Chromatography Forum

Hi all
I'm working with solid phase peptide synthesis, and when purifying the crude peptides (1000 Da - 4500 Da), I've seldom observe more than a 50% recovery.

So if I start out with 100mg of a crude mixture displaying 90% purity, I will end up with ~45mg purified peptide.

When I talk to other peptide chemists, they say they have settled with this "50%" recovery, but I just wonder where the remainder ends up.

Also when I speak to consultans from various column vendors, they seem surprised that I'm not able to obtain near quantitative recoveries.

We perform the purification on a agilent 1200 system at semi-prep scale, which in our case involves, for the most part, either a C18 10x250mm, 5um, 300A column or a C12, 10x250mm, 4um, 90A column, running A: 0.1% TFA/H2O and B: 0.1% TFA/ACN as mobile phase.

A typical purification would consist of dissolving the crude peptide in water (or up to 10% AcOH) at 5-15mg/ml, depending of the solubility.

The sample is then injected (900 uL at a time) an eluted using a linear gradient over 25 mins, fx 5-40% B, followed by a 90% B flush over 5 min and 10 mins reconditioning.

The fractions are collected by time, the fractions supposed to contain the desired product are collected and pooled and freezedried. The yield is then determined by weight.

I know there's not an simple answer to this but I would just like know what other people get out of their purification and maybe a starting point to pin point anything that I'm doing wrong.

In advance thanks
Erik

Hi Erik,

As you say, there are a number of possibilities in this situation. But if you start up with some preliminary tests the answer might become clearer.

1. Inject exactly the same amount (volume) of the same sample solution several times (f.x. 3 – 4 times) running the whole gradient each time. See if the peak area increases for each injection.
2. Following the first test, inject several blanks (solvent without actual sample) and see if there are peaks where your peptides normally elute. If so do the decrease for each injection?
3. How did you determine the 90% purity by the way? Could it be that some of the sample (no matter whether it's a peptide or other stuff) elutes with the dead volume thus “escaping” the mass balance account?

Best Regards

One additional point: For a proper mass-balance, of course the start-weight must be exact. Your crude peptide might contain residual stuff from the synthesis that's not visible in the purity determination which would mean that the actual purity of that crude peptide is lower than 90%.

Thank you for your suggestions Danko and HPLCaddict.

I think the fist thing to do is to determine the excact peptide concentration being injected. I looked in the literature and a crude peptide mixture can contain
up to 70% water and salts. I guess I've been naive assuming the crude peptide would contain a minimum of water and salts.
I'll take a look in the freezer and see if I can dick up a crude peptide containing Trp or Tyr, as the concentration then can be estimated by UV-absorbance.

I'll be back when I have figured this out

\Erik