The Basics of UV-Vis Spectrophotometry

The Basics

of UV-Vis

Spectrophotometry

A primer

1 Basic Principles of UV-Vis Measurement 3

1.1 The electromagnetic spectrum 3

1.2 Wavelength and frequency 3

1.3 UV-visible spectra 3

1.4 Transmittance and absorbance 4

1.5 Summary 4

2 How Does a Modern UV-Vis Spectrophotometer Work? 5

2.1 Instrumental design 7

3 Selecting the Optimum Parameters for your 13

UV-Vis Measurements

3.1 Optical cell selection 13

3.2 Thermostatting your samples 16

3.3 Stirring your sample 16

3.4 Measurements at low temperatures 16

3.5 Solvent transparency 17

3.6 Optimum spectral band width 18

3.7 Stray light 19

3.8 The linear range of a UV-Vis instrument 19

3.9 Other useful information 20

3.10 Wavelength or inverse centimeters 20

4 Overview of Common UV-Vis Applications 21

4.1 Identification—spectra and structure 21

4.2 Confirmation of identity 22

4.3 Quantifying a molecule 22

4.4 Kinetics 24

4.5 Color measurement 26

4.6 Structural changes of compounds 28

4.7 Protein and nucleic acid melting temperature 28

4.8 Multi-component analysis 30

4.9 Software requirements 32

5 Glossary 34

Contents

1.1 The electromagnetic spectrum

Ultraviolet (UV) and visible radiation are a small part of the electromagnetic

spectrum, which includes other forms of radiation such as radio, infrared

(IR), cosmic, and X rays.

wavelength UV light has the highest energy. Sometimes, this energy may

be sufficient to cause unwanted photochemical reactions when measuring

samples that are photosensitive.

1.3 UV-visible spectra

When radiation interacts with matter, several processes can

occur, including reflection, scattering, absorbance, fluorescence/

phosphorescence (absorption and re-emission), and photochemical

reactions (absorbance and bond breaking). Typically, when measuring

samples to determine their UV-visible spectrum, absorbance is measured.

Because light is a form of energy, absorption of light by matter causes

the energy content of the molecules (or atoms) in the matter to increase.

The total potential energy of a molecule is represented as the sum of its

electronic, vibrational, and rotational energies:

total

= E

electronic

+ E

vibrational

+ E

rotational

The amount of energy a molecule possesses in each form is not a

continuum but a series of discrete levels or states. The differences in

energy among the different states are in the order:

electronic

> E

vibrational

> E

rotational

In some molecules and atoms, incident photons of UV and visible light have

enough energy to cause transitions between the different electronic energy

levels. The wavelength of light absorbed has the energy required to move an

electron from a lower energy level to a higher energy level. Figure 2 shows

an example of electronic transitions in formaldehyde and the wavelengths

of light that cause them.

1. Basic Principles of UV-Vis Measurement

Figure 1. The electromagnetic spectrum, with the visible light section expanded.

The energy associated with electromagnetic radiation is defined as:

E = hν

where E is energy (in joules), h is Planck’s constant (6.62 × 10

-34

Js), and

ν is frequency (in seconds).

Spectroscopy allows the study of how matter interacts with or emits

electromagnetic radiation. There are different types of spectroscopy,

depending on the wavelength range that is being measured. UV-Vis

spectroscopy uses the ultraviolet and visible regions of the electromagnetic

spectrum. Infrared spectroscopy uses the lower energy infrared part of

the spectrum.

1.2 Wavelength and frequency

Electromagnetic radiation can be considered a combination of alternating

electric and magnetic fields that travel through space in a wave motion.

Because radiation acts as a wave, it can be classified in terms of either

wavelength or frequency, which are related by the following equation:

ν = c/λ

where ν is frequency (in seconds), c is the speed of light (3 ×

108

-1

), and

λ is wavelength (in meters).

In UV-Vis spectroscopy, wavelength is usually expressed in nanometers

(1 nm = 10

-9

m). It follows from the equations that radiation with shorter

wavelength has higher energy, and, for UV-Vis spectroscopy, the low (short)

Figure 2. Electronic transitions in formaldehyde. UV light at 187 nm causes excitation of

an electron in the C-O bond and light at 285 nm wavelength causes excitation and transfer

of an electron from the oxygen atom to the C-O bond.

Ultraviolet Visible Infrared

LOWER

ENERGY

HIGHER

ENERGY

-14

-12

-10

-8

-6

-4

-2

1 10

Wavelength [m]

Cosmic ray

Gamma ray

X ray

Ultraviolet

Infrared

Microwave

Radar

Television

NMR

Radio

Visible

-3

Frequency [Hz]

C O

n pi*

transition

(285 nm)

pi pi*

transition

(187 nm)

C O

electronic energy levels

vibrational energy levels

rotational energy levels

electronic transition

Figure 3. Incident light of a specific wavelength causes excitation of electrons in an atom.

The type of atom or ion (i.e. which element it is) and the energy levels the electron is

moving between determines the wavelength of the light that is absorbed. Transitions can

be between more than one energy level, with more energy i.e. lower wavelengths of light,

required to move the electron further from the nucleus.

However, for molecules, vibrational and rotational energy levels are

superimposed on the electronic energy levels. Because many transitions with

different energies can occur, the bands are broadened (see Figure 4). The

broadening is even greater in solutions owing to solvent-solute interactions.

1.4 Transmittance and absorbance

When light passes through or is reflected from a sample, the amount of

light absorbed is the difference between the incident radiation (I

) and

the transmitted radiation (I). The amount of light absorbed is expressed

as absorbance. Transmittance, or light that passes through a sample, is

usually given in terms of a fraction of 1 or as a percentage and is defined

as follows:

T = I / I

or %T = I/I

× 100

Absorbance is defined as follows:

A = –logT

For most applications, absorbance values are used since the relationship

between absorbance and both concentration and path length is normally

linear (as per the Beer Lambert law, described in section 1.9).

1.5 Summary

– UV and visible light are part of the electromagnetic spectrum

– In UV-Vis spectroscopy wavelength is expressed in nanometers (nm)

– Light can be reflected, scattered, transmitted or absorbed from matter,

and can cause photochemical reactions to occur

– Energy from incident light causes electrons to transition to different

energy levels. Electronic transitions also occur between the vibrational

and rotational energy levels of molecules

– Absorbance of light is used for most UV-Vis spectroscopy applications.

It is defined as A=-logT, where T is transmittance.

These transitions result in very narrow absorbance bands at wavelengths

highly characteristic of the difference in energy levels of the absorbing

species. This is also true for atoms, as depicted in Figure 3.

Figure 4. Electronic transitions and UV-visible spectra in molecules (I is intensity and

λ is wavelength).

I∆E

Energy level 0

Light of speciﬁc

wavelength absorbed

–

Energy level 1

Energy level 2

Energy level 3

–

Ultraviolet visible (UV-Vis) spectrophotometers use a light source to

illuminate a sample with light across the UV to the visible wavelength

range (typically 190 to 900 nm). The instruments then measure the light

absorbed, transmitted, or reflected by the sample at each wavelength.

Some spectrophotometers have an extended wavelength range, into the

near-infrared (NIR) (800 to 3200 nm).

2. How Does a Modern UV-Vis Spectrophotometer Work?

Figure 5. A UV absorbance spectrum, showing an absorbance peak at approximately 269 nm.

From the spectrum obtained, such as the one shown in Figure 5, it is

possible to determine the chemical or physical properties of the sample.

In general, it is possible to:

– Identify molecules in a solid or liquid sample

– Determine the concentration of a particular molecule in solution

– Characterize the absorbance or transmittance through a liquid or solid—

over a range of wavelengths

– Characterize the reflectance properties of a surface or measuring the

color of a material

– Study chemical reactions or biological processes.

Various types of measurements can be performed by combining different

accessories and sample holders with the UV-Vis spectrophotometer.

Different accessories exist for different measurement capabilities and

sample types, e.g., solids versus liquids, and for different measurement

conditions (Figure 6 and 7).

Figure 6. A fiber-optic probe accessory can be fitted to a UV-Vis spectrophotometer

to measure liquid samples in a range of containers.

UV-Vis spectrophotometry is a versatile technique and has been used for

close to a century in a wide range of fields. UV-Vis spectrophotometers are

in common use in material testing/research, chemistry/petrochemistry,

and biotechnology/pharmaceuticals laboratories.

265 270

0.4

0.2

0.0

Abs

Wavelength (nm)

Figure 7. A solid sample, like this

polycrystalline photovoltaic solar cell,

can be measured using a

UV-Vis spectrophotometer.

2.1 Instrumental design

Components

The key components of a spectrophotometer are:

– A light source that generates a broadband of electromagnetic radiation

across the UV-visible spectrum

– A dispersion device separates the broadband radiation into wavelengths

– A sample area, where the light passes through or reflects off a sample

– One or more detectors to measure the intensity of the reflected or

transmitted radiation

Other optical components, such as lenses, mirrors, or fiber-optics,

relay light through the instrument.

Figure 8. Schematic of the internal layout

of an Agilent Cary 5000 UV-Vis-NIR

spectrophotometer, showing the

main components. Note that this is a

high-performance instrument.

UV-Vis spectrophotometers for routine

measurements have a simpler optical design.

0.1

0.01

Spectral irradiance

200

Wavelength (nm)

400 600 800 1000

Tungsten-halogen lamp

The tungsten-halogen lamp uses a filament. When a current is passed

through the filament, it becomes heated and emits light (see Figure 10).

The lamp yields good intensity over part of the UV spectrum and over the

entire visible and NIR range (350 nm- 3000 nm). This type of lamp has

very low noise and low drift and typically has a functional life of 10,000 h.

