture
HPLC for peptides

I have to start optimising a wet lab metod to separate amino acids, 2-3 amino acid peptide and protein digest on HPLC. We have C-18 column. We should use gradient or isocratic run and which UV wavelength will be right for detection. I have just started and I also want to know about precuations to prevent column. I want to optimise prptide separation by Dry Lab afterwards.

Dr. Analytical
Dr. Analytical's picture
There are many ways to do

There are many ways to do protein and peptide separations. We will need to know more information about your method before we can recommend anything.

For example, do you have an existing method for the separation? If you do, then please provide that information. If you do not have a method, then there are many decisions to make.

First, what kind of equipment do you have? Binary or quaternary pump? Autosampler? Diode array or variable wavelength or fluorescence detector?

What kind of sample preparation will you be doing, or do you need help with that also?

Please write back with these details and then we can get started.

rohit2008
rohit2008's picture
Thanks. I am starting with

Thanks. I am starting with Insulin. I want to perform HPLC for active and denatured states of Insulin. We have quaternary pump and this is a Shimazdu machine. We have earlier performed run of insulin in HPLC and obtained one peak. That was bfore I joined here. I think isocratic runs will be sufficient for this experiment.

Dr. Analytical
Dr. Analytical's picture
If you have a method that

If you have a method that provides good retention with acceptrable peak shape, then, yes, you can use an isocratic separation. However, make sure that you have adjust your conditions so that you are getting enough retention to see a separation. You do not want your insulin peak eluting near the void time, since you will probably not be able to separate the other peak.

The only thing to worry about with an isocratic method is the presence of other components that are highly retained, such as larger proteins or polymers. These materials will stick to the column under isocratic conditions, and over many injections they will change the nature of the surface. You will often see a change in retention, peak shape and resolution. To prevent this problem, program a "cleaning" gradient, using more acetonitrile, and do this at least once a week, depending on the number of injections.

kamyabi
kamyabi's picture
you are right Dr. Analytical,

you are right Dr. Analytical,
i think if some one else has also gone it before then i dont think u need a new method, but i would like to suggest first repeat what the other person has done and see if you are getting same peak at same RT under same conditions. Some times after the excessive usage of column the Rt of peaks shifts, and that may confuse the results.

desmondo84
desmondo84's picture
HI

HI
I was reading one of your replies with interest regarding insulin analysis. I too have been using an isocratic system, with a mob phase contaiing 0.2M NaSo4, and 27% AcN. However, it's been eating my C18 RP xbridge columns! Do you think it's due to protein accumulation? Generally, problems start with a gradual increase in backpressure and resolution is totally lost. I've ran a regeneration prpotocol with 0.1% TFA in AcN going from 10 - 100% but I think my latest column is still in trouble!
Any assistan e would be greatly appreciated.
regards
Des

Dr. Analytical wrote:

If you have a method that provides good retention with acceptrable peak shape, then, yes, you can use an isocratic separation. However, make sure that you have adjust your conditions so that you are getting enough retention to see a separation. You do not want your insulin peak eluting near the void time, since you will probably not be able to separate the other peak.

The only thing to worry about with an isocratic method is the presence of other components that are highly retained, such as larger proteins or polymers. These materials will stick to the column under isocratic conditions, and over many injections they will change the nature of the surface. You will often see a change in retention, peak shape and resolution. To prevent this problem, program a "cleaning" gradient, using more acetonitrile, and do this at least once a week, depending on the number of injections.

Dr. Analytical
Dr. Analytical's picture
desmondo84:

desmondo84:
Are you using a column designed for large molecule separations? For these large molecules you should be using a column with a larger pore size - 130 or 300 A. Also, although there are separations of proteins using C18 columns, often C4 or C8 columns are preferred, because they are less retentive.

A gradual increase in pressure usually means that you are plugging the column. Most often you are simply plugging the entrance frit on the column, not the column packing. A partially blocked frit can cause peak splitting and poor resolution.

With biological samples, my big concern is solubility of the sample components in the mobile phase. If some, or all, of the components are not very soluble, they will precipitate inside the system, usually in the tubing just after the injector, or at the column frit and the first few mm of the column packing. You can prevent this problem by dissolving your sample in mobile phase first, or at least dissolving in something similar to the mobile phase (salt plus acetonitrile).

A 0.2 M salt solution sounds too concentrated for this separation. Most buffers are used at about 0.02 - 0.05 M levels, and they are usually buffers, not simple salts. The salt solutions are usually used with size exclusion columns. It is also possible that the salt is contibuting to the destruction of the column.

And finally, yes, under isocratic conditions some proteins could be collecting on your column because they are retained strongly. The best solution to this problem is to not inject those proteins at all. Use a guard column or an SPE cartridge before injection.

As you can see, there are many reasons why your columns can be failing. Please write back with some more details and ideas, and maybe we can figure it out.