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Help with Lab calculations [column chromatography]


Suzi19

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Apologies for the stupid questions but I have confused myself.

I was separating out blood components in lab- column chromatography- and now we have to calculate Na+ content of each fraction collected (in μmoles) and the protein content of each fraction in mg. Each fraction was 1.5ml.
The Na+ concentration in mM and the protein concentration in mg/ml were read off standard curves.
For the sodium, the samples were diluted (0.5ml of blood serum with 9.5ml water). Would I be right to use the full 10ml amount rather than the 0.5ml amount in my calculation? And do- n=c(changed to M) v (changed to litres) = .... then change the finial answer to micro moles.
For the protein, I have used Cm= m/v. Again 0.5ml of blood plasma plus 2ml of reagent was used- so would i use the full 2.5ml volume? For my calculation i didn't change the units- not sure if thats right or not. so for example, 5.15mg/ml= m/2.5ml. so m= 5.15 x 2.5= 12.88mg

Additionally for the protein, the standard curve line did not go to very low absorption vales, such as 0.006 so I couldn't read off protein concentration value. Could I use the Beer-Lambert Law equation? However i dont know the values of the absorption co-efficient or the path length, so I'm unsure as to how I would do this.

I now realised that it asks for moles/mg of each fraction- so i am confused as to which volumes I use for the initial calculations because concentrations were read of standard curves using the diluted volume/ plasma with reagent. I dont know whether to use the 10ml or the 0.5ml from the fraction.

 

Thankyou!

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I was not able to follow everything you said. However, I have some general suggestions. Sometimes a sample is diluted as part of its being assayed. Occasionally, a sample is so concentrated, that it has to be diluted before a portion is taken for an assay. I would first calculate the concentration of the analyte in the assay, then I would do each dilution calculation separately, assuming that there were two dilutions. The amount of substance that one possesses is usually only of interest for the initial solution. That value is found simply by multiplying the concentration of the initial solution by its volume.

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