Examining Energy and Light: Understanding Spectroscopy and Spectrophotometry

Over the last few columns, we have examined the fundamentals of chromatography, liquid chromatography, methods development, and liquid chromatography–mass spectrometry (LC–MS). In this column, we dive into the field of optical spectroscopy and spectrophotometry and their use in the analytical laboratory. Many laboratories routinely use spectrophotometry as stand-alone instrumentation or coupled with a liquid chromatography system such as a diode array detector (DAD) or UV-vis detector. This column looks the overall scope of spectroscopy and breaks down the types of optical spectroscopy including spectrophotometry as a tool for the analyst to employ in improving their analyses.

Analytical organic laboratories are often acutely focused on their chromatography, mass spectrometry, and hyphenated techniques. Chromatography is a powerful separation tool under the larger umbrella of the overall fields of spectrometry and spectroscopy. The highest level, especially for analytical scientists, is spectrometry which is the measurement of the interaction of matter with energy (in particular electromagnetic radiation). 

Under spectrometry is the important subdivision of spectroscopy, which contains the bulk of what modern scientists consider analytical instrumentation techniques. Spectroscopy is the study or measurement of the interaction of matter and electromagnet radiation resulting in spectra (wavelength or frequency of the radiation). Spectroscopy is also described as the study of color from all bands of the electromagnetic (EM) radiation spectrum and is the basis of many of the most common types of laboratory analyses from atomic absorption to X-ray fluorescence (Figure 1). 

Electromagnetic radiation are all the waves in the electromagnetic field carrying electromagnetic radiation throughout space. Electromagnetic radiation is made up of oscillating waves of magnetic and electrical fields measured by frequency and wavelength. Waves show regular repetitive changes in value where points in the wave are either in-phase (oscillate in unison) or out-of-phase (oscillating at different points not in unison). Wavelength is the distance measured between the nearest two points in phase with each other. These two adjacent peaks (or troughs) are said to be separated by a single wavelength (λ) while the distance from peak to trough is the
amplitude (Figure 2).

The number of peaks in a unit of time or space is called its frequency. When peaks, such as the wave discussed, are measured in time it is referred to as temporal frequency measured in hertz (Hz) (one event per second) and is the reciprocal of period (the duration of time for one cycle of an event such as the occurrence of peak and trough).

The Electromagnetic Radiation Spectrum

The EM radiation spectrum encompasses a wide band of energy including energy waves such as radio waves, microwaves, infrared, visible light, ultraviolet, X-rays, and gamma rays
(Figure 3). Radio waves at the end of the spectrum have the largest wavelength and the lowest frequency. The size of these radio waves is in the hundreds of meters comparable with the size of buildings. This wavelength size does not travel particularly long distances or through many obstacles. Just think of the range of AM radio stations in a city with tall buildings versus the range of FM or TV signals in the same area. On the other end of the range are gamma rays which are high frequency and so small they cannot be measured since the particles slip between the molecules of measurement devices. For this reason, these very small waves from ultraviolet to gamma rays are a risk to cells, tissues, DNA, and other molecules (Figure 3).

Many laboratory analytical techniques are focused on range of waves associated with light from infrared to ultraviolet light in the range of 100 nm to 1 mm. This energy encompasses the ultraviolet ranges, visible light, and the infrared spectrum. Light outside of visible range for humans includes infrared light in the range upwards of 700 nm. Infrared (IR) light is divided into infrared-A (700–1400 nm), infrared-B (1400–3000 nm), and infrared-C (3000 nm to 1 mm). These wavelengths of light have a longer spatial period for a periodic wave than visible light. IR radiation is used in a wide variety of applications including scientific and industrial equipment, law enforcement, and medical devices. One of the common uses for IR is in the application of contactless thermometers during the COVID-19 pandemic. 

