# Theory of UV-Visible Spectroscopy (The Basics)

## UV-Visible Spectroscopy

UV-Visible/NIR spectroscopy(UV-Vis Spectroscopy) can be divided into ultraviolet, visible, and near-infrared regions of the spectrum, depending on the wavelengths used. since its frequency is close to the overtone frequency of many natural vibrations, weak substance-specific absorption bands can be detected. It can therefore be used for non-destructive measurements, such as determining the sugar, lipid, protein content of foodstuffs and for identifying medicinals.

## Principle of UV-VIS Spectroscopy

The theory of UV-visible spectroscopy exploits the wave-like nature of electromagnetic radiation and its interaction with matter and is widely used in analytical spectroscopic instruments to identify, characterize and quantify an extraordinary wide range of molecular compounds.

When a material is irradiated with an electromagnetic wave, phenomena such as transmission, absorption, reflection, and scattering can occur and the observed spectrum shows the interaction of wavelengths with objects of a discrete dimensions, such as atoms, molecules, and macromolecules.

Absorption occurs when the frequency of the incoming light is equal to the energy difference between a molecule’s ground and excited states. The excitation of an electron from the ground state to an excited state is described as an electronic transition (Figure 1), this is the key fundamental of molecular spectroscopy

The energy difference of each ground/excited state pair corresponds to an absorption band. The relationship between the energy difference and wavelength is described by the Planck equation.

E=hν=hc/λ

where E is the energy required to promote an electron from the ground to excited state, h is Planck’s constant, ν is the wavenumber, c is the speed of light, and λ is the wavelength.

Planck’s equation demonstrates that the less energy needed to excite the electrons, the longer the wavelength of the absorption band. The absorption bands are indicative of the molecular structure of the sample and will shift in wavelength and intensity depending on the molecular interaction and environmental conditions. These bands are typically broad and featureless due to the numerous molecular vibrational levels associated with the electronic energy levels.

UV-Visible/NIR spectroscopy can be divided into ultraviolet, visible, and near-infrared regions of the spectrum. The ultraviolet region is defined as 180 to 400 nm, visible between 400 and 800 nm, and the near-infrared is from 800 to 3200 nm.  Near-infrared light is generally poorly absorbed because its photon energy is insufficient to induce electronic transitions and its frequency is greater than the natural vibration frequency of most chemical bonds.  However, since the frequency in the NIR is close to the overtone frequency of many natural vibrations, weak substance-specific absorption bands can be detected. It is convenient for a range of nondestructive measurements, such as determining the moisture, sugar, lipid, protein content of foodstuffs and for identifying medicinals.

## Beer-Lambert Law

A UV-Visible/NIR spectrophotometer measures the transmittance or the amount of light transmitted through a sample by ratioing the intensity of the incident light (I0) to the intensity of the transmitted light (I).

T= I/I0

The relationship between transmittance and absorbance is described by the following equation

abs  =   2-log⁡ I/I0 ∙100       =   2-log (%T)

Absorbance measurements are frequently used to quantify an unknown sample’s concentration by exploiting the Beer-Lambert Law that describes how light is attenuated based on the materials it passes through.  The transmittance, and therefore the absorbance, are directly proportional to a sample’s concentration, c, molar absorptivity, ε , and cuvette pathlength, l.

I=I0 e-εcl

Taking the logarithm on both sides and transforming the formula,

-log I/I0 = εcl

If the left side -log(I/I0) is defined as the absorbance A, then

A= εcl

The amount of light absorbed by the sample depends on the number of molecules interacted with. The more concentrated a sample is, the more molecules are present and the higher the absorbance. Likewise, the longer the pathlength of the cell, the greater the distance that the light travels through the sample, increasing the number of molecules interacted with and therefore the absorbance. To compare the absorbances of two solutions with either different concentrations or pathlengths, there needs to be a constant variable to normalize the data on. Additionally, to determine a sample’s concentration by measuring absorbance, the cell pathlength and the strength of the electronic transition of the chromophore must be known.  This constant or the probability of the electronic transition occurring is the molar absorptivity. Since molecules have different electronic transitions of varying strengths, the molar absorptivity will vary depending on the transition being probed and is therefore wavelength dependent.