Figure 10. Intensity spectrum of the tungsten-halogen lamp.

In UV-visible spectrophotometers using both a D

and a tungsten-halogen

lamp, either a source selector is used to switch between the lamps as

appropriate, or the light from the two sources is mixed to yield a single

broadband source.

Xenon flash lamp

Unlike the D

or tungsten-halogen lamps, which provide a constant light

source, a Xenon flash lamp emits light for an extremely short time,

in flashes. Since it emits only for a short time and only during sample

measurement, it has a long life. The sample is only irradiated with light at

the time of measurement. This short illumination time makes the Xenon

flash lamp suitable for measuring samples that may be sensitive to

photobleaching. Photobleaching can be observed on sensitive samples

when exposed to a constant long exposure by a continuous light source.

The Xenon flash lamp emits high intensity light from 185 – 2500 nm, which

means no secondary light source is required (Figure 11). The Xenon flash

lamp may be used for many years before requiring replacement, which

makes it a popular choice compared to systems using D

or tungsten-

halogen lamps. An extra benefit is that it does not require warmup time,

unlike D

or tungsten-halogen lamps.

Light sources

The ideal light source would yield a constant intensity over all wavelengths

with low noise and long-term stability of the output. Unfortunately, such

a source does not exist. Two different light sources have historically been

used in UV-visible spectrophotometers:

– The deuterium arc lamp was used to provide a good intensity

continuum in the UV region and useful intensity in the visible region

– The tungsten-halogen lamp yielded good intensity over the entire visible

range and part of the UV spectrum

More recently, a single Xenon flash lamp has been used more widely.

The use of a Xenon flash lamp as a single source has significant

advantages over the use of the two conventional lamps.

Deuterium (D

) arc lamp

The deuterium arc lamp uses arc discharge from deuterium gas and yields

a good intensity continuum in the UV region and useful intensity in the

visible region, 185 to 400 nm (see Figure 9). Although modern deuterium

arc lamps have low signal noise, noise from the lamp is often the limiting

factor in overall instrument noise performance. Over time, the intensity of

light from a deuterium arc lamp decreases steadily. Such a lamp typically

has a half-life (the time required for the intensity to fall to half of its initial

value) of approximately 1,000 hours. This short half-life means the D

lamp

needs to be replaced relatively frequently.

Figure 9. Intensity spectrum of the deuterium arc lamp.

0.1

0.01

0.00

Spectral irradiance

200

Wavelength (nm)

300 400 500 600 700

100

0.1

0.01

Spectral irradiance

200

Wavelength (nm)

400 600 800 1000

Figure 11. Intensity spectrum of the Xenon lamp.

The monochromator

All the light sources produce a broad-spectrum white light. To narrow the

light down to a selected wavelength band, the light is passed through a

monochromator. A monochromator consists of:

– An entrance slit,

– A dispersion device, to spread the light into different wavelengths

(like a rainbow) and allow the selection of a nominated band of

wavelengths, and

– An exit slit where the light of the nominated wavelengths passes

through and onto the sample.

An easy way to think about a monochromator is to think of a room, with

the sun shining through a window. The sunlight hits a prism that separates

the white light into a rainbow. The rainbow falls onto a window on the

opposite side of the room. As the prism is turned, light of different colors

i.e. different wavelengths, pass out of the room through the window.

Ideally, the output from a monochromator is light of a single wavelength.

In practice, however, the output is always a band of wavelengths.

Most spectrophotometers on the market today contain holographic

gratings as the dispersion device. These optical components are made

from glass, onto which extremely narrow grooves are precisely etched onto

the surface. The dimensions of the grooves are of the same order as the

wavelength of light to be dispersed. Finally, an aluminum coating is applied

to create a reflective surface.

Interference and diffraction of the light falling on the grating is reflected at

different angles, depending on the wavelength. Holographic gratings yield a

linear angular dispersion with wavelength and are temperature insensitive.

However, they reflect light in different orders, which overlap (see Figure 12).

As a result, filters must be used to ensure that only the light from the

desired reflection order reaches the detector. A concave grating disperses

and focuses light simultaneously.

Figure 12. How a holographic grating disperses white light into light of different

wavelengths.

Single monochromator spectrophotometers

A single monochromator spectrophotometer is used for general-purpose

spectroscopy and can be integrated into a compact optical system.

Figure 13 shows a schematic diagram of a single monochromator optical

system. A single monochromator spectrophotometer cannot select the

wavelengths of light as narrowly as a double monochromator system,

but this ability may not be required for many applications, for example

when measuring samples that have broad absorption peaks.

Figure 13. Single monochromator spectrophotometer.

DetectorSampleLight source Monochromator

Grating

First order

Second order

Figure 15. Cuvettes for measuring liquid samples. From left to right: A standard 10 mm

pathlength, 3 mL cuvette, an ultramicro cell for measuring very low volumes, and a long

pathlength cuvette for dilute solutions.

Double monochromator spectrophotometers

A double monochromator is typically found in high-performance

instruments. The two monochromators are arranged in series. The source

light is split by the first monochromator and then further split by the

second. Stray light, light that leaks into the system, is reduced, and the

spectral accuracy (the ability to accurately select a particular wavelength)

is increased. Figure 14 shows a schematic diagram of the double-

monochromator optical system.

Figure 14. Schematic diagram of a double monochromator spectrophotometer.

Sample compartment

In the sample compartment, the sample is positioned to allow the

beam from the monochromator to pass through the sample. For the

measurement of absorbance, liquid samples would typically be held

in a cuvette with a known, fixed pathlength. A cuvette is a rectangular

liquid holder as shown in Figure 15. They are made from glass, quartz,

plastic or another material that transmits UV or visible light. Standard

cuvettes have a 10 mm pathlength and are made from quartz, to ensure

good transmittance of UV wavelengths. Cheaper plastic cuvettes can

also be used, but generally do not transmit UV light so are only useful if

measurements in the visible wavelength region are required. A multitude

of cuvettes for special applications are available – from cuvettes that hold

tiny volumes of liquids through to those that have much longer pathlengths,

for use with very dilute samples.

Mono 1 Mono 2 DetectorSampleLight source

Solid samples can be held in place for simple transmission measurements.

They can also be measured at various angles of incidence. For more

complex measurements, like diffuse reflectance or transmission, other

accessories may be installed into the sample compartment.

Single beam spectrophotometer

The simplest UV-Vis spectrophotometer has a single beam optical system.

In a single-beam system, the light from the monochromator passes

through the sample and then to the detector. This simple design means

less optical components are used, and it makes it possible to reduce the

size of the instrument and thus the cost.

However, before a sample can be measured, a baseline or blank sample

must be measured. For liquid measurements, the baseline reading is

taken to allow for any absorbance of the cuvette and solvent used.

With a single beam system, the baseline needs to be measured separately

from the sample. The separate readings mean that if there is any variation

of light intensity, or system optical performance, between the baseline

and sample being read, the measurement may be less accurate.

This inaccuracy is a concern for sample measurements that take a long

time, or where the blank may vary over time. In practice this means that

a baseline/blank measurement should be run frequently and regularly

during a measurement session if using a single beam system.

Double beam spectrophotometer

Many UV-Vis systems use a double beam optical system. In the

double beam type, the light emitted from the monochromator is split into

two beams: a reference beam and a sample beam. The light is usually split

with an optical component such as a rotating wheel which has a mirrored

segment, or a half-silvered mirror called a beam splitter. Each beam enters

the sample chamber through separate optical paths. Since two beams

of the same wavelengths are available, the reference/blank and sample

can be measured at the same time. This means the sample

measurement can be corrected for any instrument fluctuations in real time.

This real time correction delivers a highly accurate measurement.

Figure 16. Schematic diagram of double beam optical system, with dual detectors.

Dual beam spectrophotometer

Another, more recent, spectrophotometer design uses a dual-beam optical

layout with a sample and reference detector. The reference detector is used

to correct lamp brightness fluctuations for each measurement, while the

solvent or blank (in the case of a solid sample) is measured in the sample

position and then subtracted from the sample spectrum after collection.

With improvements in electronics and software, this design keeps the

measurement process simple and reduces the chance of user error due to

mismatched cuvettes or incorrect sample placement. Dual beam design has

the same performance as a routine double beam instrument, while double

beam design is now typically reserved for research-grade instruments.

Sample compartment

The sample compartment of a UV-Vis spectrophotometer is typically a

black-colored box with a closing lid. The matt black inside the compartment

helps to absorb stray light that may enter the compartment.

In the sample compartment, the sample is positioned to allow the beam

from the monochromator to pass through the sample. As discussed above,

glass, plastic, or quartz cuvettes (Figure 15) are used for liquid samples.

Solid samples are held in position by a holder attached to the floor of

the sample compartment. The light can also be taken out of the sample

compartment using fiber optics. Fiber optics are useful when measuring very

large, hot, cold, radioactive, or other dangerous samples. As shown in

Figure 6, fiber optics can take the light from the spectrophotometer through a

fiber optic probe, to measure solutions outside of the sample compartment.

Alternatively, a fiber optic device that allows the measurement of light

reflectance, fluorescence or transmission through a solid sample can be used.

Detector 1Reference

Detector 2Sample

Light source Monochromator

Silicon diode (Si)

Silicon photodiode detectors (Figure 18) are extensively used as detectors

in modern spectrophotometers. Photodiode detectors have a wider

dynamic range and are more robust than PMT detectors. In a photodiode,

light falling on the semiconductor material allows electrons to flow through

it, depleting the charge in a capacitor connected across the material.

