Chromatography Forum

Hi all,

is there anyone who did some HPLC preparations?

i read some discriptions on certain column/media preotein-loading capacity. it reads " Frontal loading. 10mg/mL protein solutions. Column: ***(4.6mmID x 50mm). Conditions: loading mobile phase=80:20 water: acetonitrile with 0.1% v/v TFA. Flow rate = 1mL/min."

what is the meaning of "frontal loading"? to get some kind of frontal peak(meaning over the linear scope)??

many thanks for your kind comments.

kiknos in shanghai,china.

one friend told me ""Normally, people would load a maximum 80% of the frontal capacity, in order to prevent breakthrough (over saturating the ion-exchange bed)".

so the "frontal" means "begin to breakthrough" ?!

and the "breakthrough" reminds me of another thing!

we have "static capacity", and we also have "dynamic capacity esp. under 5% breakthrough", for some kinds of columns such as Affinity columns.

anyone has some experiences on that?

thanks again,
kiknos

kiknos,

It's very common to observe fairly dramatic differences between static and dynamic capacity. One could argue that reporting static capacity is more than little misleading because of this but for some reason it is common practice in the case of protein separations.

thanks for the reply, Chris

an expert on protein purification told me he chooses media like such way:
firstly compare the static capacity by very simple tests(making "slurry" and mix with sample...) on all media he can find;

then compare the dynamic capacity by relatively "troublesome" tests(packing column and loading and eluting and calculating) among those offering high static capacity for the sample.