HPLC Salicylic Acid

[firstyearpostgrad]

Hi,
I am trying to quantify SA in infected leaf tissue. However, as I am new to this type of work I am a little confused as to the use of an internal standard. I am spiking all my samples with a known amount of pure SA so that I can eliminate variations between injected samples. So, I have two questions:
1) How do I calculate the percentage recovery of my SA from my samples to test how efficient my extraction procedure is?
2) If all my samples (plant extracts) are spiked with pure SA, how is this showing how much SA is produced by the plant following infection - Am I not just measuring the SA that was spiked in?
If anyone can help that would be great as I am very confused!
Thanks in advance.

[CharonY]

Are you using UV detection or MS?

If it is only UV you cannot use the same compound as internal standard since, as you pointed out, you would measure the combined amount of sample+standard. In an MS you would use an isotope labeled compound where you could distinguish standard from sample.

Instead you can only use an internal standard (to judge the run quality) that is a different compound not found in the tissue.

To your questions:

1) You would generally use a matrix that is similar to your sample (ideally leaf tissue that is completely free of SA) and spike it with the known amount. Alternatively you can make a baseline subtraction (i.e. measuring the base SA level and compare it to the spiked sample). The latter is a bit less reliable, though. The recovery rate is then based on the area of a pure SA sample (i.e. you do three runs, pure SA, spiked sample, pure sample). There can be an additional error introduced by the sample prep itself, if e.g. additional UV absorbing components are present.

 

2) Yes, hence you also to run non-spiked samples.

[firstyearpostgrad]

Thank you CharonY.

I am using UV as my detection system. So as my internal standard I would use a homologue of salicylic acid or can it be anything that elutes from the column at a different time and can be distinguished from the salicylic acid. Then whatever does elute off at the expected time of salicylic acid and has the correct properties is how much has been produced by the plant due to the infection (I get this by using my caliberation curve made from known standards)?

 

Your explanation is so clear and pretty sure I understand now. Thank you!

[John Cuthber]

I think you need to read about this

http://en.wikipedia.org/wiki/Standard_addition

as well as this

http://en.wikipedia.org/wiki/Internal_standard

[CharonY]

Reading through the wiki articles should be a good basis. In addition, you really have to be clear for what the internal standard is being used. Depending on whether it is for normalize elution time or signal area, for example. In some cases an external standard is much more useful.