The amount of charge needed to recharge the capacitor at regular

intervals is proportional to the intensity of the light. The limits of detection

for silicon-based detectors are approximately 170 to 1100 nm.

The detector

A detector converts the light from the sample into an electrical signal.

Like the light source, it should give a linear response over a wide

wavelength range, with low noise and high sensitivity. Spectrophotometers

normally contain either a photomultiplier tube detector or a photodiode

detector. Other specialized detectors are found on high-performance

systems to improve wavelength coverage or sensitivity.

Each detector has a different sensitivity and wavelength range.

For systems with multiple detectors, the system will switch to the detector

corresponding to the required wavelength range for the measurement.

Photomultiplier tube (PMT)

The photomultiplier tube (Figure 17) combines signal conversion with

several stages of amplification within the body of the tube. The nature

of the cathode material determines spectral sensitivity. A single PMT

yields good sensitivity over the entire UV-visible range from 200 to 900

nm. A PMT detector provides high sensitivity at low light levels. For dilute

samples, most of the light hitting the sample will pass through to the

detector. To accurately detect small differences between blank and

sample measurements, the detector must have low signal noise at these

high light intensity levels.

Figure 17. A photomultiplier tube detector.

Figure 18. A silicon photodiode detector.

Indium gallium arsenide (InGaAs) photodiode

The InGaAs detector is a specialized detector that provides excellent

performance for the visible and the NIR wavelength range. InGaAs

detectors are available in narrow band (800 to 1700 nm) and wide band

(800 to 2500 nm) options. These detectors are useful for their linear

response and sensitivity in the near infrared region.

Lead sulfide (PbS) detector

The most common NIR detector used in spectrophotometers is the PbS

detector. This detector is sensitive between 1000 to 3500 nm. In high

performance, wide wavelength range spectrophotometers, the PbS

detector is often combined with a PMT detector for UV-visible coverage.

Where high sensitivity is required at the low NIR frequencies, a PbS

detector may be combined with a narrow band InGaAs detector.

Cathode Anode

p layer

n layer Gold block

Intrinsic region

Metal contact Photon

SiO

Selecting the most suitable sample holder, solvent and instrument

parameters is critical for the success of your measurement.

3.1 Optical cell selection

Liquid samples are usually contained in a cuvette (which is another name

for an optical cell, or just ‘cell’). Cuvettes come in a variety of designs to suit

the application. These include:

– A ‘standard’ 10 mm pathlength optical cell (refer to Figure 15).

Holding around 3.5 mL, the cell has two optical windows, parallel to

each other. Typically, the other sides are frosted or grooved to indicate

that these sides are to be used for handling the cell. The optical

windows should be kept as clean as possible and never touched.

Avoid scratching the optical surfaces when not in use. Disposable,

limited use, cells are also available. These are manufactured from

polystyrene or polymethyl methacrylate (PMMA) and cannot be used

at elevated temperatures. Polystyrene does not transmit UV light which

allows only measurements between 340 to 800 nm to be performed.

PMMA cells can be used down to 300 nm.

– For small volumes, up to around 0.5 mL, a semi-micro cell can be used.

These have similar external dimensions to a standard cell but have a

narrow channel on the inside to reduce the required sample volume.

Ultra-micro cells are also available, holding as little as 0.5 µL. The black

masking either side of the optical window (as shown in Figure 19 and

Figure 20) prevents internal reflection within the cuvette. Care needs to

be taken to ensure the cells align to the optical height of the beam in the

UV-Vis instrument. This is referred to as the z-height. The z-height is a

measurement from the base of the cuvette to the centre of the optical

path length.

Semi or ultra-micro cells are useful when sample volumes are limited.

Figure 19. “Masked” cuvettes ensure that the

optical beam is passed through the sample.

Ensuring the z-height of the cuvette is

compatible with your UV-Vis spectrophotometer

design is critical.

Figure 20. A 10 mm pathlength,

1.4 mL volume semi-micro cell.

3. Selecting the Optimum Parameters for your UV-Vis Measurements

To measure multiple liquid samples a flow-through cell can be used.

Flow-through cells are also available in a variety of internal cell volumes and

pathlengths. The cells are usually connected to a peristaltic pump and can

be connected to an autosampler. The pump pushes the sample through

tubing connected to the cell, filling the cell for the measurement. A rinse

solution is then pushed through to clean the cell before the next sample is

pumped into the cell.

Figure 21. Flow cell, with 4 x 11 mm rectangular apertures, 10 mm pathlength.

Shown with connectors and tubing.

Most optical cells are provided with a cap or lid. The cap is designed to both

reduce accidental spillage of the sample and evaporation of the sample.

The use of a cap is strongly recommended when measuring volatile or

dangerous samples.

45mm

15mm

38.5mm

8.5mm

Cell pathlengths

The pathlength is the distance that the incident light travels through a

sample. Cuvettes are available with different pathlengths. The pathlength

you should use is dependent upon the absorbance of your sample:

– Concentrated samples with high absorbance (>3 Abs) need a short

pathlength cuvette (less than or equal to 5 mm) – or they need to be

diluted. Short pathlength cuvettes can also be used to compensate for

solvents with high absorbance.

– Dilute samples with low absorbance (<0.2 Abs) need a long pathlength

cell (up to 100 mm) which will increase the absorbance reading.

This can also help reduce the level of error.

Most UV-Vis spectrophotometer systems are provided with a cuvette

holder suitable for a standard cuvette with a 10 mm pathlength. Longer

pathlength cuvettes will require an appropriate long pathlength cell holder.

Shorter pathlength cells are available in the same dimensions as the

standard 10 mm pathlength cuvette. They will fit into the standard cell

holder supplied with the instrument. Cell spacers are also available to

securely fit a smaller pathlength cuvette into the standard cuvette holder.

Material

The optical properties of the material used in the cuvette windows needs

to be considered. For the widest wavelength range quartz glass is

preferred. As quartz glass is more expensive, optical glass or plastic

(polystyrene) can be used when measurements below 340 nm

(polystyrene) and 350 nm (optical glass) are not required. Polystyrene cells

are not suitable for use at elevated temperatures. Care also needs to be

taken when using polystyrene cells to ensure that samples/solvents will

not damage the cells. Polystyrene cells are often referred to as disposable,

or plastic cell. They are easily scratched and are designed for a single use.

Table 1 shows the transmission of common materials over the UV to NIR

wavelength range.

Table 1. Transparency windows for common cuvette materials.

Material Suitable Wavelength (nm)

Quartz 170–2700

Infrasil quartz (NIR) 220–3800

Optical glass 334–2500

Polystyrene (disposable) 340–800

Cuvette matching

Some cuvettes are sold as matched pairs and are used for most UV-Vis

and UV-Vis-NIR routine analyses. Matched pairs ensure both cuvettes

provide a similar absorbance or transmission reading when empty or

filled with water. The match code is like a batch number. It reflects the

transmission characteristics of the batch (melt) of raw material that the

cuvettes are made from.

For a pair of cuvettes to be matched they must be within the acceptable

transmission tolerance at a particular wavelength. For example,

a pair should transmit within 1.5% of each other at 200 nm. Cuvette

manufacturers specify the transmission matching tolerances at measured

wavelengths for the materials they supply.

The matching codes are only of real value when comparing new cells.

Transmission characteristics change during use because of surface

contamination or wear due to cleaning processes or mishandling.

Therefore, a brand-new cell will not necessarily match an older used cell

of the same match code.

Modern production and fusing techniques have improved flatness,

parallelism, and construction tolerances. Together with more attention to

quality assurance of raw materials, this has resulted in a virtual elimination

of the need for transmission matching in regular standard high grade

quartz cells.

100

0 300 500 2000 2200 2400 2600 2800 3000 3200 nm

Transparency of different cell materials

plastic

optical glass

suprasil

Figure 22. Transparency of common optical cell materials.

Cleanliness

The oils in fingerprints are significant absorbers in the UV region.

Fingerprints left on optical surfaces can cause erroneous results.

Wipe off all fingerprints and contaminants using a clean soft cloth before

using a cell. Ensure that the cloth is free of detergents or lubricants.

Lens tissues for glasses or other uses often contain detergents or

lubricants which can affect your measurements.

Do not use an ultrasonic cleaner to clean your glass or quartz cuvettes.

Each ultrasonic bath generates ultrasonic waves at a different frequency

and if your bath operates at the resonant frequency of the cell, the cell

will break.

Fluorinated acids such as hydrofluoric acid (HF) in all concentrations

should be avoided as they will attack the quartz and glass. Strong basic

solutions (pH 9.0 and above) will also degrade the surface of the windows

and shorten the life of the cuvette.

Once a blank measurement has been made avoid cleaning the optical

windows of your cell. If cleaning is required, take a new blank measurement

before continuing your measurements.

Other cuvette handling tips

Some simple steps can be taken to ensure the longevity of cuvettes:

– Consider removing samples from cuvettes immediately after use.

This will prevent your sample from drying out and sticking to your

cuvette. Take particular care with proteins and strong dyes as they are

known to adhere to the inside surfaces of the cuvette. Remove these

sample from the cell immediately after testing and rinse thoroughly

with your solvent between samples.

– If measuring the same sample over a long period of time, keep the

sample capped and at an appropriate temperature to minimize

evaporation. Some samples may require continuous stirring.

– Clean all cuvettes thoroughly at the end of each day and either:

• store them in a suitable container after drying or,

• store the cuvettes wet in a mildly acidic solution (1% nitric acid or

hydrochloric acid), in an acid resistant beaker. Only store one cuvette

per beaker to avoid chipping the cuvette. Always rinse with copious

amounts of water immediately before use again.

