What is a Spectrophotometer?

Spectrophotometer vs Spectrometer | Spectrophotometer Components | Measuring Absorbance With Spectrophotometer | Choosing A Spectrophotometer

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A spectrophotometer is an instrument used to measure absorbance or transmittance of light after it interacts with a sample. This is an important measurement for many research applications. Absorbance and transmission measurements can be used to:

• Identify materials by mapping molecular absorption profiles.
• Work out solute concentrations in solutions.
• Detect trace impurities in samples.
• Track the progression of a chemical reaction.

Spectrophotometers differ from simpler modular spectrometers as they take individual measurements at specific wavelengths, whereas USB spectrometers measure the intensity of transmitted light at many wavelengths simultaneously.

By measuring light one wavelength at a time, spectrophotometers are more sensitive than spectrometers, and they can achieve much higher resolution.

Spectrophotometer vs. Spectrometer

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Although spectrophotometers and UV-vis spectrometers measure the same thing, they have different designs and therefore have different benefits and limitations.

Spectrometers are instruments built to separate and measure the intensity of different spectral components simultaneously. Therefore, UV-vis spectrometers disperse light using a diffraction grating, then direct this dispersed light towards an array detector.

The spatial separation of this dispersed light is measured by multiple detectors across this array (often a charge-coupled device). This way the CCD detector can measure wavelength and intensity at the same time. Often, the density of this array determines the spectrometers spectral resolution. Spectrometers require a separate sample holder and light source.

Spectrophotometers use a single pixel detector which allows higher resolution and sensitivity. They have separate compartments to disperse light (often using a monochromator) and transmit it through a sample holder with a fixed position. These individual components are built into a single unit.

Spectrophotometer Components

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Spectrophotometers contain several components housed in one system. The Ossila spectrophotometer contains:

• A broadband white light source
• A monochromator
• A sample holder (also containing focussing optics)
• A silicon photomultiplier detector

How to Measure Absorbance with a Spectrophotometer?

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Spectrophotometers use absorbance spectroscopy principles, a technique grounded in the Beer-Lambert Law. This law relates the amount of light absorbed by a sample to the absorbing properties of that sample and the path length of the light. Specifically, absorbance (A) is given by:

Where

• ε is the molar absorptivity (a constant for a given substance and wavelength)
• c is the concentration of the absorbing species
• l is the path length of the sample (usually the width of a cuvette, in cm)

Absorbance is measured on a logarithmic scale, representing the ratio of incident light intensity (I₀) to transmitted light intensity (I) as it passes through a sample:

Absorbance is a unitless quantity and is a relative measurement. But one conclusion that can be drawn from these two equations, is that even small changes in concentration can result in significant changes in absorbance.

Absorbance measurements are wavelength dependent. Molecules absorb specific wavelengths of light based on their electronic structure, giving rise to characteristic absorption spectra for every material. These spectra serve as molecular fingerprints, allowing for both qualitative identification and quantitative analysis of compounds.

To ensure accurate absorbance measurements, spectrophotometers are typically zeroed or baseline-corrected using a blank (a cuvette containing only the solvent). This corrects for any absorbance by the solvent or cuvette material itself, ensuring only the sample’s contribution is measured. Then the sample is placed in the spectrophotometer, and the absorbance spectrum is measured.

Choosing a Spectrophotometer

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