Any advices for a RP-HPLC separation???

[Karvezide]

Hello,

we are dealing with a protein mixture (MW: between 30 and 7 kDa) isolated from serum, whose major component is a protein that contains 243 AA. We have used a gradient method with a RP-18 endcapped column (Chomolith, 5µm, 4,6*100 mm) to separate the protein mixture.

The result is quite miserable: particularly the peak shape of the major protein (eluted at 21-24 min, at 22 min maximal, distorted, brodening, tailing Peakshap). We thought that the major peak should be somehow heterogeneous, perhaps due to the isoforms of this major protein. Furthermore, the baseline drift is significantly variable and we can't achieve the baseline separation for many peaks.

We have tried a serial connection of two columns, decrease the flow rate (from 1.2 ml/min to 0.5 ml/min), or increase the temperature (from 37°C to 45°C). But unfortunally they diden't work.

Can anyone give us some advices, how to improve this seperation? If the column is not suit for it, which columns sould be helpful?

A small summary of the separation method:

Mobile Phase: A: 0.1% Trifluoroacetic Acid in water / B: 0.1% Trifluoroacetic Acid in Acetonitrile
Gradient: (Time,%B),(0, 28 ),(30, 58 )
Detector: UV at 214 nm
Flow rate: 1.2mL/min
Column Temp: 37°C
Column: Chromolith C18e 300 Å, 5 µm, 4.6*100 mm
Sample solvent: 3 M Guanidinium-HCl
HPLC equipment: Hitachi with autosampler (7°C)


Thanks a lot in advance

[Bryan Evans]

Often times, ODS is not the optimal phase of choice for protein
separation. Improved peak shape / recovery can sometimes
be obtained by using shorter alkyl chain group (there are
other factors as well).

Intrada WP-RP was designed for the separation of highly hydrophobic
proteins. Below are some applications:

Proteins (670-1 kDa): http://www.silvertonesciences.com/files/TI289E.pdf
Interferon: http://www.silvertonesciences.com/files/TI263E.pdf

[Bryan Evans]

Some quick things you can try:

- Increase column temp to 65C (it's not uncommon for folks to use
higher temperature for proteins)

- If not done already, dilute protein in 0.1% TFA

- Inject lower sample mass on to the column - see if that helps
with the separation.

[Noser222]

An isocratic hold at the beginning of the method may help.

If you have substantial baseline drift, are you sure you are allowing enough time between injections to re-equilibrate? At the least, you should not do another injection until the baseline has returned to where it started.

[Uwe Neue]

There are several reasons for the problem. First, you are likely to be much better off with a packing with a larger pore size, 300 A. Packings with this pore size are also typically designed for ppetide/protein separations and will do better than a random packing. Look for the Biosuite line or the Delta-pak line at Waters. Furthermore, it is occasionally also better to use a shorter chain for protein separations. Both lines have a shorter-cahin option. Look for applications that are similar to yours and select the right packing.

[Kostas Petritis]

In terms of the baseline drift, I would suggest that you detect at higher wavelengths (254 or 280 nm) as you almost always have some aromatic amino acids in your sequence. Otherwise they are some ways to limit the drift at 214 nm as it has been described in previous threads...

[Karvezide]

Thanks a lot for the replies and the suggestions.

I usually allow a column equilibration with 5 column-volume eluents before each new injection. Is it enough?

I have also another question:

How can I examine the homology of a peak? Can I collect the peak and then inject in the same column? Or must I use another type of column?

Thanks!!

[goxy43]

Hi,
I hate to put it this way, but: chromolith is not the best column for separation of peptides and proteins. It is great for small molecules. Also, I do not agree with Uwe in terms of particle size and pore size. In my experince, the separation of proteins will certainly be difficult with a small pore size but peptides of the size you have mentioned are separated quite effective. I do the peptide separation with 3 µm particles and 100 A pore size. I think that your problem is simple the column you are using. i have tested these columns in all formats and can say that the only operating monolithic column for peptide and protein separation is the one sold by LC Packingy/Dionex.
When using monolith, pay attention to the fact that the optimal flow rate strongly differs in comparison to packed columns of the same ID and length.