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Designers of the screens used on mobile phones, tablets, laptops and

televisions strive to make them as thin as possible. Reducing the thickness

by even tens of microns can make a significant difference to reducing the

overall size of a device. These screens use light emitting diodes (LED) and

liquid crystal displays (LCD) to control the colors of the display, with a back

light reflector to provide the illumination. Back light reflectors are usually

made from a transparent polymer material with a reflector film applied to one

surface. They look like a sheet of plastic mirror. Measuring the reflectance

and transmission of the back light reflector is a critical part of developing

a new design. It is also an important quality control measurement during

their manufacture. The reflectance and transmission of a back light reflector

is measured with a UV-Vis spectrophotometer. The back light reflector

is mounted vertically in the sample compartment and rotated around its

central axis, allowing the measurement of its reflectance and transmission at

different angles of incidence.

Here are the details of the measurement

3.3 Stirring your sample

Stirring of a thermostatted sample is important and ensures that both

solution and temperature homogeneity is always maintained. Stirring is

particularly important for viscous samples or to ensure consistently mixed

solutions when studying a chemical reaction within the cuvette.

The effectiveness of stirring to achieve thermal (and chemical) homogeneity

is strongly dependent upon the sample, solvent, and viscosity of the

solution. It is important to note that viscosity changes with temperature

and this may influence stirring efficiency, and the measurements, when the

temperature is ramped over time.

To stir solutions a magnetic stirring bead or star, is placed at the bottom of

the sample cuvette. Specially designed cuvettes with a recessed base are

available. This circular recess contains the stirring bead and increases the

stirring efficiency.

Care should be taken when developing the analytical method to ensure that

the stir speed is suitable for the solutions. If the stirring speed is too slow

the sample may not mix properly. If the speed is too high, air bubbles can be

trapped in the sample, causing erroneous results.

It is recommend that test experiments are conducted on all samples to

find the optimum stir speed for your experiment. When measuring liquid

of similar viscosities to water, stir speeds of 800 to 900 rpm generally

yield the best temperature uniformity within standard cuvettes. Lower the

stirring speed for higher viscosity samples and increase the speed for lower

viscosity samples.

3.4 Measurements at low temperatures

When measuring samples at lower than ambient temperature,

condensation may form on the outside of cuvettes. This can interfere with

the measurement. Condensation can be prevented by purging the sample

compartment of the UV-Vis spectrophotometer with a clean dry gas.

Some systems have specialized purging ports to allow entry of the gas

into the sample compartment without introducing any light. An alternative

for measuring cool samples is to use a fiber optic dip probe. A fiber optic

coupler is inserted into the sample compartment and directs the light from

the system through a fiber optic cable, through a dip probe directly inserted

in the sample (as shown in Figure 6). The light is then directed back to the

detector through a return fibre optic cable. For high throughput sampling

this technique may be preferred when multiple samples are being measured

at a fixed temperature.

3.2 Thermostatting your samples

Many samples can be measured at room temperature, but there are some

circumstances that require samples to be heated or cooled. These include:

– Cooling of volatile samples to reduce evaporation

– Heating of viscous samples to improve sample handling or

homogeneity

– Samples that are sensitive to chemical change when heated

– Observing changes in samples as they are heated or cooled.

UV-Vis spectrophotometers can be fitted with accessories to control the

temperature of samples. The simplest temperature control systems are

suited for fixed temperature measurements. Typically, these systems use

a thermostatted water circulator to pass heated water through a manifold

holding the sample cuvette. For more precise temperature control a Peltier

heater/cooler is embedded into the sample manifold. Peltier devices allow

greater temperature control and allow temperature ramping measurements

to be undertaken. An air-cooled Pelter system requires less maintenance

than a water-cooled Peltier system or a water circulation system. Water

circulating systems need periodic maintenance, including checking water

hoses for leaks and topping up of the coolant solution. Another advantage

to the Peltier system is their quiet operation as no pumping of coolant

solution is needed.

When using either temperature control options your system should provide

you with temperature monitoring. As a minimum the system should report

the temperature of the sample manifold. This is particularly important

for an external water heated system. Heat losses from the temperature

set on the water bath may occur between the circulator and the sample

manifold. For Peltier controlled systems the sample manifold temperature

is monitored providing feedback to keep the temperature stable.

When temperature control is critical, taking measurements of the sample

directly provides a more accurate reading. Small temperature probes

are inserted into the sample, inside the cuvette. The probes are carefully

positioned out of the light path. When monitoring the temperature directly

in the samples your UV-Vis control software should allow you to record the

temperature of each cuvette at each measurement.

3.5 Solvent transparency

When measuring liquid samples, or dissolving solid samples for UV analysis,

solvent transparency needs to be considered. Solvents are selected based

on sample solubility, stability, pH requirements, and the UV-visible cut-off

wavelength. For aqueous soluble compounds water is an excellent choice

as it allows measurement throughout the UV wavelengths. The use of

organic solvents does limit the effective useable UV wavelength range.

When selecting a solvent, consider both the solubility of your sample in the

solvent as well as the transparency of the solvent in the wavelength range

of interest (as shown in Figure 23).

Figure 23. Transparency ranges of common solvents in the UV region. While water is

preferred for UV analysis, another solvent may be required if your sample is not soluble

in water.

Useful transparency ranges of common solvents in the UV region

Acetone

Tetrachloroethylene

M-Xylene

Toluene

Benzene

N.N-Dimethylformamide

Ethyl Propionate

Carbon Tetrachloride

Ethyl Formate

Butyl Acetate

Ethyl Acetate

Methyl Formate

Chloroform

1.2-Dichloromethane

Dichloromethane

Glycerol

Dioxane

Hexane

Iso-Octane

2.2.4-Trimethylpentane

Acetonitrile

Cyclohexane

Methanol

Ethanol

Methyl Cyclohexane

Iso-Propyl Alcohol

Water

190 nm 210 230 250 270 290 310 330 350 370

One molecule with a very important role

The protoporphyrin molecule forms the basis of life for many plants and

animals. The body stabilizes the molecule by inserting zinc atoms into its

ring structure in immature red blood cells (reticulocytes). As the reticulocytes

mature, the zinc is replaced by iron. The mature red cells then combine with

globin-forming hemoglobin, the oxygen carrying molecule in the blood of

many animals and humans.

In plants, magnesium is inserted into the protoporphyrin ring to form

chlorophyll, one of the primary compounds needed for photosynthesis.

Protoporphyrin absorbs UV light – no surprise there, given its importance in

photosynthesis. The molecule also fluoresces. These characteristics make

protoporphyrin ideal for analysis with spectroscopy. In fact, it forms the basis

of the primary screening test for childhood lead poisoning (1).

1. Clinical and Laboratory Standards Institute, Erythrocyte Protoporphyrin Testing; Approved Guideline,

Volume 16, No. 8, 1996

270 310 350 390

Molecular band width

Spectral

Band Width

½ peak height

Wavelength (nm)

Absorbance (A)

As a guideline the SBW should be set at one tenth of the molecular band

width of the sample (examples in Table 2).

3.6 Optimum spectral band width

When measuring a sample consideration should be given to the

measurement resolution required. Most solid or liquid samples analyzed

by UV-Vis spectroscopy have naturally broad peaks, in the order of 20 nm

or more from side to side. It is good practice to use an instrument with a

spectral bandwidth (SBW) setting approximately one tenth of the natural

bandwidth of the analyte. The SBW of the instrument is defined as the width

of the band of light at one-half the peak maximum (as shown in Figure 24),

and sometimes referred to as full width at half maximum (FWHM).

The SBW of the UV-Vis spectrophotometer is related to the physical slit

width of the monochromator design.

Figure 24. Spectrum A shows a peak maximum close to 345 nm. The spectral bandwidth

is shown. The spectral slit width of the UV-Vis spectrophotometer will always be narrower

than the required spectral band width.

Depending on the spectrophotometer design, the physical slit can be either

a fixed or variable width. For most mid-range UV-Vis spectrophotometers,

a fixed spectral bandwidth of 1.5 nm is common and sufficient for resolving

the peaks of most liquid and solid samples. Using a larger SBW allows more

light through the sample and can give better quality data and less noise, but

will not resolve narrow or close together sample peaks. Using a smaller SBW

will provide better resolving power but can result in increased data collection

times to achieve the same data quality due to less light reaching the sample.

High performance or research grade spectrophotometer systems are more

frequently designed to allow the user to select the slit width, and thereby

adjust the resolution of the system. This is useful when measuring more

challenging samples. The slit width can be maximized to allow greater light

throughput in highly absorbent samples where high resolution of the peak is

not necessary. Greater light throughput to the detector allows better method

repeatability, accuracy, and precision of the results. When high resolution is

required the slit width can be reduced (as shown in Figures 25 and 26).

Abs

440.0

430.0 450.0 460.0 470.0 480.0 nm

4.0 nm

2.0 nm

0.2 nm

3.000

2.400

1.800

1.200

0.600

0.000

Observed Absorbance

Figure 25. Overlaid scans, each measured using a different instrument slit.

As the slit widens the signal-to-noise ratio improves, however resolution decreases.

Table 2. Recommended spectral band width settings for common UV-Vis

measurement types.

