Overview

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• Source: Microbioz India


• Date: 25 Jan,2024

A technique known as UV-Vis Spectroscopy has a wide range of uses, it is used to measure the concentration of substances in a sample by measuring the absorption or transmission of ultraviolet and visible light through the sample. Nevertheless, as with any analytical method, there are obstacles that come with it.

Here are some common problems encountered during UV-Vis spectroscopy analysis and their possible ways out:

Chemical impurities in the sample:

1. Challenge: Chemical impurities in samples can interact with the analyte.
2. Solution: Use solvents and containers of high purity. Filter any particulates from samples.

Baseline variation:

1. Challenge: Change in the baseline signal affects accuracy of absorbance measurements.
2. Solution: Optimize the instrument conditions; check for impurities in solvents, ensure stable environment (temperature and humidity). Perform regular baseline correction.

Also read:

Exploring the Role of UV Vis Spectroscopy in Pharmaceutical Analysis

UV/VIS Spectroscopy for Determination of Nucleic Acids (DNA and RNA)

Instrument calibration issues:

1. Challenge: Calibration that is inaccurate results into wrong determinations of concentration.
2. Solution: Instrument should be calibrated using standard reference materials that have known concentrations. Regularly check and recalibrate the instrument to maintain accuracy.

Spectral Interference:

1. Challenge Overlapping absorption spectra from different constituents in the sample
2. Solution: Select wavelengths where interference does not arise. Use chemometric techniques or mathematical algorithms to deconvolute overlapping spectra.

Solvent absorption:

1. Challenge Some solvents may absorb within UV-Vis range thereby interfering with measurements
2. Solution: Choose solvents that do not interfere with analysis . Subtract solvent contribution using blank solution

Scattering effects:

1. Challenge: Measurement can be affected by scattering of light by particles present in the sample
2. Solution: Minimise scattering effects using filtered cuvettes; centrifuge or filter samples to remove particles

Photobleaching:

1. Challenge: Some samples might undergo photodegradation when exposed to light for long periods
2. Solution: Minimize time of exposure, use less intense light or measure in the dark where possible. Also, protective coatings can be used for light-sensitive samples.

Temperature Variations:

1. Challenge: Changes in temperature affect both viscosity and concentration of the sample.
2. Solution: Control and monitor the temperatures of the sample and the instrument. If necessary, use a temperature controlled cuvette holder.

Poor Signal-to-Noise Ratio:

1. Challenge: The signals may be weak for low concentration samples.
2. Solution: Increase cuvette path length; take more concentrated sample or increase instrument sensitivity. Take multiple scans and average them thus improving signal to noise ratio.

Non-linear Calibration Curve:

1. Challenge Absorbance concentration relationship is not linear
2. Solution: Choose an appropriate calibration curve model; if possible, apply standard addition method especially in complex samples

These challenges can be overcome through regular maintenance, appropriate calibrations and careful preparation of samples to obtain reliable results in UV/Vis Spectroscopy analysis. Furthermore, understanding specific properties of analyzed samples is crucial for successful use of this technique.