Introduction to Spectrophotometry

Overview

Spectrophotometry is a technique used to measure how much light is absorbed by a solution at different wavelengths. It is essential in biology for quantifying concentrations of light-absorbing molecules, such as pigments, in solution. The instrument used for these measurements is called a spectrophotometer.

• Purpose: To determine the absorbance and action spectra of photosynthetic pigments.

• Applications: Used in labs to analyze pigment concentration and properties.

Light and Electromagnetic Radication

Electromagnetic Spectrum

Understanding the electromagnetic spectrum is crucial for spectrophotometry. Light is a form of electromagnetic radiation (EMR), which consists of waves of energy that travel through space. EMR includes a range of wavelengths, from very short gamma rays to long radio waves.

• Visible Light: The portion of EMR that is visible to the human eye, ranging from approximately 380 nm (violet) to 750 nm (red).

• Wavelength: The distance between two consecutive peaks of a wave, denoted by the Greek letter lambda (λ).

• Energy Relationship: Shorter wavelengths have higher energy; longer wavelengths have lower energy.

Example: The colors of visible light (ROYGBIV: Red, Orange, Yellow, Green, Blue, Indigo, Violet) correspond to increasing energy and decreasing wavelength.

Interaction of Light with Matter

When electromagnetic radiation encounters matter, it can be reflected, transmitted, or absorbed. The specific interaction depends on the properties of the material and the wavelength of the light.

• Reflection: Light bounces off the surface.

• Transmission: Light passes through the material.

• Absorption: Light is taken up by the material, often resulting in a color change.

Example: Chlorophyll absorbs most red and blue light, transmitting and reflecting green light, which is why plants appear green.

Spectrophotometers – Principles of Operation

How a Spectrophotometer Works

A spectrophotometer measures the amount of light absorbed by a sample at specific wavelengths. It separates light into its component wavelengths and passes it through a sample, measuring the intensity of transmitted light.

• Light Source: Produces white light.

• Monochromator: Prism or grating selects a specific wavelength.

• Sample Holder: Holds the solution in a cuvette.

• Detector: Measures transmitted light and calculates absorbance or transmittance.

Example: Measuring the absorbance of a blue dye (DCPIP) at 625 nm to determine its concentration.

Units of Measurement

Transmittance and Absorbance

The spectrophotometer displays two scales: transmittance and absorbance.

• Transmittance (T): The proportion of light that passes through the sample. Defined as:

• It: Intensity of transmitted light

• I0: Intensity of incident light

• Percent Transmittance (%T):

Example: If and , then and .

• Absorbance (A): The amount of light absorbed by the sample. Defined as:

Example: If , then

The Beer-Lambert Law

Relationship Between Absorbance and Concentration

The Beer-Lambert Law states that the absorbance of a solution is directly proportional to the concentration of the absorbing substance, the path length of the light through the solution, and the molar absorptivity of the substance.

• A: Absorbance (unitless)

• ε: Molar absorptivity (L·mol-1·cm-1)

• c: Concentration of the solution (mol/L)

• l: Path length of the cuvette (cm)

Example: By measuring absorbance at a known path length and molar absorptivity, the concentration of a solute can be determined.

Sample Preparation and Blanking

Importance of Blanks

Blanks are used to calibrate the spectrophotometer and account for absorbance by solvents or containers. A blank solution is identical to the sample solution in all aspects except it does not contain the analyte of interest.

• Purpose: To set the spectrophotometer to zero absorbance, ensuring only the analyte's absorbance is measured.

• Procedure: Use the blank to calibrate before measuring samples.

Example: A blank containing only buffer is used before measuring DCPIP solutions.

Using the Spectrophotometer & Colorimetric Measurement of DCPIP

Operation of the Unico 1100RS Spectrophotometer

The Unico 1100RS spectrophotometer is used for colorimetric measurements, allowing quantification of absorbance or transmittance at specific wavelengths. The procedure involves warming up the instrument, preparing samples and blanks, and measuring absorbance.

• Step 1: Warm up the spectrophotometer for at least 15 minutes.

• Step 2: Prepare DCPIP solution and buffer.

• Step 3: Pipette solutions into cuvettes and place in the sample holder.

• Step 4: Use the blank to calibrate the instrument.

• Step 5: Measure absorbance of samples at 625 nm.

Example: Measuring absorbance of DCPIP at different concentrations to create a standard curve.

Standard Curves and Unknown Concentrations

Creating a Standard Curve

A standard curve is generated by measuring the absorbance of solutions with known concentrations. This curve is then used to determine the concentration of unknown samples by comparison.

Procedure: Measure the absorbance of each tube at 625 nm, using tube 1 as the blank. Plot absorbance versus concentration to create the standard curve.

Application: Use the standard curve to determine the concentration of unknown samples by matching their absorbance values.

Additional info:

• Opaque or cloudy samples cannot be analyzed in standard spectrophotometers due to scattering of light.

• Digital spectrophotometers can report measurements in both %T and A, depending on user needs.