Representative Compound Peak nm Band Width nm Optimum (SBW) nm

Amino Acids

tryptophan

279

4.5

tyrosine 275, 195 40, 10 4.0, 1.0

phenylalanine 258 2.2 0.2

Nucleotides

adenosine

260

2.8

thymine 265 30 3

Proteins

cytochrome c, oxidized

410

2.5

rhodopsin 500, 278 ~90, 25 9, 2.5

ribonuclease 278 20 2

Pigments and Dyes

ß-carotene

480 35

3.5

chlorophyll a 660 20 2

Coenzymes

Nicotinamide adenine dinucleotide

260

3.5

NADH 340, 260 50, 25 5, 2.5

Simple Organics

benzene, vapor

253

<<0.1

<<0.01

benzene, solution 253 2 0.2

anthracene 375 3 0.3

When optimizing spectral resolution, the data interval of the collection

also needs to be considered. A minimum of three data points across the

peak should be collected. While a smaller data interval can provide better

resolution there will be a trade-off between how long the data takes to

collect and the data interval.

Figure 27. The maximum absorbance of peaks will be lower and the curve will flatten as

stray light in the instrument increases.

Figure 28. A UV-Vis system with poor stray light performance will show deviations from

Beer’s Law. This makes concentration calculations unreliable.

3.8 The linear range of a UV-Vis instrument

Both instrument design and the measurement parameters used will

determine the maximum absorbance an instrument will be able to measure

at a specific wavelength. At high absorbance very little light is reaching the

detector which decreases the signal to noise ratio (refer to the characteristic

'fringe' on the spectrum in Figure 29). Understanding the limits of your

system allows you to avoid measuring samples or performing calibrations

which are outside the capabilities of your instrument. For liquid samples,

diluting the sample is a way to get the measurement into the linear range of

the instrument. Alternatively, you can use a short pathlength cuvette.

Figure 26. These two scans demonstrate the effect on resolution of varying the SBW.

At a SBW of 15 nm (upper graph), little structural detail is observed in the spectrum.

At a SBW of 3 nm (lower graph), the peaks are much more defined.

3.7 Stray light

Stray light or stray radiant energy (SRE) is defined as the percentage of

radiation reaching the detector whose wavelengths are outside the selected

spectral band. It is caused by poor instrument design (light leaking into the

instrument from the laboratory lights or daylight through windows, or the

light not being well separated by the monochromator) or from damage to

the instrument. Most systems are provided with instrument performance

checks that identify stray light issues. This is done using a test solution.

The solutions used to test stray light levels are non-transmitting at the

indicated wavelengths (they do transmit at other wavelengths), so the

observed transmittance is due only to stray light.

Stray light causes decreased absorbance readings and changes the

observed peak shape (as shown in Figure 27). As a result, stray light causes

deviation from the Beer-Lambert law (as shown in Figure 28), making

concentration measurements unreliable. The stray light performance

of a UV-Vis instrument also determines the maximum absorbance the

instrument can measure.

1.0 2.0 3.0 4.0

10% Stray Light

Beer’s Law

1% Stray Light

0.1% Stray Light

2.0

1.0

0.0

3.0

Concentration

Observed Absorbance

0-10 10

2.0

1.0

0.0

3.0

Relative Wavelength (nm)

Absorbance

True (Gaussian)

0.01% SRE

0.1% SRE

1% SRE

1200.00 1600.00 2000.00 nm

2.0000

1.0000

Abs

0.0000

1200.00 1600.00 2000.00 nm

2.0000

1.0000

Abs

0.0000

Figure 29. As sample absorbance increases progressively less light reaches the detector.

This increases noise in the results and an obvious spiky signal will be observed in

scanning mode.

3.9 Other useful information

Absorbance (A or Abs) is frequently measured in UV-Vis spectroscopy

due to the linear relationship between concentration and absorbance as

described by the Beer-Lambert law. For other applications, the percentage

of light transmitted or absorbed may be more meaningful. When comparing

the optical properties of a material for example it may be more useful to

compare the percent transmission or absorbance difference.

Most UV-Vis spectrophotometer systems will enable you to convert your

collected data between the commonly used parameters. The relationship

between these parameters is shown in Table 3.

3.10 Wavelength or inverse centimeters

Most UV-Vis measurements are reported against wavelengths measured

in nanometres (1×10

−9

m). in some older literature the reciprocal length

or wavenumber (cm

-1

) is used. Wavenumber is often used in infrared (IR)

spectroscopy measurements. Using a wavenumber scale is useful as

it conveys the change in energy levels of the incident radiation. A lower

wavelength gives a larger wavenumber and a higher energy (as shown in

Table 4).

The use of wavenumber for infrared spectroscopy also allows for easier

visualization of spectral differences as the wavelength gets progressively

shorter.

For UV-Vis spectroscopy, wavelength is generally preferred as a convenient

way to visualize the displayed spectrum over a spectral range.

Most UV-Vis spectrophotometer systems will enable you to collect a

spectrum in either wavelength or wavenumber.

Table 3. The relationship between percent transmission and absorbance can be hard to

visualize. The table shows that a sample measuring 7 Abs transmits just 0.00001% of the

light through the samples.

%T T Abs %A LogA

100 1.0 0 0 –

50 0.5 0.3 50 -0.52

10 0.1 1 90 0

1 0.01 2 99 0.3

0.1 0.001 3 99.9 0.48

0.01 0.0001 4 99.99 0.60

0.001 0.00001 5 99.999 0.70

0.0001 0.000001 6 99.9999 0.78

0.00001 0.0000001 7 99.99999 0.85

Table 4. Conversion between wavelength (nm)

and wavenumber (cm

-1

λ nm cm

-1

3300 3030

3000 3300

2500 4000

2000 5000

1500 6666

1000 10000

800 12500

600 16667

400 25000

200 50000

175 57143

400 500 600 700 800 900 1000

3.5

3.0

2.5

2.0

1.5

1.0

0.5

4.0

0.0

2 mgml

3 mgml

5 mgml

7 mgml

10 mgml

12 mgml

14 mgml

16 mgml

18 mgml

21 mgml

25 mgml

4.1 Identification—spectra and structure

UV-visible spectra generally show only a few broad absorbance peaks.

Compared with techniques such as infrared spectroscopy, which produces

many narrow peaks, UV-visible spectroscopy provides a limited amount of

qualitative information. With only a few broad peaks, it’s difficult to identify

a compound based on a characteristic spectrum.

Most absorption by organic compounds results from the presence of π

(that is, unsaturated) bonds. A chromophore is a molecular group usually

containing a π bond. When inserted into a saturated hydrocarbon (which

exhibits no UV-visible absorbance spectrum), it produces a compound with

absorption between 185 and 1000 nm. Table 5 lists some chromophores

and the wavelengths of their absorbance maxima.

Table 5. Selected chromophores and the wavelength of their absorbance maxima.

Chromophore Formula Example λmax (nm)

Carbonyl (ketone) RR’C=O Acetone 271

Carbonyl (aldehyde) RHC=O Acetaldehyde 293

Carboxyl RCOOH Acetic acid 204

Amide RCONH

Acetamide 208

Ethylene RCH=CHR Ethylene 193

Acetylene RC=CR Acetylene 173

Nitrile RC=N Acetonitrile <160

Nitro RNO

Nitromethane 271

The presence of an absorbance band at a particular wavelength often is a

good indicator of the presence of a chromophore. However, the wavelength

position of the absorbance maximum is not fixed but depends partially on

the molecular environment of the chromophore and on the solvent in which

the sample is dissolved. Other parameters, such as pH and temperature,

also may cause changes in both the intensity and the wavelength of the

absorbance maxima.

4. Overview of Common UV-Vis Applications

Conjugating the double bond with additional double bonds increases

both the intensity and the wavelength of the absorption band. For some

molecular systems, such as conjugated hydrocarbons or carotenoids,

the relationship between intensity and wavelength has been systematically

investigated. Transition metal ions also have electronic energy levels that

cause absorption of 400–700 nm in the visible region.

FTIR spectra can be used to identify compounds

Fourier Transform Infrared (FTIR) spectra contain a lot more detail than

UV-Vis spectra. A spectrum like the one shown here (red) can be matched

against a library of FTIR spectra to identify the compound. In this case,

a pharmaceutical—salicylic acid (the library spectrum of which is shown

in blue).

4.3 Quantifying a molecule

Beer’s law

If 100 photons of light enter a cuvette and only 50 emerge from the other

side, the transmittance is 0.5, or 50 %. If these 50 photons then pass

through an identical cuvette, only 25 will emerge, and so forth. Figure 31

shows the plot of transmittance against path length of the cuvette.

4.2 Confirmation of identity

Although UV-visible spectra do not enable absolute identification of an

unknown, they are used to confirm the identity of a substance through

comparison of the measured spectrum with a reference spectrum.

Where spectra are highly similar, derivative spectra may be used.

As shown in Figure 30, the number of bands increases with higher orders

of derivatives. These complex derivative spectra can be useful in qualitative

analysis, either for characterizing materials or for identification purposes.

For example, the absorbance spectrum of the steroid testosterone shows

a single, broad, featureless band centered at around 330 nm, whereas the

second derivative shows six distinct peaks. The resolution enhancement

effect may be of use as well in identifying an unknown. Figure 30 shows

a computer simulation. When two Gaussian bands with a 40 nm natural

spectral bandwidth (NBW), separated by 30 nm, are added in absorbance

mode, a single band with a maximum midway between the two component

bands results. The two components are not resolved. Using a fourth

derivative of the spectrum, these two bands are clearly visible, with maxima

centered close to the λ

max

of the component bands.

Figure 30. Resolution enhancement using derivative analysis. The original, overlapping

peaks resulted in a single, broad peak. By taking the 4th derivative of the spectrum,

a spectrum with much higher resolution of the peaks results.

Figure 31. Transmittance and path length—the Bouguer-Lambert law.