Aside from transmission and absorption, UV-Visible spectroscopy can also measure the reflectance of a sample, or how effective a surface is in reflecting the total amount of incident light. Reflection occurs when light strikes a material’s surface and causes a change in the direction of the light waves. There are two types of reflectance: specular and diffuse, where the sum of the two components is the total reflectance. Specular reflectance is light reflected at same angle as the incident beam and diffuse reflectance is light reflected in a different direction to the incident light, shown in Figure 4.

## UV-Visible Spectroscopy Instrumentation

The principle of measurement for the UV-visible spectrophotometer is relatively straightforward and consists of a light source, a wavelength dispersive element, sample, and detector.

Monochromator

The monochromator itself houses the mirrors, slits, and grating. panchromatic light from a light source is introduced into the monochromator through the entrance slit and collimated onto a diffraction grating which is rotated to select discrete wavelengths. The light is then refocused by another mirror onto the exit slit so that can be adjusted  to control the spectral bandwidth (SBW). The light is then refocused by another series of mirrors and directed to the sample where it is either transmitted, absorbed, or reflected.

There are two types of optical arrangements: single and double beam. In the single-beam configuration, the monochromator, sample, and detector are arranged in series and the obtained monochromatic light with intensity I0 irradiates a sample and the transmitted light with intensity I is detected. Here, I/I0 is the transmittance. Although the optical system is simple, it is easily affected by fluctuations in the light source, and a blank measurement is required for each measurement. In a double beam configuration, monochromatic light is divided into two beams by a fixed or dynamic beam splitter, and the individual beams pass through a sample and a reference and detected, shown in figure 6. The light source intensity changes with time so the reference beam monitors the lamp energy and accounts for energy differences from voltage fluctuations, lamp drift, and stray light. By splitting the optical path, the incident and transmitted light can be measured simultaneously, compensating for the effects of light source fluctuations. Therefore, the measured absorbance is the ratio of the sample beam to the reference beam.

The image below in Figure 7 represents the sample compartment for a single and double beam instrument. In the double beam instrument, the photometric value is the ratio of the sample to reference beam so any fluctuations in the light source are canceled out. However, in a single beam instrument since there is only one beam, a ratio of the intensities cannot be acquired and the influence of the light source fluctuations can be seen in the spectra on the right, depicting the light intensity as a function of time for a single beam (red) and double beam (blue) instrument. As time progresses, the signal intensity in the single beam instrument begins to decrease while the double beam spectrum provides a consistent baseline.

Light Sources

When selecting and evaluating an instrument, the type of light source used will have an effect on UV-Visible/NIR measurements. A few things to consider are: (1) the operational wavelength range required for the application or where the sample’s chromophore absorbs, (2) the required light throughput, (3) the stability of the source, and (4) the cost and lifetime of the source.

JASCO spectrophotometers use deuterium and halogen light sources. The deuterium lamp is used for the UV region from 190 to 350 nm while the halogen lamp covers a much broader spectral range from 330 and 3200 nm. Both the deuterium and halogen lamps used are continuous sources, although the D2 is also a line source. In continuous sources, the arc created excites the molecules enclosed in the vacuum to a higher energy state. The relaxation of the electrons back to the ground state emits photons and as the electrons return to the ground state, the excitation process restarts, providing a continuous source of light. Continuous sources therefore provide a uniform amount of light through the monochromator to the sample. While this constant output of light can potentially lead to photobleaching of light sensitive samples, shutters can be implemented so the sample is only irradiated with light during the measurement itself.

Grating

The grating is a dispersive element used to select the wavelengths required to probe the electronic transitions of a sample’s chromophores. It is rotated to the wavelengths selected and diffracts the light into several beams. The direction that light is diffracted depends on the angle and wavelength of the incident beam, and the grating’s groove (or line) frequency, or the number of grooves on the grating per millimeter.

