calculating dilution factor - Chromatography Forum

calculating dilution factor

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Hi all.
I have a question about calculating concentration of HCP in fractions collected from an AIEX run and I can't get my head around it.. i think it's a pretty simple problem but i'm not sure my logic is correct.
background info: CHO harvest feed is injected onto a 1ml AIEX column using an Akta avant. 2ml fractions are collected.
I take my harvest and dilute it 1:4 in buffer A.
I inject 30ml into the 1ml column (at 1ml/min).
I get a wide FT flat peak, an elution peak and a tiny peak at the end (strip). (see chromatogram below)
I'm collecting 2ml fractions, so I get 15 fractions for the FT, 7 for the Eluate and 1 for the Strip.
I want to measure the amount of HCP in in the 3 peaks, so what i did and I pooled the fractions together (for each peak) and used an assay to measure HCP. The results I'm getting are too low though, and i'm afraid that i'm not taking into account the dilution correctly.
how would you do it? Do i need to multiply the result times the dilution factor? how would you calculate the dilution factors?

thank you!
You're worrying too much!

You have measured some amount (we'll call it "X") that came from 30 mL injected. That means the concentration in what you injected was 0.033X / mL . That was a 1/4 dilution, so your original concentration was four times that: 0.13X / mL
-- Tom Jupille
LC Resources / Separation Science Associates
+ 1 (925) 297-5374
hi Tom thank you for your reply.
if that's the case, than it's even lower than i was expecting..
let me give you some measured values:

HCP in undiluted Harvest: 0.1mg/ml

HCP in FlowThrough: 0.001mg/ml
HCP in Eluate: 0.002 mg/ml
HCP in Strip: 0.003 mg/ml

the mass balance doesn't make sense to me..
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