Lambert (1760) generally is credited with the first mathematical expression

of this effect, although it now appears that Bouguer first stated it in 1729.

The equation is:

T= I/I

= e

–kb

Where:

– I

is the incident intensity

– I is the transmitted intensity

– e is the base of natural logarithms

– k is a constant

– b is the path length (usually in centimeters).

Beer’s law is identical to Bouguer’s law, except that it is stated in terms of

concentration. The amount of light absorbed is proportional to the number

of absorbing molecules through which the light passes. Figure 32 shows a

plot of transmittance against concentration.

400

500 600

400

500 600

1.5

0.0

0.5

1.0

0.0

-5.0x10

-6

5.0x10

-6

4th derivative

Wavelength (nm)

Absorbance (A)

1.0 2.0 3.0 4.0

100%

50%

25%

Transmission

Path length

100% 50% 25% 12.5% 6.75% 3.125%

Figure 32. Transmittance and concentration—Beer’s law

Combining the two laws gives the Beer-Bouguer-Lambert law:

T= I/I

= e

–kbc

Where c is the concentration of the absorbing species (usually expressed in

grams per liter or milligrams per liter). This equation can be transformed into

a linear expression by taking the logarithm and is usually expressed in the

decadic form:

A = –logT = –log(I ⁄ I

) = log(I

⁄ I) = ∑bc

Where A is the absorbance and ∑ is the molar absorption or extinction

coefficient. This expression is commonly known as Beer’s law. Figure 33

shows a plot of absorbance against concentration.

The extinction coefficient (ε) is characteristic of a given substance under

a precisely defined set of conditions, such as wavelength, solvent, and

temperature. In practice, the measured extinction coefficient also depends

partially on the characteristics of the instrument used. For these reasons,

predetermined values for the extinction coefficient usually are not used

for quantitative analysis. Instead, a calibration or working curve for the

substance to be analyzed is constructed using one or more standard

solutions with known concentrations of the analyte.

For electronic transitions, the difference in energy between ground and

excited states is relatively large. Therefore, at room temperature, it is highly

likely that all molecules are in the electronic ground state. Absorption and

return to ground state are fast processes, and equilibrium is reached very

quickly. Thus, absorption of UV-visible light is quantitatively highly accurate.

The simple linear relationship between absorbance and concentration and

the relative ease of measurement of UV-visible light have made UV-visible

spectroscopy the basis for thousands of quantitative analytical methods.

Assuming Beer’s law is obeyed for the zero-order spectrum, a similar linear

relationship exists between concentration and amplitude for all orders of

derivative spectra:

Zero order: A = εbc

First derivative: dA/dλ = (dε/dλ)bc

nth derivative: d

A/dλ’ = (d

ε/ dλ’)bc

at λ, where A is absorbance, ε is the extinction coefficient, b is the sample

path length, and c is the sample concentration.

For single-component quantification, the selection of wavelengths is

more difficult with derivative spectra than with absorbance spectra since

both positive and negative peaks are present. The even-order derivatives

have a peak maximum or minimum at the same λmax as the absorbance

spectrum, but for the odd-order derivatives, this wavelength is a zero-

crossing point. Taking the difference between the highest maximum and

the lowest minimum gives the best signal to noise (S/N) but may result in

increased sensitivity to interference from other components.

For accurate results, the sample to be analyzed must contain only the

absorbing component for which the calibration has been performed.

If the sample is a solution, a pure sample of the solvent should be used as

a blank. It may be possible to correct for an interfering component with a

second wavelength.

100%

50%

25%

Transmission

Concentration

100% 50%

25%

12.5%

6.75%

3.125%

100%

0.5

0.0

1.5

1.0

Absorbance (A)

Concentration

Figure 33. The Beer–Bouguer-Lambert law describes a linear relationship between

absorbance of incident light and the concentration of the molecule.

4.4 Kinetics

Analysis of reaction kinetics is fundamental for understanding how

reactions occur in chemistry and biochemistry. UV-Vis spectrophotometry

is an ideal technique for this application as the sample is not destroyed.

It can be used when the change in reactant or products produces a change

in absorbance at a specific wavelength over time. With a fast scanning

UV-Vis system, multiple scans can be taken during the reaction to allow the

reaction to be visualised and aid in the selection of wavelengths selected

for the rate calculation.

Single point kinetics

Single point kinetics analysis is the simplest method of determining a

reaction rate. A single wavelength is selected, usually the maximum

absorbance of the analyte of interest, and, after initiating the reaction, the

absorbance is continuously monitored at that wavelength. This results in a

plot of absorbance versus time, as shown for four samples in Figure 34.

Monitoring the change in absorbance over time allows you to study a

reaction – when the absorbance stops changing, this is usually an indication

that the reaction is complete. Figure 34 shows four different samples over

30 minutes.

The advantage of a single point measurement is that for fast reactions near

continuous data can be collected as the system does not need to move to

another wavelength during measurement. The data collection speed of the

instrument and the signal averaging time is the limiting factor in the amount

of data that can be collected.

Multiple wavelengths can also be monitored for kinetics measurements.

This may provide you with an insight into the creation of a reaction product

at one wavelength and depletion at another.

Concentration measurements

One of the most common applications for the UV-Vis spectrophotometer is

for simple quantification of concentration. Coupled with a fiber optic dip probe

accessory, a UV-vis spectrophotometer can be used to take measurements

directly in the sample container, without the need to decant to a cuvette.

Find out more.

Figure 34. Monitoring the change in absorbance over time allows you to study a reaction

– when the absorbance stops changing, this is usually an indication that the reaction is

complete. This graph shows four different samples over 30 minutes.

Scanning kinetics

Scanning a wavelength range over time can provide additional information

when performing kinetics measurements. Apart from providing a visual

impression of the reaction as it occurs (as shown in Figure 34), this

measurement allows the flexibility to select any number of wavelengths

over the scan range to perform a reaction rate calculation. This could

include analysing the consumption of reactants or the production of

reaction products as the reaction progresses. When performing scanning

kinetics it is important to ensure that the UV-Vis system can rapidly scan

the wavelength range selected. Slower scanning will limit the amount of

data collected at any wavelength during the reaction.

Rapid-mix kinetics

To monitor the reaction rate or two rapidly reacting solutions may require

the use of a specialised rapid-mix or stopped flow accessory. When fitted to

a UV-Vis spectrophotometer, these accessories provide accurate delivery of

two or more solutions to a flow cell inside the UV-Vis, where mixing occurs.

The stopped flow accessory triggers the UV-Vis system to start the analysis

as soon as the solutions are mixed in the flow cell. Options include the ability

to change the mixing ratio of the solutions and the ability to thermostat both

the flow cell and reactants. As the collection speed is critical it is important

to select a UV-Vis system with a data collection rate that is fast enough to

collect a series of data points within the reaction time frame. The example

shown in Figure 36 shows a reaction monitored over three seconds.

20105 25 30

2.0

1.0

3.0

Time (mins)

Absorbance (A)

0.12

0.13

0.14

0.15

1 2 30

Time (sec)

Abs

Figure 35. Scanning kinetics: UV-vis spectrophotometers provide powerful insight into

reaction chemistry. The impact of temperature on chemical reactions can be observed

quickly and easily with rapid scanning at multiple temperatures. Here’s a scanning

kinetics experiment.

Figure 36. Using a modern UV-Vis spectrometer and a rapid mix accessory allows

measurement of reactions over the time span of mere seconds. Here’s an example

experiment.

4.5 Color measurement

Color is an important property of a material. The color of matter is related

to its absorptivity or reflectivity of specific wavelengths of light. The human

eye sees the complementary color to that which is absorbed, as shown in

Figure 37 and Figure 38.

Kinetics measurement considerations

The rate of a reaction can be influenced by temperature. For this reason,

it can be important to maintain a sample at a constant temperature,

for example, body temperature (37 degrees) is usually selected for

biological reactions. Peltier cooled/heated cuvettes or water thermostatted

cuvette holders are commonly used with a UV-Vis system for this purpose.

These accessories can either keep a sample at a specific temperature or

change (ramp) the temperature over time.

The temperature accuracy and reproducibility need to be considered

carefully, as slight variations in temperature or the temperature change rate

can have a significant impact on results. Some systems provide the ability

to measure the temperature of the samples from directly within the

cuvette (rather than just measuring the temperature of the cuvette holder).

When combined with feedback to the temperature control system, this

typically provides better temperature control of the sample. Temperature

probes in the sample can also be used to record the sample temperature

along with absorbance data.

Consistent and reproducible stirring of samples is also important. When

measuring reactions at room temperature, stirring ensures the reactants

are mixed consistently during the reaction. For thermostatted samples,

stirring ensures there is no temperature gradient across the sample.

When measuring complex systems, the use of a fiber optic probe may be

preferred. A fiber optic cable allows the light from the spectrophotometer

to be directed to a sample outside the UV-Vis spectrophotometer. This can

be useful for measurements of a flowing manufacturing process, where the

sample is at extremes of temperature or pressure, or when the sample is

physically unable to fit inside the spectrophotometer sample compartment.

A demonstration of kinetics experiments can be viewed on the

agilent.com site.

Figure 37. Transmission and color. Much like the spectrophotometer detector, our eye

sees the light transmitted through or bounced off a surface. We perceive this as the color

of the object.