The spacing between the grooves determines the diffraction order of the light, or how many beams are diffracted at that particular wavelength, as well as the spectral resolution. A diffraction order of 0 means the incident light angle and the diffracted angle are roughly the same while a diffraction order of 1 is twice the incident wavelength.  Wider groove spacing means fewer orders of diffraction, resulting in higher the light throughput. Aside from using a grating with large groove spacings, filters are commonly used to remove any higher orders of diffraction from the grating. Light diffracted from the grating is refocused by another mirror onto the exit slit which is adjusted to accommodate for the dispersive properties of different wavelengths of light.

Bandwidth

While the monochromator is set to a specific wavelength, the light emerging is not perfectly monochromatic, but contains a range of wavelengths. As seen in figure 8, the total energy at the exit slit of the monochromator at a specific wavelength has the intensity distribution of an isosceles triangle. The peak of this triangle is the target wavelength and the spectral bandwidth is the full width half max (FWHM) of the triangle. The bandwidth should be set to 1/10 of the sample peak’s FWHM.

The spectral bandwidth is directly related to the slit widths of the instrument and the relationship between the slit width (Δx) and the bandwidth (Δλ)  is expressed by the following formula

∆λ=(d∙cos⁡β)/(n∙f) ∆x

where d is the groove spacing of diffraction grating, β is the diffraction angle, n is the diffraction order, and f  is the focal length. When comparing instrument performance, it’s more appropriate to discuss spectral bandwidth rather than the slit width since the spectral bandwidth accounts for the grating resolution and differences between different types of gratings.

To differentiate peaks in a spectrum, the bandwidths and therefore the slit widths, need to be adjusted. The instrument’s ability to separate light into defined wavelength regions is known as spectral resolution. The narrower the bandwidth and therefore the slits, the better the resolution. However, since less light will pass through narrower slits, the spectrum will have more noise. Likewise, wider slits and bandwidths increase the light throughput and therefore the signal, but produce poorer peak resolution. The goal is to find a balance between the required resolution and the desired signal to noise. Figure 9 illustrates the measured absorption spectra using bandwidths of 1 and 5 nm. As the bandwidth increases, the peaks collapse and broaden.

It is also important to note that different bandwidths should and can be specified depending on the wavelength region being probed. The near infrared region has less light throughput than the visible region, so the bandwidth can be set to a larger value in the NIR to allow for more light to reach the sample.

Detectors

Detectors are used to measure the transmitted or reflected light from a sample and convert it into a signal. The type and material of the detector will determine the sensitivity and wavelength range of the data that can be acquired. While photomultiplier tubes and silicon photodiodes are sensitive in the ultraviolet and visible wavelength ranges,  Lead sulfide (PbS) photoconductive cells and indium gallium arsenide (InGaAs) photodiodes are used to measure the near-infrared region of the spectrum. However, all the detectors mentioned below exploit the photoelectric effect where light or photons that are incident on a material result in the emission of electrons.

In a photomultiplier tube (PMT) detector, photons are incident on the photocathode surface which produces electrons. The initial electrons travel through the tube where they hit a series of plates or dynodes that amplify the number of electrons for every dynode that’s hit via secondary emission. The multiplied secondary electrons are collected at the anode, sent to an external circuit, and converted to the output signal. PMTs have a wide spectral response, high signal to noise output, and high stability.

A silicon photodiode is a semiconductor device that exploits the photoelectric effect to convert light into an electrical current. When the incident photons’ energy is larger than the bandgap of silicon, the photons are absorbed and the electrons in the valence band are excited to the conduction band, creating holes in the initial valence band. The photodiode is made up of a p- and a n- junction and a depletion region. An applied electric field in this depletion region pushes the positive holes towards the n-junction while the negative electrons move towards the p-junction, building up areas of highly positive and negative charges and thus producing a photocurrent. Photodiodes have a quick response time, a slightly broader spectral range than a PMT, and low noise.  While silicon photodiodes are less sensitive than PMT detectors in the UV and visible regions, they are a cheaper alternative for applications not requiring high sensitivity.

JASCO spectrophotometers use two detectors for the NIR region: a PbS detector and an InGaAs detector. While the InGaAs detector is a photodiode, the lead sulfide detector is a photoconductive cell that operates similarly to a photodiode. However, unlike photodiodes, in a photoconductive cell, the resistance decreases as the incident light intensity increases and the measured output is linearly proportional to the input incident light power.

