The building blocks of UV Vis spectroscopy including Color Scales, Fundamentals, Instrumentation, and Calibration
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UV Vis spectroscopy is a scientific technique used to measure the amount of light that is absorbed or transmitted by a sample at different wavelengths of ultraviolet (UV) and visible (Vis) light.
The process involves shining a beam of UV Vis light through the sample and measuring the amount of light that passes through it. By analyzing the pattern of absorption and transmission of light, scientists can identify and quantify the components of the sample.
A unique relationship exists between the substance and its UV Vis spectrum when a substance absorbs the maximum light at a specific wavelength. This relationship can be used for:
Qualitative analysis, i.e., determining the presence of certain substances.
Quantitative analysis, i.e., determining the amounts of certain substances.
UV Vis spectrophotometry is commonly used in many fields of science, including chemistry, biology, and physics, to study the properties of materials and their interactions with light. It is also widely used in industry for quality control and analysis of materials such as drugs, food, and cosmetics.
This page will give you essential knowledge about UV Vis Spectroscopy and its applications.
Get your answers about the UV Vis fundamentals and applications in the following sections:
UV Vis spectroscopy is a type of absorption spectroscopy in which a sample is illuminated with electromagnetic rays of various wavelengths in the ultraviolet (UV) and visible (Vis) ranges. Depending on the substance, the UV or visible light rays are partially absorbed by the sample. The remaining light, i.e. the transmitted light, is recorded as a function of wavelength by a suitable detector. The detector then produces the sample's unique UV Vis spectrum (also known as the absorption spectrum).
To learn more about basics of UV Vis spectroscopy, download the METTLER TOLEDO guide “Spectrophotometry Applications and Fundamentals"
When light hits an object, it can be absorbed by the object, typically because the wavelength of the absorbed light corresponds to an electronic excitation in the object. The remaining light is transmitted, i.e. it passes through the object.
In a spectrophotometer the transmittance is measured by dividing the intensity spectrum of light transmitted through a sample (I) by the intensity spectrum of light transmitted through the blank (I0).
Absorbance/Transmittance Converter
=
Absorbance (A), also known as optical density (OD) is the amount of light absorbed by the object and can be expressed as follows
Transmittance (T)
To learn more about fundamental knowledge on UV Vis spectroscopy techniques, download the guide, “Spectrophotometry Applications and Fundamentals”.
What Is the Beer-Lambert Law?
The Beer-Lambert Law states that the amount of energy absorbed by a solution is proportional to the path length and concentration. Put simply, a more concentrated solution absorbs more light than a dilute solution does.
The mathematical statement of Beer's law is:
A = ϵ.d.c
Where ϵ = molar absorptivity, d = path length and c = concentration. Molar absorptivity is a unique physical constant of the sample that relates to the sample’s ability to absorb light at a given wavelength. ϵ has the unit as L·mol-1·cm-1.
To learn more about basics of UV Vis spectroscopy, download the METTLER TOLEDO guide, “Spectrophotometry Applications and Fundamentals”
What Is the Difference between Scanning and Array Spectrophotometers?
A UV Vis spectrophotometer is an instrument designed to measure the absorbance in the UV Vis region using the Beer-Lambert law. It measures the intensity of light passing through a sample solution in a cuvette and compares it to the intensity of the light before it passes through the sample.
The main components of a UV Vis spectrophotometer are a light source, a sample holder, a dispersive device to separate the different wavelengths of the light, and a suitable detector.
Scanning Spectrophotometer
Conventional scanning spectrophotometers work on the principle of taking consecutive transmittance measurements at each defined wavelength. The light is split into different wavelengths by a diffraction grating. A sample cuvette is placed between the diffraction grating and the detector.
Array Spectrophotometer
In an array spectrophotometer, the sample is illuminated by a continuum, i.e. all spectral components of light at once, thus it absorbs light of different wavelengths simultaneously. The transmitted light is then diffracted by a reflection grating. This instrumentation helps to acquire the UV Vis spectrum faster than it can be obtained using a traditional scanning spectrophotometer.
As compared to a scanning spectrophotometer, an array spectrophotometer has no moving parts.
To find out more, download “Array versus Scanning”. The white paper compares the two established UV Vis spectrophotometer setups and assesses their performance and benefits.