Figure 38. The wavelengths of light associated with different colors (left) and the color

of absorbed light and the associated complementary color the human eye sees (right).

red blue-green

orange greenish blue

yellow green purple

green red-purple

bluish green red

greenish blue orange

blue yellow

violet yellow-green

Absorbed color Complementary Color

650-780

595-650

560-595

500-560

490-500

480-490

435-480

380-435

Wavelength (nm)

800

700

600

500

400

Light source Absorbance

by sample

Detector

(eye)

In practice, both the generation and sensation of color are highly complex

and depend on many factors, including:

– The spectrum of the light falling on the object (consider the difference

in colors seen at sunset versus the middle of the day)

– The surface structure of a solid material (the scales of a fish or the

feathers of a bird are two examples where the physical structure of the

surface changes the color seen)

– The viewing angle (some surfaces, such as pearlescent paints, change

color as the angle you are viewing the surface at changes)

Specialized color measurement systems, such as the CIE L*a*b, and

instrumentation to measure color have been developed. When equipped

with the appropriate software, most spectrophotometers can be used to

measure color. Color perception is also influenced by the surface and its

ability to produce specular (mirror like) reflectance or diffuse (scattering)

reflectance. Because of these factors, color measurement may require

special accessories which allow specular and diffuse reflectance to be

collected and observed at different viewing angles.

A color measuring instrument will take the UV-Vis spectrum of a sample

and convert it into three color coordinates that locate the color in three-

dimensional color space (refer to the image in the side panel). The three

coordinates define the sample’s lightness, chroma and hue. Lightness is

a measure of how light or dark a color is. Chroma is a measure of ‘color

purity’, and hue is the dominant spectral color -similar to the colors seen in

a rainbow.

As well as being used for color matching measurements e.g. measuring

paint colors on a manufactured item, a UV-Vis spectrophotometer can also

be used to measure a change of color in a solution. UV-Vis measurements

are often used for this purpose to assess whether a reaction has taken place

or is proceeding, without visual inspection. Color based assays are one of

the widest used applications for UV-Vis spectrophotometry.

Measuring color

UV-Vis spectrophotometers are used to measure the color of liquids and

solids. By measuring the visible light that is transmitted through a solution or

material, or the light that is reflected from a surface, the color of the liquid or

material can be calculated. A series of three numbers is used to indicate the

coordinates of the measured color in a ‘color space’. Find out more.

How black is black? Ask a deep-sea fish

Researchers have found many deep-sea fishes with ultra-black skin – that

which reflects less than 0.5% of incident light. The fish use their ultra-black

skin to remain undetected while they hunt for prey. In the deep, dark sea

creatures often use the glow of bioluminescence to help them see their prey

or their attackers. Ultra-black skin absorbs the light of bioluminescence,

allowing the fish to remain out of sight.

Researchers from the Smithsonian National Museum of Natural History

and Duke University measured the reflection of the fish skin, which absorbs

99.5% of light. This compares to the superb bird of paradise (99.95%) and the

blackest material ever made - Vantablack, which absorbs 99.96%.

The mechanism used by the fish to absorb light could have applications in

solar panels, telescopes, camera and camouflage systems.

Read the research results

4.6 Structural changes of compounds

UV-visible spectroscopy can be used to determine many physicochemical

characteristics of compounds. These measurements can identify a

compound or determine specific properties.

Conformational studies

UV-Vis spectroscopy can provide insight into protein structure. UV-Vis

spectrophotometry is also non-destructive, so precious samples will not be

sacrificed. This makes UV-Vis an ideal for use prior to analysis by techniques

such as LC or mass spectroscopy. This is demonstrated in the comparison

of an innovator and biosimilar monoclonal antibody pair. Find out more.

4.7 Protein and nucleic acid melting temperature

UV-Vis spectroscopy is commonly used in the life sciences for analysis

of biomolecules such as proteins and nucleic acids. The absorbance

spectra of proteins is due to the absorbance of the aromatic amino acids

tryptophan, tyrosine, and phenylalanine. Multicomponent analysis can be

used to determine how many of each aromatic amino acid are present in

an intact protein.

A protein at room temperature has a specific tertiary structure or

conformation that in turn creates a specific electronic environment for the

aromatic amino acids. Another application of UV-Vis spectroscopy exposes

proteins to heat or chemical denaturants. This will, at a certain temperature

or concentration, cause the protein to unfold or melt and lose its structure.

Innovator

Biosimilar

[GdnHCl] (M)

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

0 1 2 3 4 5 6 7

a/b Ratio

260 280 300 320 340

-0.04

-0.02

0.02

Wavelength (nm)

d2 (Absorbance)/d2 (nm)

In this process, the electronic environment of the aromatic amino acids

changes, which in turn results in spectral changes or shifts.

Figure 39. The strands of the DNA double helix unwind as the DNA is heated. This increases

the absorbance of UV light at 260 nm. As the DNA is cooled, the strands rejoin.

Deoxyribonucleic acid (DNA) in its native state comprises two strands

of deoxyribose molecules helically wound around the same axis.

The strands are linked by hydrogen bonds between the purine and

pyrimidine bases—adenine is joined to thymine (A-T), and guanine to

cytosine (G-C). These bases are primarily responsible for the UV absorbance

of DNA and the other types of nucleic acids, with a peak maximum at

260 nm. As in any multicomponent system, the observed absorption of any

nucleic acid molecule should equal the sum of the individual absorbances:

DNA

= A

adenine

+ A

guanine

+ A

cytosine

+ A

thymine

However, the observed absorbance is always significantly less than

expected because the hydrogen bonding between the bases changes their

electronic environment. When a molecule is heated, the hydrogen bonds

break, the double helix unwinds, and the absorbance increases so that it

approaches that expected from the sum of all bases (refer to Figure 39).

This denaturation process is known as melting or thermal melt. In a thermal

melt experiment, the temperature of a double stranded nucleic acid solution

is increased in a stepwise fashion, and the absorbance at 260 nm at each

temperature is measured and plotted as a melting curve (as shown in

Figure 40). The midpoint of the temperature range over which the melting

occurs is the T

value. The T

value of a particular nucleic acid sample

depends primarily on the percentage of G-C pairs in the sample, each of

which contains three hydrogen bonds (in contrast, each A-T pair contains

two hydrogen bonds). The higher the percentage of G-C pairs in the sample,

the higher the observed T

Figure 40. Measuring the absorbance at 260 nm, whilst increasing the temperature results

in this characteristic graph of a DNA ‘melt’. The change in absorbance indicates the multiple

transitions as the DNA helix unwinds.

To perform protein and DNA melt analyses the UV-Vis spectrophotometer

must have a means to change the sample temperature accurately and

reproducibly. Recent advances in spectrophotometric instrumentation offer

significant reductions in elapsed times for thermal melt measurements,

as well as higher temperature accuracy than previously possible. UV-Vis

thermal melt analysis systems are available with integrated in-cuvette

temperature probes, which can be used to accurately control the

temperature of the solutions during the experiment. In-cuvette stirring is

also provided to ensure samples are heated homogeneously. When there

are large numbers of samples for analysis a multicell holder is built into or

806040

0.50

0.40

0.45

0.55

Temperature (°C)

Absorbance (A)

Once nucleation

has occurred,

renaturation is rapid

due to zippering.

Temperature increase

causes the bases to

unstack, and hydrogen

bonds break.

When the solution

is returned to lower

temperatures,

the DNA strands

start to reconnect.

Duplex

DNA

Partially

unwound

DNA

Totally

denatured DNA

(separate strands)

A T

G C

the absorbance of standards of known concentrations of pure

components are measured to determine the extinction coefficient for

each component at each wavelength selected. The absorbance of

the mixture at each wavelength is the sum of the absorbance of each

component at that wavelength, which in turn depends on the

extinction coefficient and the concentration of each component.

Thus for two components x and y, the equations are:

A′

(x + y)

= A′

+ A′

= e′

+ e′

and

A″

(x + y)

= A″

+ A″

= e″

+ e″

Where:

– A′ is absorbance at wavelength ′

– A′′ is absorbance at wavelength ′′

– e′ is molar absorptivity at wavelength ′

– e′′ is molar absorptivity at wavelength ′′

– c is concentration

– b is path length.

These equations are easily solved to determine the concentration of each

component. If measurements were always perfect, accurate results

could be obtained even for complex mixtures of components with very

similar spectra. In practice, however, measurement errors always occur.

Such errors can significantly affect the accuracy of results when there

is major spectral overlap. Figure 41 shows a simulated two-component

mixture with no overlap of the spectra at the absorbance maxima.

230 240220210200

0.5

1.0

0.0

Wavelength (nm)

Absorbance (A)

x y x + y

Nucleic acid thermal melt

analysis

Sample temperature control is a key

requirement of the UV-Vis thermal

melt system. Rapid and reproducible

heating can be assisted by the use

of an in-cuvette temperature probe.

Find out more.

provided with the instrument. Most Peltier driven temperature accessories

require a circulating water bath to cool the Peltier elements. Again, recent

innovations have enabled the use of air-cooled peltiers in the cell block.

The required temperature range is typically between 0 and 110 °C and along

with the inbuilt temperature monitoring and stirring provide the ability to

ramp temperatures of the sample during analysis as rapidly as possible.

4.8 Multi-component analysis

Multicomponent analyses using UV-visible spectra have been performed

for almost as long as single-component analyses. However, because

the techniques used in multicomponent analysis often gave incorrect

results they were not widely applied. A well-designed modern UV-Vis

spectrophotometer yields more precise data, and modern curve-fitting

techniques give more accurate results and—perhaps more importantly—

indicate when results are incorrect.

Principle of additivity

According to Beer’s law, absorbance is proportional to the number of

molecules that absorb radiation at the specified wavelength. This principle

is true if more than one absorbing species is present. All multicomponent

quantitative methods are based on the principle that the absorbance at

any wavelength of a mixture is equal to the sum of the absorbance of

each component in the mixture at that wavelength.