Figure 14 shows the D* values for different detectors as a function of wavelength. The higher the D*, the better the sensitivity of the detector. The InGaAs detector shown in blue is more sensitive than the PbS detector (green) but has a shorter wavelength range. While there are other InGaAs detector options (orange) with extended wavelength ranges, the sensitivity of those is even less than the PbS detector option.

Figure 15 illustrates the difference in signal to noise between the two NIR detectors. The more sensitive InGaAs detector (blue) shows higher S/N than the PbS detector (green). Ultimately deciding between the two comes down to the application and prioritizing sensitivity, wavelength range, and cost.

Stray Light and Photometric Linearity

Stray light is any light that does not fall under the Gaussian distribution at a specific wavelength. Since UV-Visible spectrometers measure the ratio of the incident light to the transmitted light, any light that does not ‘reach’ the detector is considered absorbed by the sample since the instrument detector cannot differentiate between absorbed and stray light.  As shown in Figure 16 on the left, at higher sample concentrations when more light is absorbed, consequently less light is transmitted. The introduction of stray light causes the measured absorbance at low transmission to deviate from the Beer-Lambert Law, contributing to inaccurate photometric values that are lower than the calculated or true absorbance (Figure 16, right).

Double-monochromator instruments are used to combat the effects of stray light. While single monochromator instruments have a single set of slits and one grating, double monochromator instruments have two gratings, two sets of slits, and additional mirrors. The extra optical components reduce the effects of stray light in the sample spectrum, decreasing the noise and allowing highly absorbing or scattering samples to be adequately measured.

The range of absorbances that can be accurately measured before deviating from true sample absorbance is known as the instrument’s photometric linearity and is directed affected by stray light and therefore the monochromator’s optics. Since the absorbance is directly proportional to sample concentration, the larger the photometric range, the great the sample concentration can be measured. Figure 17A illustrates the absorbance spectra for a single- (top) and double- (bottom) monochromator instruments. Both instruments have a wide photometric range that span the entire wavelength range of the instrument. However, in the double monochromator, the photometric range is roughly 2 absorbance units greater than the single monochromator instrument. The linearity of the double-monochromator is shown in Figure 17B, where the absorbance is plotted as a function of sample concentration for nickel (II) sulfate and ranitidine hydrochloride.

## UV-Vis Spectroscopy Experimental

Choosing a Cuvette

When choosing a suitable cuvette to use for your application, we need to consider the material of the cuvette and the volume of sample required. Figure 18 shows the transparency of different cuvette materials. For samples that absorb below 350 nm, quartz or UV disposable cuvettes are necessary. However, it’s advised to compare the disposable and quartz cuvettes options. Disposable cuvettes are made of plastics that still absorb so if the sample absorbs strongly and a higher photometric range is required, quartz cuvettes are a better choice. Disposable cuvettes are also not an option for the near-infrared measurements since the material absorbs above 1000 nm.

Cuvettes are also broken down into macro, micro, and sub-micro volumes. Assuming the pathlength is 1 cm, (the standard for most UV-Visible measurements),  macro cells typically require 2.5-4 mL of sample and micro cells require 250 to 1000 mL of sample. Here the cuvette walls are tapered to accommodate smaller sample volumes. Sub-micro cells can hold 10 to 250 mL. However, these volumes will change with the pathlength of the cell, so longer pathlengths require more volume than shorter pathlengths.  Micro and sub-micro cells also have self masking options, where the cuvette walls are black. The windows of micro and sub-micro cells are typically smaller than the standard beam dimension to accommodate smaller sample volumes. While the bandwidth of the instrument and therefore beam dimensions can be reduced, any light incident on the cell walls that does not pass through the sample can introduce stray light effects, resulting in inaccurate absorbance values and a reduction in photometric linearity. The z-height is also another important characteristic of cuvettes. The z-height is the height from the base of the cell to the center of the light beam and will differ for different instrument manufacturers. The z-height for the JASCO V-700 Series spectrophotometers is 15 mm.

