Calibration curves

This guide will describe the process for preparing a calibration curve, also known as a standard curve. 

What is a calibration curve? 

Calibration curves are often used in many fields, including analytical chemistry and biochemistry. A calibration curve is used to determine the concentration of an unknown sample, to calculate the limit of detection, and the limit of quantitation. The curve is created from the instrumental response to a set of standard samples at a range of concentrations. The data are then fit with a function to enable the prediction of unknown concentrations.

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What is an ultraviolet-visible (UV-Vis) spectrophotometer?

A UV-vis spectrophotometer is an instrument used to measure the transmission and absorption of light to determine the concentration of an analyte in solution. 

The UV-Vis spectrophotometer consists of a light source, a wavelength selector, a detector, and a computer. The light source may be a single xenon lamp, or a tungsten or halogen lamp and a deuterium lamp. The wavelengths of light required for analyte detection may be selected using a monochromator or a variety of different filters.

How does a UV-Vis spectrophotometer work?

The UV-Vis light passes through the sample and reaches the detector. The transmittance is measured and used to calculate the absorbance. An absorption spectrum is obtained with absorbance plotted on the vertical y-axis and wavelength plotted on the horizontal x-axis.

What you will need to make a calibration curve:

To successfully make a calibration curve, you’ll need the following items. Here's a detailed explanation of each, including why they are important:

1. Personal Protective Equipment (PPE):

Safety is paramount in any laboratory setting. Proper PPE includes:

• Gloves: To protect your hands from exposure to hazardous substances or contaminants.
• Lab coat: To prevent accidental spills on your clothing and skin.
• Eye protection: To safeguard against splashes or spills that could harm your eyes.
• Face mask (optional): If working with volatile solvents or powders that could generate fumes or airborne particles.

2. Standard Solution:

This is a solution with a known concentration of the analyte you wish to measure. The standard solutions are critical for creating reference points on the calibration curve. Multiple concentrations are typically prepared to span the expected range of analyte concentrations in unknown samples.

3. Solvent:

The solvent is used to prepare both the standard solutions and dilute unknown samples. It should be compatible with the analyte and the instrument (e.g., UV-Vis spectrophotometer). For example:

• Deionized water for aqueous solutions.
• Organic solvents like ethanol, methanol, or acetonitrile for hydrophobic analytes.

4. Pipette and Tips:

Precision pipettes and their corresponding tips are used for accurate measurement and transfer of liquids, particularly small volumes, during the preparation of standard solutions and samples. Ensure pipettes are calibrated to avoid errors in solution preparation.

5. Volumetric Flasks or Microtubes:

• Volumetric flasks: Used to prepare standard solutions with precise volumes. They are critical for ensuring accuracy in the preparation of calibration standards.
• Microtubes: Useful for smaller-scale experiments or when working with minute sample volumes.

6. UV-Vis Spectrophotometer:

The UV-Vis spectrophotometer is the instrument used to measure the absorbance of the standard solutions and unknown samples. The absorbance values are used to construct the calibration curve by plotting absorbance against concentration.

7. Cuvettes:

Cuvettes are the sample holders placed inside the spectrophotometer. They are typically made of glass, quartz, or plastic and must be compatible with the wavelength range of the spectrophotometer. For UV light measurements, quartz cuvettes are preferred due to their transparency at shorter wavelengths.

8. Computer:

A computer is essential for:

• Operating the spectrophotometer.
• Collecting and recording absorbance data.
• Plotting the calibration curve using software (e.g., Microsoft Excel, Origin, or specialized spectrophotometer software).

Optional Tools and Accessories:

• Vortex mixer: To ensure thorough mixing of solutions.
• Analytical balance: For precise weighing of solutes if preparing standard solutions from a solid.
• Ultrasonic bath: To remove air bubbles from solutions or to thoroughly dissolve solutes.

By having these items on hand and understanding their purpose, you can confidently prepare and execute the process of creating a reliable calibration curve.

Step 1: Make a concentrated stock solution

Prepare a concentrated stock solution of the standard by weighing the solute and transferring it to a volumetric flask with solvent.
See this step-by-step guide for making aqueous solutions.

Step 2: Make the standards for the calibration curve

Perform a serial dilution

Label a series of volumetric flasks or microtubes. A minimum of five standards are recommended for a good calibration curve. 

Pipette the required volume of the standard into the first flask or microtube. Change the pipette tip, add the required volume of solvent to the same flask or microtube, then mix. 

Repeat this process by pipetting from the previous solution to the new flask or microtube and adding solvent.

For more details, see the step-by-step guide to serial dilutions.

Prepare the samples and unknowns

Transfer the standards to cuvettes. 

Transfer the unknown samples to cuvettes. The unknown samples should have the same buffer and pH as the standards.

Step 3: Run the standards and samples in the spectrophotometer  

Place each standard in the UV-Vis spectrophotometer and obtain a reading. Obtain between three and five readings for each standard and record the data in a spreadsheet.

Repeat with the unknown samples.

Step 4: Plot the data

Plot the data with absorbance on the y-axis and concentration on the x-axis. 

If samples were measured in triplicate, use this data to determine the standard deviation and add error bars. 

Step 5: Examine the calibration curve

Examine the plot. The calibration curve should look linear and have a section that is non-linear—this is the limit of linearity (LOL), a sign that instrumental detection is nearing saturation.

Fit the data to a linear regression

Use statistical software to fit the data to a linear regression. 

The output is the following equation: 

y = mx + b

Where m is the slope (the units are absorbance/µm), and b is the y-intercept (the units are absorbance).

Obtain a coefficient of determination (R²)

The coefficient of determination (R²) quantifies goodness of fit—the square of the correlation coefficient between actual and predicted Y values. R² is typically a fraction between 0.0 and 1.0, with 1.0 being a perfect fit.