Colour is everywhere. Every chemical compound absorbs, transmits, or reflects light over an electromagnetic spectrum in wavelengths. When light passes through any solution a section of it is absorbed. Spectrophotometry allows both qualitative and quantitative analysis. As the concentration of a substance increases light absorption increases, and light transmission decreases.
Spectrophotometry is used in chemistry, biochemistry (for enzyme-catalysed reactions), physics, biology, and clinical studies (examining haematology or tissues). It allows scientists to analyse different samples without having any skin contact as the samples are contained in a small tube called a cuvette or in case of the Photopette, measurements are done directly in the sample container without having to transfer it.
Spectrophotometry is a standard and inexpensive technique to measure light absorption or the amount of chemicals in a solution. It uses a light beam which passes through the sample, and each compound in the solution absorbs or transmits light over a certain wavelength.
Spectrometry is measured by a spectrophotometer; an instrument that is made up of two instruments – a spectrometer and a photometer. The spectrometer produces the light of the wavelength and the photometer measures the intensity of light by measuring the amount of light that passes through the sample.
In addition to those two components, spectrophotometers consist of a light source, a monochromator, a sample chamber containing a cuvette, a detector (such as a photomultiplier tube or photodiode) to detect the transmitted light, a digital display and a data analysis software package.
Spectrophotometers rely on light sources to operate. Because of the wide range of samples, light sources can vary in nature, and use a wide spectrum of wavelengths, including visible, UV and IR.
The monochromator (such as a prism or grating) inside the machine refracts the light into a single spectrum and disperses polychromatic light into the essential wavelengths. A grating divides the light available into different segments. Gratings are common in spectrophotometers that use UV, visible and infrared regions.
The sample chamber is where the operator inserts the sample for analysis. Samples are typically placed into a cuvette made of a material such as glass or quartz.
The detector is the light-receiving element that absorbs the energy of the incident light. Examples of typical spectrophotometer detectors include photomultiplier tubes and photodiodes. They convert the light energy into an electrical signal, which is converted into an absorption figure.
Modern day spectrophotometers typically have a digital display built into the instrument. This gives operators an accessible way to change instrument settings, set up method parameters and see results. It has no effect on the way the instrument works however.
Alongside digital displays, most spectrophotometers have the ability to do any calculations and analysis. Once all the method parameters have been set up within the instrument, data and results are output once the method is complete.
In the spectrophotometer, the number of photons absorbed by a solution is called the absorbance readout. The longer the path-length that the light must travel through a solution prior to it reaching the detector, the greater the chance of a photon being absorbed.
Different compounds absorb best at different wavelengths. A UV-visible spectrophotometer uses light over the ultraviolet range (185 – 400 nm) and visible range (400 – 700 nm) of the electromagnetic radiation spectrum. Whereas an IR spectrophotometer uses light over the infrared range (700 – 15000 nm).
Ultraviolet (UV) and visible (VIS) spectroscopy show electronic transitions in atoms and molecules, to measure this a spectrophotometer is used. Compounds that absorb in the visible region are coloured, whereas ones that absorb only in the UV region are colourless.
UV-VIS spectrophotometer usually use two light sources. A deuterium lamp is used for the UV region and a tungsten lamp for the VIS region. These lights reach the monochromator via a mirror. The wavelength for red light is between 700 and 750 nm and blue between 400 and 450 nm. If the wavelength is shorter than 350 nm it is UV and has more energy.
There are generally two types of spectrophotometers: a single beam, and double beam. Single beam spectrophotometers use a single beam of light – visible or UV – which passes through a sample in a cuvette. Light intensity is measured before and after the light passes through the sample, and using Beer-Lambert’s Law (see further below), the concentration of the analyte can be calculated.
Double beam spectrophotometers work in a similar way to single beam spectrophotometers but with a key difference. The initial light source is split into two; one beam passes through the sample, and the other through a reference solution or the solvent. The ratio of the two light beams then corresponds to the absorbance of the sample.
Single beam spectrophotometers are generally more compact and have a higher dynamic range but the optics in a double beam can permit higher levels of automation, better precision and can correct for background absorption of the solvent.
