Getting Started in HPLC

Section 4E. Calibration

   
Now we are ready to talk about the actual process of calculating results from an LC analysis. The chromatogram gives us two important pieces of information for each band: retention time and peak size (either area or peak height). The retention time tells us what compound the band represents. The peak size can be converted into the concentration of that compound in the sample that was injected into the LC system.


 
For example, we might make up solutions of methyl paraben (a food preservative) at three different concentrations and inject each of these into our LC system: In each case a peak is seen at the same retention time (3.65 minutes), as expected for different samples of methyl paraben. However the size of the peak in each sample is different - a higher concentration of methyl paraben is seen to yield a larger peak in each case. In order to calculate the concentration of methyl paraben in a sample, we need to be able to measure peak size.


Food Additive Std; 10 ppm Methyl Paraben Food Additive Std; 30 ppm Methyl Paraben Food Additive Std; 50 ppm Methyl Paraben

 
   
The size of the peak is most frequently determined as peak area. We seldom measure peak areas manually - it is just too tedious and imprecise. Instead we rely on the data system. Data systems work by slicing up the peak as shown on the right. Then the height of each slice is measured and all the slice-heights are added together to give the area. Finally, a "correction trapezoid" is computed to account for the offset between the baseline and electronic zero; the area of this trapezoid is subtracted from the raw area to give the actual area under the peak. The data system can also measure peak height by using the height of the tallest slice.

Integrators do the best job of measuring peak height or area when the peak is well resolved and the baseline is relatively level.


   
In the absence of a data system, we can measure the peak height manually with a ruler, simply recording the distance from the baseline to the top of the peak. The units of peak height will cancel out when we calculate the concentration of the compound giving the peak. That is, whether we measure peak height in centimeters, millimeters, or % of full scale, you can forget the units (just measure ALL peaks with the same units).


   

Now our series of methyl paraben samples yield the following information, once our integrator has done its job (or we have measured the individual peak heights manually): The next step is to use these data to obtain a "calibration plot" (sometimes also called a "calibration curve") so that we can analyze any sample for methyl paraben.


ppm methyl paraben
30 ppm methyl paraben
50 ppm methyl paraben

Calibration data for methyl paraben showing retention times (tR), peak area, and name for each peak in the calibration runs

   
Calibration curves are obtained by plotting peak size on the y-axis versus sample concentration on the x-axis - for a series of samples with known concentrations. Using the data above for the 10, 30, and 50 mg/L samples of methyl paraben, we can generate the plot shown here. We see a straight line through the data points, and the line passes through the zero-zero point of the graph - or through the origin.


   
Calibration plots like these are called "linear" and yield the best analytical results. They also permit the simplest approach to sample analysis - as we will see in the following section. The straight-line plot shows that peak size increases proportionately with sample concentration. If we double the concentration, we also double peak size.


 
   
 

 


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Last revised: April 06, 2001.