On the other side of visible light is ultra-violet radiation, which is also classified into three groupings: ultraviolet-A (UVA) from 315–400 nm; ultraviolet-B (UVB) from 280–315 nm; and ultraviolet-C (UVC) from 100–280 nm. UVA was frequently used in artificial tanning before it was discovered to cause formation of free radicals and reactive oxygen. UVB is absorbed to a large extent (along with UVC) by the Earth’s atmosphere. UVB and UVC cause the reaction leading to the production of ozone. UVB is a double-edged sword, it is needed for the production of ozone and the production of vitamin D in the body, but it is also the leading cause for sunburn and DNA damage leading to cancer. UVB is very photoreactive and can cause unwanted DNA changes in the photoreactive cells of the epidermis such as melanocytes and basal cells. UVA has the longest wavelength and penetrates beyond the skin’s hypodermis into the deeper dermis and can also change the DNA of skin cells, but not to the same degree as UVB radiation.

Most animals including human beings can only perceive light within the range of 400–700 nanometers, which also gives us the perception of colors when they are absorbed by objects and reflect back as the opposite color. We see the color red when light of many spectrums hits something like an apple and all of the other wavelengths of visible light are absorbed while the reds are reflected back for the human eye to see.

Energy and Matter Interactions

There are numerous interactions between EM and matter including absorption, transmission or refraction, reflection, and emission. Absorption occurs when matter transforms EM energy into internal energy (such as thermal energy) through an absorber. Transmission (refraction) is a function of the amount, wavelength, and angle of which energy (or light) pass through matter. Reflection is the measurement of the amount of energy reflected matter and emission is the change in energy state as the incident energy passes through matter. All of these processes become the basis for some form of detection or spectroscopic measurement technique (Figure 4).

Other types of spectroscopy interactions include elastic scattering (similar to reflection) where the incident beam is scattered within the target material rather than just reflected. Inelastic scattering also involves the measurement of scattering but is part in a change in wavelength. Impedance is the slowing of the transmitting of energy. Resonance spectroscopy is characterized by radiant energy being a radiating field between quantum states of the material. Finally, there is nuclear spectroscopy that utilizes the nuclei to determine the properties of matter. 

Spectroscopy techniques are divided into atomic and molecular spectroscopy depending on the target to be measured and the material being tested (Table I). Atomic spectroscopy studies the energy and matter interactions between atoms. Most of the atomic spectroscopy techniques are applied to the study of elemental composition. The instruments under atomic spectroscopy include atomic absorption (AA), X-ray fluorescence (XRF), and inductively coupled spectroscopy (ICP). Molecular spectroscopy studies the interaction of energy and matter between molecules and is most often found in techniques measuring organic molecules and includes instruments such as Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), UV, and visible light spectroscopy. 

Spectrometry instruments are often further divided and defined by the type of interactions they produce and the type of energy that is measured. For example, X-ray fluorescence (XRF) measures X-rays and the amount of fluorescent energy a material emits during exposure. Some techniques focus on a narrow band of interactions (such as emission only) or a small part the EM spectrum (like X-rays) while other techniques monitor an array of interactions and wavelengths on the spectrum. 

In addition to all these spectroscopy techniques that have been discussed, there are spectroscopy detectors that are coupled with other chromatography or mass spectrometry techniques into hybrid or hyphenated analytical systems. In some cases chromatography systems such as capillary electrophoresis (CE), gas chromatographs (GC), or liquid chromatographs (LC) are coupled to mass spectroscopy (MS), flame ionization detectors (FID), UV-vis spectrophotometers (UV-vis), refractive index detectors (RID), and evaporative light scattering detectors (ELSD) which each in their own way measure the interactions of energy and matter to produce a result. 

Techniques utilizing the interaction of the energy bands included in the wavelengths of “light” are frequently referred to as spectrophotometers. The basic pathway for a spectrophotometer contains an energy or light source which is then sometimes split or isolated by a filter or monochromator before encountering a sample. The energy interacts with the sample and the energy that is transmitted, absorbed, emitted, or reflected is measured by a detector (Figure 5).

Spectrophotometer Configurations

Spectrophotometers are a common, easy-to-use spectrometer found in many organic analytical chemistry laboratories. They often work on the simple principal of comparing the absorbance or transmission of light through a liquid sample compared to a blank or a standard material. The difference or change from the blank or standard material is used to calculate concentration or a number of other results. 

The most widely used form of spectrophotometers are UV or UV-vis spectrometers operating in the UV and visible wavelengths of light from 180–800 nm. There are several options and configurations for spectrophotometers including number of pathways (that is, beams) detectable and the types of modes of detection performed.