Spectrophotometers measure absorbance (A) and transmittance (T). The intensity of light (I0) measures photons per second. When light passes through a blank sample, it does not absorb light so is symbolised as (I). Scientists use blank samples without chemical compounds as a reference. They contain everything that is in the sample cuvette, except the one material which absorbance is being measured.
To calculate the transmittance the following equation is used:
Transmittance (T) = It/I0
It = Light intensity after passing the cuvette (transmitted light)
I0 = Light intensity before passing the cuvette (incident light)
Absorbance (A) = – log10 T = – log IS/IR
The Beer-Lambert Law (sometimes just referred to as Beer’s Law) is the relationship between the attenuation of light, through a substance, and the properties of the substance.
The Beer-Lambert law indicates that the amount of light that is absorbed by a substance is proportional to the amount of the sample concentration. It is also determined by the amount of solute that is present. But to fully understand the Beer-Lambert Law, understanding the relationship between absorbance and transmittance is of importance.
The sample molecules or ions in a solution can be detected and quantified using a spectrophotometer and The Beer-Lambert Law with this equation: A = ƐCL
A = absorbance of light at a specific wavelength
Ɛ = molar extinction coefficient (the absorbance of 1 mole of a substance dissolved in 1 litre solvent)
C = the molar concentration of a sample
L = the optical path length of a sample
To measure absorption of a sample, you need to know the values of the three factors – molar extinction coefficient, molar concentration and optical path length.
The molar extinction coefficient is a value at which a chemical species attenuates light at a given wavelength. The SI unit is m2/mol, but sometimes expressed as M-1 cm-1 or L mol-1 cm-1.
You can obtain the molar extinction coefficient for your target sample from literature sources.
Concentration refers to the concentration of the sample. This is simply the molar concentration, and measured as mol/L.
Path length refers to the distance that light travels through the sample as absorption is directly proportional to distance of light traveled. This is determined by the size of your cuvette.
Whilst determining the absorption from Beer-Lambert’s Law is relatively straightforward, make sure to use the correct units, or convert them correctly to avoid any errors in your end result.
Spectrophotometry is an incredibly robust technique that has been adopted by many areas of science, industry and manufacturing. Below, we look at some of the ways spectrophotometry gets used and why.
Understandably, the process of manufacturing pharmaceuticals is carefully monitored to ensure the end product is exactly what it says it is.
Where possible, using spectrophotometry is a quick and effective method to perform QC testing on raw materials, intermediates and final products. By measuring samples and comparing them to samples, it is possible to understand whether the correct compound has been manufactured and whether any impurities are present.
Due to the low sample size typically required for spectrophotometry, it also makes it an incredibly cost effective method, where raw material costs are high.
Water quality is incredibly important for industry, manufacturing and consumption, yet it can be hard to determine quality without some form of testing. Spectrophotometry provides a non-destructive method of analyzing water for quality, clarity and purity. Typically, water is measured on the APHA or Hazen scale, which was initially introduced to measure waste water, but can be applied to ‘purer’ samples also.
Measuring water quality has important applications, such as determining the presence of heavy metals in drinking water, determining the concentration of pollutants in wastewater and validating water purity for laboratory testing or manufacturing processes.
What is the difference between a spectrometer and spectrophotometer?
Although similar words, both actually have different meanings. A spectrometer is just one part of a whole spectrophotometer, and is the part that is mostly responsible for measurement. A spectrophotometer is the word used to describe the whole instrument.
What is the difference between absorption and optical density?
Absorption and optical density may be used interchangeably, but they mean different things. Absorption measures the amount of light that hits the detector, whereas optical density measures the amount of attenuation, or the loss of light intensity. Therefore, both measurements are different, however, most spectrophotometers can produce both results.
What are the units of absorption?
Absorption has no units and should be reported as a figure only. However, it is common to see absorption reported with the units ‘a.u.’, which typically stand for ‘arbitrary units’, but sometimes in the case of absorption, are mistakenly referred to as ‘absorption units’.
What is photospectrometry?
Photospectrometry is simply another name for spectrophotometry, however, it is less commonly referred to. Spectrophotometry is the correct term to use to describe the method of measuring absorption.