RP-HPLC chromatograph interpretation

dear all,

from an RP-HPLC peak report, is it possible to determine the actual concentration of the compund being tested? how? what is the formula? the peak report contains the following information: retention time, area, height, and width.

thanks!:)

The raw peak information from a chromatogram (area, height) is just that; the raw data to be used for other calculations.

You cannot determine the amount of a substance present without first analyzing one or more standards of known concentration. Once you know the relationship between peak area and amount analyzed (called a "response factor"), then you can calculate the quantity in an unknown.

Many data systems will give you an "Area Percent" report, which is just the percentage of the total that each peak represents. This also has limited meaning, since different compounds may have much different response factors.

To summarize, to get an amount value, you will need a standard. More questions? Just write.

Thank you for taking time to answer my query. And yes i do have more questions. :)

Actually I have a standard and the retention time of my "unknown" is within the peak range of that standard. With this, I can say that I have more or less the same substance, right?

The thing is, I'm not satisfied with just saying that I have the same substance in my unknown (qualitative). What I am after now is to report the actual amount of the substance in my sample. You said that I can calculate the quantity in an unknown once I know the response factor. So now, my more important question is how to determine such response factor? What is the formula, if there is any? And how exactly do I use this response factor to compute the quantity of the substance in an unknown?

Again, thanks so much! :)

Well, we could spend days talking about this, but I will try to keep it a little shorter. :-)

The response factor equation for the standard is:

RF = (Peak Area Std)/(Amount Injected Std)

You can use either concentration or mass, but be consistent in all calculations. It would be best to do several injections and get an average RF.

Now, for your unknown, assume the RF is the same (it's the same compound), and you know the peak area, so you just rearrange the equation for Amount Injected.

Amount Injected (Sample) = (Peak Area Sample)/RF

Again, multiple injections will get a better value.

This is called "Single Point Calibration" because you are only using one standard. It is a good approximation if standard and sample are close in concentration. If not, the accuracy will suffer and you will have to take another approach.

Now, as to whether or not your sample has the same compound: well, that is always something we struggle with. If the retention times are the same (or very close) and the peak shape is the same, we usually ASSUME that it is the same compound. But we really can't say with complete certainty. It is best to have some other information, like an absorbance spectrum or mass spectrum, or some other knowledge of the sample.

This should get you started.

Okay, got it! Your instructions were very clear and concise :) But let me just clarify, in the formula for RF, you were referring to the Peak Area and not the Area %, right?

As to the compound in my sample, I'm pretty confident that I have the same compound as the standard because I only have a single prominent peak in my sample very close to the retention time of the standard. But then again, I really need to firm up my results with further characterization. I intend to do ESI-MS, CD, and FTIR in the next few months. And I'm sure more questions will arise as I am very new to these.

Thanks v. much, Sir! I really appreciate your help. :)

You are correct, that you use peak area (or height), not the percent value. The area percent results really have no meaning except to tell you the percentage of the total. There are some specific examples in pharmaceutical analysis where you might be able to get some useful information, but in the general case, no.

Also, I would strongly urge you to get some training in HPLC and analytical chemistry. There are many things to learn; more than I can teach you in this forum. You need to learn how the instrument works, how to use the software, how to troubleshoot problems, and how to set up calibration systems and review results.

There are many resources for training available, including some that I offer. But it is very important that you get it from someone.

You did not mention which country you live in, but you should be able to find some good training that is not too far away. The manufacturer of your instrument may offer classes in your country, or a nearby country. And you can look for a Chromatography organization that may offer training also.

Good luck with your learning. We hope to hear from you again.

Ah right! I really appreciate all your suggestions and I will start looking for trainings right away, or as soon as I finish my thesis. I'm an MS Biology student from the Philippines.

Thank you again Sir. :)

[color=deeppink][/color] % Purity= (Area of sample/Area of Std)* con.Std/Con.Sample*DF*100

Good day to all!

Well, Ms. Charity, if i may suggest some info regarding quantitation in the HPLC, if you are using a PC-driven LC, it is sure that the software you are using has the ability to perform the Calibration of your standard solutions. I don't know how many levels of concentrations of the standard you are going to use but through that. you can have the quantitation calculation. Just see if you can ask th supplier of your LC can teach you on how to do it.

I hope this will help.

Hi,

     I have an important question about the RF(response factor). Whatare the parameter on the the response factors depends. I mean to ask.....Can i theoritically calculate the RF of a substance (for a perticular hplc system) knowing molecular properties like the extinction coefficient. Somewaht i feel that given a hplc system the RF should just be propertional to the extinction coefficient.  Am i right?

Critical suggestions and coments are most welcome and will be appreciated.