Solvent Selection

Solvents and substrates should be carefully selected for UV-Visible/NIR experiments. If the sample is liquid, it should be soluble in the solvent selected and assist in maintaining sample stability. Ideally, the solvent or substrate should be transparent in the wavelength range where the sample’s chromophore absorbs, to reduce any additional absorbance that could potentially reach the limits of the instrument’s photometric range.

Response and Scanning Speed

The response is the amount of time that the data is integrated over or the length of time the detector collects photons before transferring the signal to the A/D converter for processing. The square root of the response is proportional to the signal to noise, so the longer the response the better the S/N. Increasing the response will have a more substantial effect when a sample’s signal is small since there is less light throughput.

The scanning speed determines how quickly the monochromator scans through the specified wavelength range to acquire data points at the specified data pitch. When used in continuous scan mode, the scanning speed must be selected with an appropriate response to prevent distortion in the measured spectrum. The following guideline can be used when selecting the response and scanning speed

Response × Scanning speed <  FWHM/10

where FHWM is the full width at half the peak height of the target peak.

How to Perform Baseline Measurements

A baseline or background measurement is used to account for the solvent or buffer and the cell. For liquid samples or samples that are dissolved or diluted in solution and use a cuvette, the solvent and cuvette can absorb and reflect light which can lead to inaccurate absorbance values and lower signal to noise. This is especially important for quantitation measurements. In Figure 19, the sample, solvent, and solvent subtracted sample spectrum is shown and illustrates that neglecting to acquire a solvent spectrum can lead to incorrect identification of sample peaks and absorbance values.

While all JASCO UV-visible/NIR spectrophotometers are double-beam instruments and have a reference beam, the main purpose of it is account for fluctuations in the light source, filter, light source and detector changes, and light attenuation of the sample, not solely for baseline measurements. The equation in Figure 20 shows that since the reference light intensity R* is canceled when calculating percent transmittance, the same result is obtained regardless of the presence or absence of a solvent on the reference light beam side. Therefore, for most samples, the baseline measurement of the solvent can be acquired with the solvent in the sample beam position, and then the sample is swapped with the solvent cuvette and the sample measurement acquired, also shown in Figure 20.

That being said, the reference beam does occasionally serve a purpose for baseline measurements. For samples with strong absorbances, the solvent can be placed in a cuvette in the reference beam for attenuation (rear-beam attenuation) to balance the intensity of the reference and sample beams since the detector is measuring low light intensities from the highly absorbing sample and very bright light intensity from the reference. Solvents whose properties change over time, such as oxidizing agents, should also be placed in the reference beam for both baseline and sample measurements. In this case, two matching cuvettes are used where the solvent is placed in both the reference and sample beam for the baseline measurement, shown in Figure 21. For the sample measurement, the sample is swapped into the sample beam position and the spectrum acquired.

Rear Beam Attenuation

In the double-beam instrument, the reference beam can be attenuated by a neutral density filter with a transmittance of 1% to expand the photometric range. The addition of a neutral density filter to the reference beam balances the absorption by the sample so that the difference in the intensity of the reference and sample beams is not as great.  This method is effective for samples with an absorbance of 3 or more. Figure 22 shows the absorbance spectrum of a highly absorbing sample without (left) and with (right) rear beam attenuation. The spectrum on the left becomes noisy and the absorbance maxes out at 10 AU, exceeding the photometric range of the instrument. However, when a 1 OD neutral density filter is placed in the reference beam, the actual absorbance of the sample and full photometric range of the instrument can now be acquired, along with a significantly better signal to noise ratio at lower wavelengths.

Integrating spheres

An integrating sphere is a spherical cavity whose inner wall is coated with a highly reflective material such as barium sulfate (Figure 23). In an integrating sphere, light undergoes multiple reflections until the reflected light enters the detector. The presence of a sample can change the optical path, as in the cases of scattering due to sample turbidity, non-planar samples, and thick samples. An integrating sphere is indispensable when the position and size of the light beam received by the detector change due to a change in the optical path, and the transmittance cannot be measured.

