Spectrophotometry is a method to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through a sample solution. The basic principle is that each compound absorbs or transmits light over a certain range of wavelength. This measurement can also be used to measure the amount of a known chemical
substance. Spectrophotometry is one of the most useful methods of quantitative analysis in various ×elds such as chemistry, physics, biochemistry, material and chemical engineering
and clinical applications.
Every chemical compound absorbs, transmits, or reØects light (electromagnetic radiation) over a certain range of wavelength. Spectrophotometry is a measurement of how much a chemical substance absorbs or transmits. Spectrophotometry is widely used for quantitative analysis in various areas (e.g., chemistry, physics, biology, biochemistry, material and chemical engineering, clinical applications, industrial applications, etc). Any application that deals with chemical substances or materials can use this technique. In biochemistry, for example, itis used to determine enzyme-catalyzed reactions. In clinical applications, itis used
to examine blood or tissues for clinical diagnosis. There are also several variations of spectrophotometry such as atomic absorption spectrophotometry and atomic emission spectrophotometry.
A spectrophotometer is an instrument that measures the amount of photons (the intensity of light) absorbed after it passes through sample solution. With the spectrophotometer, the amount of a known chemical substance (concentrations) can also be determined by measuring the intensity of light detected. Depending on the range of wavelength of light
source, it can beclassi×ed into two different types:
UV-visible spectrophotometer: uses light over the ultraviolet range (185 – 400 nm) and visible range(400-700nm)of electromagnetic radiation spectrum.
IRspectrophotometer: uses light over theinfrared range(700-15000nm)of
electromagnetic radiation spectrum.
In visible spectrophotometry, the absorption or the transmission of a certain substance can be determined by the observed color. For instance, a solution sample that absorbs light overall visible ranges (i.e., transmits none of the visible wavelengths) appears black in theory. On the other hand, if all visible wavelengths are transmitted (i.e., absorbs nothing), the solution
sample appears white. If a solution sample absorbs red light (~700 nm), it appears green becausegreen is the complimentary color of red. Visible spectrophotometers, in practice, use a prism to narrow down a certain range of wavelength (to ×lter out other wavelengths) so that
the particular beam of light is passed through a solution sample.
Devices and mechanism
Figure 1 illustrates the basic structure of spectrophotometers. It consists of a light source, a collimator, a monochromator, a wavelength selector, a cuvette for sample solution, a photoelectric detector, and a digital display or a meter. The detailed mechanism is described below.
A spectrophotometer, in general, consists of two devices; a spectrometer and a photometer.A spectrometer is a device that produces, typically disperses and measures light. A photometer indicates the photoelectric detector that measures the intensity of light.
Spectrometer: It produces the desired range of wavelength of light. First a collimator (lens)
transmits a straight beam of light(photons)that passes through a monochromator (prism) to split it into several component wavelengths (spectrum). Then a wavelength selector (slit)transmits only the desired wavelengths, as shown in Figure1.
Photometer: After the desired range of wavelength of light passes through the solution of a sample in cuvette, the photometer detects the number of photons that is absorbed and then sends a signal to a galvanometer or a digital display, as illustrated in Figure1.
Beer-Lambert Law (also known as Beer’s Law) states that there is a linear relationship between the absorbance and the concentration of a sample. For this reason, Beer’s Law can only beappliedwhen thereis a linear relationship. Beer’s Law is written as:
A is the measure of absorbance(no units),
ϵ is the molar extinction coef×cient or molar absorptivity (or absorption coef×cient),
l is the path length, and
C is the concentration.
The molar extinction coef×cient is given as a constant and varies for each molecule. Since absorbance does not carry any units, the units for must cancel out the units of length and concentration. As a result, has the units: L·mol ·cm. The path length is measured in centimeters. Because a standard spectrometer uses a cuvette that’s 1cm in width, is always assumed to equal 1 cm. Since absorption, , and path length are known, we can calculate the
concentration of the sample.