Spectrophotometers are characterized as either single beam, containing only one pathway from source to detector and therefore only have one sample pathway similar to Figure 5, or a double beam (split) system splits the energy source prior to interacting with the sample and diverts to a second sample cuvette which most often contains a blank, reference standard or sample (Figure 6).

There are two basic types of a double-beam configuration. Alternating in-time configurations have a series of converging mirrors that bring the beam back to one detector. The beam is directed at alternating times between the two cuvettes and the detection occurs alternately as well. Simultaneous in-time configurations split the beam between the two cuvettes but do not direct the resulting beams to a single detector and instead have multiple detectors that can operate simultaneously during scans. 

Some spectrophotometers have preprogrammed settings and allow for only the monitoring of selected or a single wavelength (fixed wavelength). This configuration usually employs some filter or slit which can be programed to allow only single wavelengths to be exposed to the sample. In contrast, scanning and array spectrophotometers allow for an entire range, multiple sets of wavelengths, or scanning subsequent wavelengths along an entire range of wavelengths. A scanning spectrometer uses a tunable monochromator to isolate individual wavelengths of light from the incident beam before interacting with the sample. An array spectrophotometer allows for the simultaneous scanning of a range and has no filter or monochromator; however, there is often a focusing slit to confine the incidence beam. This configuration allows the entire focused incident beam to interact with the sample and the data from individual wavelengths occur at the detector (known as reverse optics) (Figure 7).

Spectrophotometry Lamps

The incident energy or light is provided by several different types of lamps including halogen, xenon, deuterium, and light-emitting diodes (LED). Halogen lamps (also known as tungsten or quartz lamps) are similar to incandescent light bulbs and cover the wavelengths of visible light from about 320–1100 nm. In this lamp, the filament heats up and emits light when a current flows through it. The bulb is filled with inert gas to prevent evaporation of the tungsten filament. A halide is also contained in the lamp to create the halogen cycle to return evaporated tungsten to the filament and reduce blackening of the bulb. Instruments with only halogen lamps can only detect wavelengths of the visible light. Generally, halogen lamps last about 2000 hours and are comparatively low cost.

Deuterium lamps (D2) operate in the range of 190–370 nm and are often paired with halogen lamps to cover the UV-vis spectrum. D2 lamps operate at high temperature and require specialized quartz housings that can increases cost. A D2 lamp is a discharge light source with deuterium sealed in a bulb. A hot cathode is used to deliver a steady arc discharge. These lamps require a large power supply that adds to the cost. D2 lamps last about 1000+ hours.

Xenon lamps are discharge lamps and high energy sources just like the D2 lamp, but xenon lamps cover the entire UV-vis range and more from 185–2000 nm and eliminate the need for two types of lamps. Xenon gas is sealed in the bulb with a tungsten electrode. The lamp operates at 80 Hz, which can prolong their life but comes with increased costs higher than halogen or deuterium lamps. LED lamps produce single wavelengths of light with only small variations in bandwidth. The positive side means no monochromator is needed for some spectrophotometers.

Each type of lamp has its own benefits and drawbacks including range, cost, and lamp life. Some technological or programming advances for spectrophotometers can increase lamp life by limiting lamp emissions to set time periods. One feature called press-to-read (PTR) ensures the lamp is only ‘on’ when needed to increase lamp life.

Spectrophotometry Measurement Modes and Calculations

In all the configurations of spectrophotometers there are often multiple modes in which a system can be operated. Many multi-mode instruments can operate in standard fixed wavelength or multiwavelength modes and some in scanning modes as well. The data can then be used for different types of calculation such as for transmittance, absorbance, concentration, and kinetics.

Transmittance (T) is the calculation of the different of intensity (I0) energy from the incident beam to the resulting beam (I) (Figure 8). Transmittance is usually expressed in units of percent.

The next type of measurement mode is the negative log of transmittance called absorbance (Equation 1).

When a sample in solution is placed in a transparent cuvette the light intensity is found to be proportional to the sample concentration and the length of the pathway of the cuvette (Figure 9). This is called Beers Lambert Law.