There are two types of measurements that can be made in an integrating sphere: diffuse transmission and diffuse reflectance. In diffuse transmission measurements (Figure 24), the incident light enters the sample compartment, where a mirror directs it through the sample sitting at the entrance of the sphere. The transmitted light is reflected by the diffuse surface of the integrating sphere and eventually makes its way through a third aperture to reach the detector. Diffuse reflectance measurements are obtained by placing the sample at the reflectance port of the integrating sphere, shown in Figure 24. Diffuse reflectance measurements are typically obtained for powder samples or samples with rough surfaces. Baseline measurements using an integrating sphere are straightforward: a ‘white’ standard (barium sulfate or Spectralon) plate is placed at the reflection of the sphere and the entrance port  is left empty.

The spectra in Figure 25 show the transmittance of a sample using the standard cell holder (in blue) compared to spectra obtained using an integrating sphere, shown in green. Since only the direct transmittance was obtained using the standard cell holder, a significant portion of the transmittance was lost without the use of an integrating sphere.

Aside from a 60 mm diameter integrating sphere, JASCO also offers a 150 mm diameter sphere. Historically the larger sphere was required for international measurement guidelines like ASTM due to its port fraction, which is smaller than the 60 mm sphere. The port fraction describes the number of ports or apertures relative to the diameter of the sphere. This port fraction is also related to radiance produced from the multiple reflections in the sphere’s cavity. The smaller the port fraction, the better the sphere can integrate the radiant flux due more reflections or bounces. However, more reflections introduces more noise into the spectrum.   The smaller 60 mm diameter sphere can now be used for ASTM measurements.

Additionally, in order to remove any potential specular component if only the diffuse reflectance is needed, a beam trap is added to the sphere to allow the specular component to exit the sphere. Specular components are typically acquired for shiny sample finishes while rougher surfaces have more diffuse reflectance. To measure the total reflectance of a sample, the specular component needs to be included and the beam trap is removed. Figure 26 illustrates the reflectance for a matte and glossy sample with and without the specular component. For the matte finish, the specular component adds relatively little to the total reflectance of the sample, but for the glossy sample, the inclusion of the specular component significantly increases the reflectance of the sample.

It is also important that the reference plates are kept clean and in good condition so their reflectance characteristics remain constant and do not contribute to incorrect reflectance values. Figure 27 shows the reflection spectrum of a dirty and clean white plate used as the reflection standard. If the standard is dirty, the reflectance, particularly in the ultraviolet region, decreases. If such a plate is used as a reference, the reflectance measured for a sample will be abnormally high.

## UV-Vis Spectroscopy Webinar

This introductory webinar provides a review of UV-Visible theory and instrumentation basics, as well as a guide to best practices and getting good data, including information on:

• How different instrument components affect measurement results
• The difference between double beam and double monochromator instruments
• How to correctly perform a baseline measurement
• Integrating spheres and their applications

Powerpoint slides can be downloaded here.

## UV-Vis Spectroscopy Applications

#### Analysis of the Melting Temperature and Thermodynamic Parameters of a Nucleic Acid using a UV-Visible Spectrophotometer

This application note reports the evaluation of the melting temperature and thermodynamic parameters of a nucleic acid sample. The absorbance at 260 nm was measured for different sample concentrations while ramping the temperature2) using a V-700 Series UV-visible spectrophotometer with PAC-743 Automatic 6/8-position Peltier cell changer, which can measure up to eight samples simultaneously.

#### Water Analysis using a UV-Visible Spectrophotometer with a 30 cm Cell

This application note introduces a new method to analyze superficially clean water using a UV-Visible spectrophotometer with a 30 cm pathlength cell. The extended sample compartment is dedicated to the 30 cm cylindrical cell which provides precise measurements of samples with extremely low absorbance values that cannot be measured using a standard 10-100 mm path length cell. By placing an integrating sphere in the extended compartment, all transmitted light can be detected and integrated to achieve a maximum absorption signal.

#### Evaluation of UPF for Sun Protection Fabrics

This application note evaluates UPF, UPF rating, and UVA and UVB transmittance of sun protection fiber products using a UV-Visible spectrophotometer and the UPF calculation system. Fluorescence measurements were also obtained to corroborate the UV-Visible results.