The simple approach to multicomponent analysis is based on

measurements at a number of wavelengths—equal to the number

of components in the mixture. The wavelengths chosen are usually

those of the absorbance maximum of each component. For calibration,

Figure 41. A two-component mixture with little spectral overlap.

In contrast, Figure 42 shows a simulated two-component mixture with

significant overlap of the spectra at the absorbance maxima.

With little spectral overlap With substantial spectral overlap

A’(x + y) = 1.1 + 0.0 = 1.1 A’(x + y) = 0.6 + 0.5 = 1.1

A’’(x + y) = 0.0 + 0.9 = 0.9 A’’(x + y) = 0.4 + 0.5 = 0.9

For a mixture of x and y where cx = cy = 1, the measured absorbances

should be:

If a 10 % error occurs in the measurement of A′

(x + y)

and A′′

(x + y)

, that is,

A′

(x + y)

= 0.99 (- 10 %) and A′′

(x + y)

= 0.99 (+ 10 %), the quantitative calculation

yields the results shown in Table 6:

Least squares method

The effect of random noise can be reduced by using additional spectral

information, that is, a series of data points can be used for quantification

instead of only two. In this so-called overdetermined system, a least

squares fit of the standard spectra to the spectrum of the measured

sample yields quantitative results (1,2). Figure 43 depicts a spectrum for

the two-component mixture shown in Figure 42 with a 10 % random error

at each measurement point.

Table 6. Comparison of multicomponent analysis results for examples with little and

substantial spectral overlap.

Little

spectral overlap

Substantial

spectral overlap

Component

Nominal

concentration

Calculated

concentration

% error

Calculated

concentration

% error

x 1 0.9 -10% 0.0 -100%

y 1 1.1 +10% 1.98 +98%

Table 7. Comparison of multicomponent analysis results from simple simultaneous

equations and least squares methods.

Using 210 and

230 nm only

Using 200–

240 nm

Component

Nominal

concentration

Calculated

concentration

% error

Calculated

concentration

% error

x 1 0.0 -100% 1.003 +0.3%

y 1 1.98 +98% 0.995 -0.5%

Figure 43. Mixture spectrum with 10 % random error at each wavelength.

With 21 data points (2 nm intervals over 200 to 240 nm), the quantitative

results from the least squares method have an error of < 1 % compared

with an error of approximately 100 % from the usual measurements at

two wavelengths, as shown in Table 7.

230 240220210200

0.5

1.0

0.0

Wavelength (nm)

Absorbance (A)

x y x + y

230 240220210200

0.5

1.0

0.0

Wavelength (nm)

Absorbance (A)

Figure 42. A two-component mixture with significant spectral overlap.

Instrumental requirements

Single-component quantification is normally performed by measuring

with the same instrument a standard or series of standards followed by

an unknown. This calibration process should eliminate instrumental bias,

making absolute wavelength accuracy and absolute photometric accuracy

relatively unimportant. On the other hand, photometric reproducibility is

essential for precise results. If measurements are performed only at the

absorbance maximum, wavelength reproducibility is also of little importance

because the rate of change of absorbance with wavelength is low. However,

if a wavelength on the side of the band is used, wavelength reproducibility

becomes particularly important. Finally, the instrumental linear range is

critical, as the calibration process relies on a linear relationship. Accurate

multicomponent analyses require excellent signal to noise performance,

especially if the simple simultaneous equations method is used. In the least

squares method, data from the sides of absorbance bands is incorporated

into the calculation, making excellent wavelength reproducibility essential as

well. Moreover, because more data is required, fast scanning is necessary

for productivity.

4.9 Software requirements

Specialized multicomponent software is available to help with the creation

of data models to analyse collected data. These software packages

can either be incorporated into the instrument software control and

reporting software or as stand-alone packages. Most UV-Vis systems

available can export data in a standard format that can be imported into

a multicomponent software package for processing.

References:

1. Kisner, H.; Brown, W.; Kavarnos, G.“Multiple analytical frequencies and

standards for the least-squares analysis of serum lipids. Anal. Chem. 1983,

55, 1703.

2. Maris M.; Brown, C.; Lavery, D. Nonlinear multicomponent analysis by

infrared spectrophotometry. Anal. Chem. 1983, 55, 1694.

3. Zwart, A.; van Kampen, E.; Zijlstra, W. Multicomponent analysis of

hemoglobin derivatives with a reversed-optics instrument. Clin. Chem.,

1984, 30, 373.

This method enables the analysis of more complex mixtures and of simple

mixtures of components with similar spectra. The residual from the least

squares calculation is a good indicator of how well the standard spectra

fit the sample spectra and is therefore a good indicator of the probable

accuracy of the results.

An example of multicomponent analysis is the quantification of five

hemoglobins in blood with minimum sample preparation (3). Figure 44

shows the absorption spectra of hemoglobin derivatives. This analysis

was previously performed using various analytical techniques, including

spectroscopy and titrations.

Figure 44. Absorption spectra of hemoglobin derivatives.

Other methods

Other statistical approaches to multicomponent analysis include the

partial least squares (PLS), principle component regression (PCR), and

multiple least squares (MLS) methods. In theory, these methods offer some

advantages over those described above, however the calibration process

can be much more complex.

Sample requirements

The simple simultaneous equations and least squares methods yield

accurate results only if calibration is performed using pure standards or

mixtures of standards for each component in the sample that contributes

to the UV-visible spectrum. The unknown sample must not have any

additional absorbing capacity.

650600550500

0.1

0.2

0.0

Wavelength (nm)

Absorbance (A)

Oxyhemoglobin

Carboxyhemoglobin

Sulfhemoglobin

Hemoglobin (pH 7.0-7.4)

Deoxyhemoglobin

absorbance: 1. characteristic of a substance to absorb light 2. Unit for light

absorbance, represented as A or Abs.

arc (lamp): Creates light by an electric or voltaic arc through an inert gas.

baseline: This is a measurement collected under the same parameters

as the sample measurement, but without the sample in place. A blank is

usually used for a baseline measurement as this allows the contributions of

the instrument, the solvent, the cuvette etc to be subtracted from the final

sample measurement.

blank: The solvent or substrate of the sample, without the absorbing

species. When measuring liquid samples, this will be the solvent (often

water) in a cuvette. The absorbance of the blank can then be subtracted

from that of the sample to determine the absorbance due purely to the

sample.

carotenoids: Chromophore linked to photosynthesis in some plants, algae

and some bacteria.

chromophore: Part of a molecule which absorbs light

CRM: abbreviation. Certified reference material. In reference to standards

supplied that have been certified to a primary standard for comparison.

cuvette: Commonly referred to as a cell, the cuvette is the container

that holds liquid samples. Cuvette are available in different volumes and

pathlengths. The cuvette material determines their optical transparency.

dispersion: In optical design refers to the ability of the optical device to split

light into its constituent wavelengths. E.g. white light on a prism creates a

rainbow effect through dispersion.

fluorescence: A form of luminescence and characteristic of some

molecules to absorb light at a frequency and emit short lived light of another

wavelength.

GMP: abbreviation. Good manufacturing practice. Commonly used in

pharmaceutical and other regulated manufacturing industries.

5. Glossary

holographic (optics): Holographic optics are created by etching an

interference pattern on an optic surface. Holographic optics can be used in

place of lenses, mirrors, and other optical devices. Their design makes them

easy to accurately replicates and small and light.

noise: In spectrophotometer terms, noise refers to the background

electrical signal contributed by the instrument itself. If this is too great,

it can overshadow the measurement signal, making it hard to differentiate

between the two signals. An easy way to think about this is when you are

looking at the stars from a city location, compared to a remote location.

The background light (‘noise’) contributed by the city lights makes it hard

to see the stars. In a remote location, there is little background light, so the

light from the stars can be easily seen.

peltier: A Peltier is a heating/cooling device operated by a thermoelectric

coupling. The device transfers heat from one side of the device to the other.

It can provide accurate temperature control of a sample.

pharmacopeia: Regulatory document listing pharmaceutical details and

required or recommended testing procedures for the pharmaceutical

industry.

phosphorescence: A form of luminescence related to fluorescence.

It is characteristic of some molecules to absorb light at a frequency and

emit a delayed, light of another wavelength.

photochemical (reactions): Chemical reaction caused by the absorption

of light.

photosensitivity: Sensitivity of a substance to react when exposed to light.

QA/QC: abbreviation. Quality control or quality assurance

qualitative (measurement): Measurement providing non-numerical

information about the sample, e.g. identification of a molecule in solution

quantitative (measurement): Measurement resulting in a numerical value.

e.g. concentration.

rare earth oxides: Holmium, didymium and samarium oxides are referenced

by standards organizations and pharmacopoeia to be used for wavelength

validity measurements.

reflection: Describes the path of light a sample which is deflected at the

angle of incidence.

scattering: Effect of light bouncing from a surface at random angles.

SOP: abbreviation. Standard operating procedure. Document written to

ensure measurement can be formed safely and repeatably.

spectrum: plural, spectra. Range of wavelengths. The electromagnetic

spectrum. Also refers to a usually graphical output of wavelength vs

intensity (or absorbance, as measured by a spectrophotometer).

SST: abbreviation. System suitability tests. Test to determine the system is

fit for purpose.

transmittance: Percentage of incident light that is transmitted through

a sample.

zero: This is equivalent to the 'Tare' function on a set of scales – it sets the

instrument reading to 0 Abs.

DE.8623263889

This information is subject to change without notice.

Published in the USA, July 23, 2021

5980-1397EN

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