HPLC calibration curve: Everything You Need to Know

HPLC calibration curve: Everything You Need to Know


  • Post By : Kumar Jeetendra

  • Source: Microbioz India, Editorial

  • Date: 04 Mar,2023

In analytical chemistry, the concentration of an unknown sample solution can be found out using a calibration curve. It is an experimentally generated graph with the concentration of solution plotted on the x-axis and the observable on the y-axis. Calibration curves are primarily used to determine the concentration of an unknown sample. Standard solutions with properties similar to the sample to be measured are created and their absorbance is measured here. Because the concentration of the standard solutions is known, the measured absorbance can be plotted against it.

HPLC Calibration Curve:

About HPLC:

The acronym HPLC stands for High Performance Liquid Chromatography. “Chromatography” is a separation process, “chromatogram” is the separation’s output, and “chromatograph” is the device used to perform chromatography.
Among the different technologies created for chromatography, columns and high-performance pumps for supplying solvent at a constant flow rate are among the most important components of chromatographs. As associated technologies got more advanced, the system formerly known as High Performance Liquid Chromatography was shortened to “LC.” Ultra High-Performance Liquid Chromatography (UHPLC), which is capable of high-speed analysis, has also grown in popularity in recent years.

HPLC can only be used to examine substances dissolved in solvents. HPLC separates substances dissolved in a liquid sample and permits qualitative and quantitative investigation of the sample’s components and their concentrations.

A calibration curve is required in order to determine the concentration of the analytes in the sample.

An example calibration curve with concentration values on the x-axis and the area under the curve on the y-axis. Image Credit : django-wiki

Many concentrations are measured to generate a linear line, which is the calibration curve. These concentrations have both lower and greater concentrations than the expected concentration of the analyte in the actual sample. When the analyte concentration in the actual sample is unknown, it is customary to generate a calibration curve using at least eight different sample concentrations. Since the expected analyte concentration in the actual sample is known, the number of sample concentrations required for the calibration curve can be decreased.

How to prepare HPLC calibration curve:

HPLC methods require calibration in order to produce quantitative findings. HPLC Calibration comes in a variety of forms, each with its own advantages and disadvantages. The goal is to pick the best one and to avoid the many minor mistakes along the way, as no one wants a nearly perfect solution.

  1. Step 1: Make some preliminary, qualitative assumptions about what kinds of factors might be present in your sample. Let’s pretend the sugars glucose, galactose, and hydroxymethylfurfural (HMF) are present in your sample.
  2. Step 2: Provide a reference solution that accounts for all of the parts, both in total and individually. A stock solution of 6 g/L that combines glucose, galactose, and HMF can be used to generate five different standard solutions via serial dilution. Use concentrations of 6 g/L glucose, 6 g/L galactose, and 6 g/l HMF for the separate ingredients. This procedure reveals the retention time (the time needed to leave the column) of each component in the sample.
  3. Step 3: Third, analyse all standard solutions by High-Performance Liquid Chromatography (HPLC), making note of the peak area and retention time for each component.
  4. Step 4: Create the calibration curve (graph), which is the plot of peak area vs concentration, in Excel or another appropriate programme. Create a straight line that best fits your data in your graph. Assure that the coefficient of determination or correlation is very near to 1.
  5. Step 5: Five, analyse your sample using High-Performance Liquid Chromatography (HPLC), making note of the peak area and retention duration. The concentration of each component in your sample can then be calculated using the calibration curve you created in Step 4.

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