Chromatography Forum

I am new to the HPLC kindly suggest me how to calculate the unknown concentration from the standard curve of AUC vs Concentration.Suppose I inject 10ul of standard dilutions and obtain a standard graph.Then how should i determine the unknown concentration from the area under curve obatined.
Kindly let me know the link for some calculation problems of HPLC.

If you are doing it graphically, you run your sample and measure the area. Then go to your calibration plot and find that area on the vertical (y) axis. Move horizontally at that level until you hit the calibration line, then drop down vertically at that point to the horizontal (x) axis. That value on the x-axis is the amount. Here's a slide from our "Fundamentals of HPLC" course:



To do it computationally, divide the area by the slope of the calibration line (this assumes that the calibration line passes through zero).

If you are doing it graphically, you run your sample and measure the area. Then go to your calibration plot and find that area on the vertical (y) axis. Move horizontally at that level until you hit the calibration line, then drop down vertically at that point to the horizontal (x) axis. That value on the x-axis is the amount. Here's a slide from our "Fundamentals of HPLC" course:



To do it computationally, divide the area by the slope of the calibration line (this assumes that the calibration line passes through zero).

From this unknown concentration i want to measure the total amount in my samples.
Suppose I had prepared a formulation(250mg) containing or encapsulating unknown amount of drug.I take 5 mg of the formulation containing x amount of drug and disperse it in 5 ml of lysing medium.Then i inject 10ul of the above dilution into the HPLC system which gives an area under curve Y.From the standard curve plotted earlier i calculate the unknown concentration.so is it the concentration in 10ul or 5 ml.then how to calculate the total amount of drug encapsulated in 250 mg of my formulation.Kindly help me.

Concentration = amount / volume.

Therefore: amount = concentration x volume