A standard type of result is obtained by calculating concentration with either a factor or a standard curve. In this type of calculation, the concentration of a sample is determined by multiplying an absorbance (Abs) value by a specific factor. This type of calculation uses Beers Lambert Law where the concentration is proportional to the absorbance (Equation 2).

The extinction coefficient (ε) is the characteristic of a substance that tells how much light is absorbed at a particular wavelength per concentration, molarity, or pathlength. The extinction coefficient is sample specific and describes how much the sample can absorb in L/(cm*mol) or mL/(cm*g). If the factor is now known, then the concentration of a sample can be determined by standard curve where known concentrations standards are measured, and a standard curve is created.

Some other modes of data include kinetic measurements where the absorbance of a sample is measured over time for changes. The change in absorbance is plotted giving rise to details regarding reaction rates. In the case of these types of measurements, internal standards can also be added to compensate for any losses or variations and correct for bias. 

There are a number of other specifications—such as accuracy, resolution, and bandwidth—that should be considered in any spectrometers. In chromatography systems these terms refer to the size and retention time of peaks, but in spectrometers it refers to the resolution and accuracy of the measurement around each wavelength being measured.

Sometimes, a spectrophotometric system’s function is enhanced by the accessories that aid measurement including high-throughput sampling systems like multicuvette holders and changers, and sippers (pumping systems allowing for either very accurate or continuous sample introduction), or temperature-controlled accessories for temperature sensitive samples. 

Most basic spectroscopy systems are equipped to perform routine analysis and experiments. Higher end instruments can provide more functionality but increase cost and the learning curve for operation. The selection of a spectrophotometry system ultimately is a tradeoff of functionality, target, resolution, and price.

Final Thoughts

The selection of spectroscopy instrumentation is dictated by the type of samples, target elements or analytes, and analytical detection level needed. In some cases, the ability to collect or recover the sample after analysis may play a role in the selection of a technique. Many mass spectroscopy methods and instruments which change or consume the sample are known as destructive methods. Methods that can sample in situ, or where sample is not changed or consumed by the process are non-destructive and may be used to collect analytical fractions or recover valuable materials. 

Organic samples or targets are best analyzed by molecular spectroscopy techniques whereas inorganic samples or targets are the focus of atomic spectroscopy. Samples with color or chromophores will interact with light energy waves while other analyte functionalities will react with different wavelengths of energy and produce separate interactions that can be measured. The selection of the appropriate analytical technique depends on all these factors to achieve the most accurate results possible for your targets.

Further Reading

1. B.T.I. Corp, S. Patel, and M. Lares, Hamamatsu. "A Guide to Selecting Lamps," https://www.photonics.com/Articles/A_Guide_to_Selecting_Lamps/a44487 (accessed 2021 -05 -21).
2. A. Hind, Agilent 101: An Introduction to Optical Spectroscopy. 7.
3. D. Sahin, Atomic Spectroscopy; IntechOpen, 2019. https://doi.org/10.5772/intechopen.89269.
4. A. Sapkota,. "22 Types of Spectroscopy with Definition, Principle, Steps, Uses" https://microbenotes.com/types-of-spectroscopy/ (accessed 2021 -05 -25).
5. V.V. Rosen, Atomic Spectroscopy: Basic Principles and Instruments, 10(10), 55 UTC.
6. ACTTR Inc., "What Are The Types of Lamps in An UV-Vis Spectrophotometer?" https://www.acttr.com/en/en-faq/en-faq-uv-vis/219-en-faq-what-kind-of-uv-vis-lamps.html (accessed 2021 -05 -21).
7. "Glossary of UV Vis Spectrophotometry," Biochrom http://www.biochrom.co.uk/content/1/91/glossary-of-uv-vis-spectrophotometry.html (accessed 2021 -05 -21).

ABOUT THE COLUMNIST

PATRICIA ATKINS is a Senior Applications Scientist with Spex, an Antylia Scientific company and has been a member of many cannabis advisory committees and working groups for cannabis including NACRW, AOAC and ASTM.

How to Cite this Article

P. Atkins, Cannabis Science and Technology 4(8), 22-30 (2021).