Why uv vis spectrophotometer

After the light has passed through the sample, a detector is used to convert the light into a readable electronic signal. Generally, detectors are based on photoelectric coatings or semiconductors. A photoelectric coating ejects negatively charged electrons when exposed to light. When electrons are ejected, an electric current proportional to the light intensity is generated. When semiconductors are exposed to light, an electric current proportional to the light intensity can pass through.

After the electric current is generated from whichever detector was used, the signal is then recognized and output to a computer or screen. Figures 2 and 3 show some simplified example schematic diagrams of UV-Vis spectrophotometer arrangements.

Figure 2: Schematic diagram of a cuvette-based UV-Vis spectroscopy system. Figure 3: Schematic diagram of a cuvette-free UV-Vis spectroscopy system. UV-Vis spectroscopy information may be presented as a graph of absorbance, optical density or transmittance as a function of wavelength.

However, the information is more often presented as a graph of absorbance on the vertical y axis and wavelength on the horizontal x axis. This graph is typically referred to as an absorption spectrum; an example is shown in Figure 4.

Figure 4: An example absorption spectrum taken from a UV-Vis spectrophotometer. The sample examined was expired hemoglobin dissolved in neutral pH phosphate buffer. The absorbance A is equal to the logarithm of a fraction involving the intensity of light before passing through the sample I o divided by the intensity of light after passing through the sample I. The fraction I divided by I o is also called transmittance T , which expresses how much light has passed through a sample.

As a consequence, A has no units. Sometimes AU is used to indicate arbitrary units or absorbance units but this has been strongly discouraged. Beer—Lambert's law is especially useful for obtaining the concentration of a substance if a linear relationship exists using a measured set of standard solutions containing the same substance. Equation 1 shows the mathematical relationships between absorbance, Beer—Lambert's law, the light intensities measured in the instrument, and transmittance.

Equation 1: A set of equations showing the relationships between absorbance A , Beer—Lambert's law, the light intensities measured in the instrument, and transmittance.

The term optical density OD is sometimes incorrectly used interchangeably with absorbance. OD and absorbance both measure the amount of light intensity lost in an optical component, but OD takes into consideration loss from light scattering whereas absorbance does not. If very little light scattering is present in a measurement, then OD may be approximated directly using absorbance and Beer—Lambert's law may be used.

Knowing the experimental conditions during measurements is important. Cuvettes designed for a 1 cm path length are standard and are most common. Sometimes, very little sample is available for examination and shorter path lengths as small as 1 mm are necessary.

Where quantitation is required, absorbance values should be kept below 1, within the dynamic range of the instrument. Two simple possible solutions to this problem are to either dilute the sample or decrease the path length. If the instrument was absolutely perfect in every way, the baseline would have zero absorbance for every wavelength examined. In a real situation, however, the baseline spectrum will usually have some very small positive and negative absorbance values.

For best practice, these small absorbance values are often automatically subtracted from the sample absorbance values for each wavelength of light by the software to obtain the true absorbance values. Depending on the purpose of the analysis, the construction of a calibration curve may be desirable. Building a calibration curve requires some data analysis and extra work but it is very useful to determine the concentration of a particular substance accurately in a sample based on absorbance measurements.

There are however, numerous circumstances in which a calibration curve is not necessary including OD measurements for bacterial culturing, taking absorbance ratios at specific wavelengths for assessing the purity of nucleic acids or identifying certain pharmaceuticals.

In UV-Vis spectroscopy, the wavelength corresponding to the maximum absorbance of the target substance is chosen for analysis. This choice ensures maximum sensitivity because the largest response is obtained for a certain analyte concentration.

Note that two maximum absorbance peaks are present in the Food Green 3 dye, a smaller maximum absorbance peak at nm and a more intense maximum absorbance peak at nm. To gain maximum sensitivity when calculating an unknown concentration of Food Green 3, the maximum absorbance peak at nm was used for analysis.

Standard solutions across a range of known concentrations were prepared by diluting a stock solution, taking absorbance measurements and then plotting these on a graph of absorbance versus concentration to build a numerical relation between concentration and absorbance.

A calibration curve was created using a least squares linear regression equation. The closer the data points are to a straight line, the better the fit. The y intercept in the straight line equation was set to zero to indicate no absorbance when no dye was present. The equation shown in Figure 5 is used to calculate the concentration of Food Green 3 variable x in an unknown sample based on the measured absorbance variable y.

Figure 2. Figure 3. Hindi Version. Old Website. Basic Principle: The Beer-Lambert law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length Figure 2. Outlining UV-Vis Spectrophotometers A UV-Vis spectrophotometer measures the intensity of light transmitted through a sample compared to a reference measurement of the incident light source.

Related Product. Share This Post:. Please wait while you are redirected to the right page Please share your location to continue. How to Choose Solvent for my Application? The ideal solvent for the preparation of sample solutions: dissolves all types of compound, completely transparent at all wavelengths, nonflammable and nontoxic.

Standard Plastic Rectangular Cells - disposable. Four-Clear-Sided Plastic Rectangular Cells for fluorimetry as well as for spectrophotometry - disposable. Flow Cells. Distilled water. Ethanol absolute. Health Hazard.