Chromatography Forum

Hi!

I am new in using HPLC, and for my thesis I did this analysis with HPLC. I already have the results of the components in the vials in g/L. How can I back calculate the concentrations to my original sample? Please help!

The original sample was treated before the HPLC analysis, I needed to acidify them to precipitate some solids and filtrate the sample before running them in the HPLC. I have already calculated the concentration of the samples after the treatment. How do I now calculate the concentration of the components in the original samples if I have the concentration in g/L in the vials?

Thanks!

I must be missing something here: you're doing a thesis and don't know how to do this, or am I just reading this wrong?

You know the final concentration in the vials. You also know what steps (masses and volumes) you used to prepare those vials. Simply back-calculate through those steps. CPG may have been a bit blunt in his post, but the fact is that this is high-school level chemistry.

Give some data if you need help in it.

Wouldn't the concentration be the same? Or there about. The OP has only added acid to precipitate solids.

What volume of acid into what volume of sample?

Go easy guys. It may have been "high-school level" for you, but for many of us putting what we learned from out-dated, soiled textbooks while the less interested kids set the gas taps alight; to practical applications isn't always easy.

The method I use is this:

I draw out a little sketch of everything that happened to the sample between when it was urine/a plant leaf/etc. right up to when it was an HPLC vial to be injected. I draw a leaf, and little arrows going to a tube, and from one tube to another, and tubes with pellets, supernatants, etc., and all the things I'm adding to each tube, so that I can write down the volume that was present at each stage.

This means that I might start with 1g solid material at the left of my picture, and show how this became 3mL of solution, to which I added 1mL something else (now I have 1g in 4mL), then I took out 1mL (which because of my drawing I know contains 1/4g = 0.25g material) and made it up to 10mL etc. etc. etc... then at the end of my diagram, I know that the final hplc vial contained 10mg/mL of original stuff. Now you have an answer from your instrument of 2.5mg/mL analyte, so you know that the actual concentration of analyte in your final sample is 2.5mg/mL...per...10mg/mL or 0.25mg/mg dry weight (or whatever; note, the values and units here are totally wild, not an indication of amounts of things you ought to be putting into an hplc!).

The alternative approach with my drawing is to start at the bottom with a measured value of 2.5mg/mL analyte, and work out how much I have in each tube working backwards until I know the concentration in the very first solution I made. From this I can work out the amount in that very first solution, and because I know how much experimental material went into it, I know the amount per g or per mL of that experimental material.

I really strongly recommend doing the little drawings. You don't have to make the eppendorf tubes too beautiful, but it really helps me make sure that my sums exactly repeat what really happened on the lab bench. Working step-by-step is far safer than trying to write out one huge equation that calculates the right answer, even if you feel a bit silly writing things like 1mM = 1 mmole/L = 1 umole/mL = 1nmole/uL etc. etc.