I want to determine the concentration of a pollutant from a water sample. I have HPLC graph of standard (pollutant) and sample. Also the concentration of the standard is known.
You can get a calibration curve from different dilution of standard and from calibration curve calculate concentration of pollutant in water sample. Calibration curve will have on x-axis concentration and on y-axis peak area.
You can get a calibration curve from different dilution of standard and from calibration curve calculate concentration of pollutant in water sample. Calibration curve will have on x-axis concentration and on y-axis peak area.
you can also use standard addition. You need to spike known amount of your standard in the sample and build a calibration curve.
http://en.wikipedia.org/wiki/Standard_addition
In case you are using HPLC-MS you may use also an isotopic standard.
If the same molecule is isolated from the tissues of an aquatic organism like fish. How could i get the concentration of it in its body
If you want quantify the same molecule in the fish, I think the best solution is to perform a calibration curve in the matrix of fish because the matrix impacts both on the response of the molecule (matrix effect, ionization...) and on the effectiveness of the extraction procedure (recovery). if you perform a calibration curve in the fish matrix, all these parameters are taking into account, and the concentration given thanks to the calibration curve will be correct !!!
Depend on the nature of the molecule. Some estimations are possible, but mainly depend on the molecule of interest which can be present (or not) in different concentration in different cell type, circulatory system, fat etc. etc. between a single organism. In this case, a total body concentration is quite a meaningless value, and it is usually more common to refer to observed concentration in different cell type/compartment/organs etc.
If the molecule is present only in a particular cell type/compartment/organs within the organism than a possible estimation can be based on the percentage of specific cell type in the organism.
In fact, I think the calibration curve must be performed on the matrix which must be analysed. therefore, if the aim is to quantify the molecule in the kidney, the calibration curve must be performed in the kidney of fish, if the molecule is in the gonad, the calibration curve must be performed on the gonad... In this way, in our laboratory, we performed calibration curve in rat testis in order to quantify hormones and pesticides in rat testis...
The matrix used for the calibration curve must be identical (if it is possible) to the real matrix
I always use a calibration curve of 5 points... And if you use a surrogate standard you must add it in the calibration curve...then you calculate the concentration for interpolation and the recovery...what kind of pullant are you interested in?
Prepare a standard curve of the standard sample with different concentrations made from a parent stock solution. Plot the AUC against concentration and get the straight line equation. Once you get this, any sample having the standard compound can be run in same conditions and its concentration can be calculated from the standard curve. If you know AUC, you will get concentration by putting the values in the equation of straight line.
You need to know which contaminants are present in order to prepare a set of standards and later develop the calibration curve. To validate the calibration curve I would recommend you the following book: Kromidas, Validierung in der Analytik, Wiley-VCH (in German).
I think, for your HPLC analysis is need to have calibration curves. These curves were established using a minimum of five standard concentrations, each containing IS. The ratio of the peak area of each compound to that of the IS was used for quantification.
Identify the matrix and it should be same when you want to find the unknown.
I think you can use the concentrations (at least 5 levels of concentration to be done in triplicate) of the standard you have to plot a calibration curve. After which you will get a line equation which will help you calculate for x which will be the same as the amount of your sample. I believe the HPLC will give you the area of the sample.
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