Lab Calculations: Calculating Na+ & Protein Content of Fractions

[Suzi19]

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 that's 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 don't 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 realized 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.

Thankyou!

 

[Bystander]

Would I be right to use the full 10ml amount rather than the 0.5ml amount in my calculation?

Each fraction was 1.5ml.

Does not say "per diluted fraction."

And do- n=c(changed to M) v (changed to litres) = ... then change the finial answer to micro moles.

What are you asking here?

0.5ml of blood plasma plus 2ml of reagent was used- so would i use the full 2.5ml volume?

Does not say "per added reagents."

 

[Suzi19]

Does not say "per diluted fraction."

What are you asking here?

Does not say "per added reagents."

Hi! Sorry I realized its quite hard to understand what I'm talking about. I think I've figured it out though.

For the sodium- so, it was diluted by a factor of 20. so multiplying the concentration of the diluted sample by 20 gives me the concentration in 0.5ml. I can then find out the number of moles etc for the 0.5ml volume, and times up to 1.5- so that I have the right amount of moles in the 1.5 ml fraction.

For the protein, I have changed it to use the original 0.5ml volume, then times up to 1.5ml. so multiplying by three. (which is the same as just multiply by 1.5ml in the first place, I believe)

For the equation, I just wanted to check what I was doing was right.

The only problem I have now is my Beer-Lambert law. We used 1cm cuvettes i believe and it was 550nm absorbance- so would these be my values in the equation?

 

[Bystander]

in the equation?

In what equation? You've said you didn't get any measureable absorbance.

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.

 

[Suzi19]

In what equation? You've said you didn't get any measureable absorbance.

I was going to try and use the Beer Lambert Law equation to try and get an answer. Yes I know- I was just going to try using the Beer Lambert Law equation, but because the straight line on he standard curve didn't go to such low values, will I just need to say that the absorbance was 0, along with the protein concentration value?

 

[Bystander]

You did see some absorbance? In which case, you are interpolating between zero and whatever your lowest standard value happens to be --- you were running versus a "blank?"

 

[Suzi19]

You did see some absorbance? In which case, you are interpolating between zero and whatever your lowest standard value happens to be --- you were running versus a "blank?"

Yes there was some absorbance, but too low to read off the standard graph given to us. Yes we had a blank solution- The spectrophotometer was zeroed against water.

 

[Bystander]

too low to read off the standard graph

"Too low," or just requires interpolation?

 

[Suzi19]

"Too low," or just requires interpolation?

Well, my value doesn't fit to the straight line on the graph. Thats why I was wondering if I could use the beer lambert law equation to figure out the concentration from the absorbance value I got- or if the value really is too low then I would just have to put zero.