I find that I typically lose about 5-15 % when running prep HPLC on fairly stable molecules, but can easily lose more without trying too hard
.
It's always worth remembering that preparative chromatography usually means longer time in solvent, and longer evaporation times. The sample quantities will make losses more visible than on analytical columns. If you always loose 10% on an analytical system, you may never notice.
Typically, the two common causes of my Prep HPLC losses are:-
1. Decomposition.
If you know the major decomposition peak, and you see it smearing toward the parent peak, you could have on-column decomposition, which I fortunately don't encounter often.
The more common form is decomposition in the loading solvent or mobile phase, which can be detected by performing replicate injections with a significant time period between and checking peak area. I establish a baseline point by ensuring the first injection is < 5 minutes after dissolving the sample.
The most common source of decomposition losses is concentration of the eluate after elution from the preparative column. I always try to analyse the eluate as soon as it leaves the column, and calculate how much analyte is present. It's often during evaporation where recovery drops from 90% to 50%.
Many molecules croak if they are rovaped down from mobile phases that have modifiers such as TFA present. Ensure temperature and/or light aren't degrading your product. You should check stability to allow for the longer time/temperature enviroment of preparative HPLC solvent volumes.
2. Precipitation
The major analyte isn't soluble in your mobile phase once it's separated from the muck or injection solvent. In preparative HPLC, this is usually accompanied by backpressure increase, and completely-blocked prep columns aren't uncommon.
Having $0.5 million worth of cGMP pharmaceutical product on a column which has almost blocked after 15 hours of increasing back pressure and decreasing flow is fun only if you're a disinterested spectator.
A more polar solvent at the end of a run may not redissolve precipitates. You may have to raise the temperature, make major modifications to the mobile phase ( such as changing to pure ethyl acetate ), and greatly decrease the solvent flow, to achieve dissolution.
Other causes, such as prep columns and solvents being different to analytical conidtions are less common. You should always try to maximise the quality of the preparative column and solvents, as trace impurities can wreck havoc when large volumes are concentrated.
Always ensure all your samples are actually loaded onto the column, and not being left behind in flasks or injector loops, by binding. I've also managed to fill loops in the injected position, and watched the sample run into the injector waste line. If you have large losses, analyse injector wastes in case a valve ( or operator
) isn't working correctly.
Please keep having fun,
Bruce Hamilton