A spectrophotometry instrument must perform according to its specification for critical UV Vis measurements, especially in clinical, pharmaceutical or industrial quality control. Therefore, performance verification must be carried out regularly. Calibration results must also be recorded and stored.
Download “How Should UV Vis Labs Do Spectrophotometer Calibration” for insight into the importance of calibration relative to UV Vis chapter revisions found in the Ph. Eur. 10 and USP42 NF37.
Major Parameters to Be Calibrated for a UV Vis Spectrophotometer
The major parameters to be calibrated for a UV Vis spectrophotometer are shown in the following table.
Performance test
Certified reference material (CRM)
Instrument Test Parameter
Acceptance criteria
USP 42 NF 37
Ph. Eur. 10
Wavelength accuracy &
repeatability
Ho(ClO4)3: 4 % Ho2O3 in 10 % v/v HClO4
Blank: Air
14 wavelengths
(240 nm – 650 nm)
Xe: 2 wavelengths (260.6, 528.6 nm)
UV (200 – 400 nm): ± 1 nm
Vis (400 – 780 nm): ± 2 nm
(S.D.) < 0.5 nm
UV (< 400 nm):
± 1 nm
Vis (> 400 nm):
± 3 nm
Photometric
accuracy &
repeatability**
K2Cr2O7 in 0.001 M HClO4
Blank: 0.001 M HClO4
60 mg/L
0 A – 2 A,
235, 257, 313, 350 nm
For absorbance ≤ 1A
Accuracy : ± 0.010A
Repeatability:
S.D. ≤ 0.005 A
For absorbance > 1A
Accuracy: ± 1%
Repeatability:
S.D. ≤ 0.5%
Accuracy: ± 0.010 A or ± 1 %, whichever is greater
Nicotinic acid in
0.1 M HCl
Blank: 0.1 M HCl
12 mg/L
0.26 A – 1.6 A
213, 261 nm
Photometric linearity
K2Cr2O7 in 0.001 M HClO4
Blank: 0.001 M HClO4
6 – 200 mg/L, up to 3.0 A,
235, 257, 313, 350 nm
All measured filters fulfill photometric accuracy acceptance criteria
R2> 0.999
Nicotinic acid in
0.1 M HCl
Blank: 0.1 M HCl
6 – 60 mg/L, up to 2.5 A
213, 261 nm
Stray light according to procedure A
(SFRM)
1.2 % w/v KCl/H2O;
10 mm path length
Blank: 1.2 % w/v KCl/H2O, 5 mm path length
Amax at 198 nm
≥ 0.7 A
(NA)
Stray light according to procedure B (SWM)
1.2 % w/v KCl/H2O;
10 mm path length
Blank: H2O, 10 mm path length
Amax at 198 nm
≥ 2.0 A
≥ 2.0 A
Resolution
0.02 % v/v toluene in n-hexane
Blank: n-hexane/
n-heptane (Ph. Eur. 10)
Amax,269/Amin,267
>1.3
Levels are stated in the respective monograph
** No specification of Photometric Repeatability (Precision) in Ph. Eur.
S.D. - Standard deviation
3. The Science of Colors
Basics of Color Measurements
Colors make our world more interesting. When we see an object, the light reflected from the object enters our eyes and is collected by several types of photoreceptors in the retina. Depending upon photoreceptor sensitivity, different people may perceive the same color differently.
To accept the accuracy of a specific color universally, numerical values must be assigned. In short, measurement equipment such as spectrophotometers and colorimeters deliver color results as values to ensure color-determination accuracy and repeatability.
Spectrophotometers quantify color data by collecting and filtering wavelengths transmitted through a sample. A mathematical equation is applied to the spectral data to map the color onto a color scale.
A CIE (Commission internationale de l'éclairage) color scale is defined using three parameters: hue, chroma and lightness.
Hue is the dominant color of an object. Primary and secondary colors combined make hue.
Chroma, also known as saturation, describes how vivid or dull a color is.
Lightness is the luminous intensity of the color (whether it is dark or light).
Each CIE color system uses three coordinates to locate a color on a scale. The three primary color scales are Tristimulus CIE XYZ, CIE L*a*b*, and CIE L*u*v*.
For example, when a color is expressed in CIE L*a*b*:
L* defines lightness
a* denotes the red/green value
b* denotes the yellow/blue value
Using the CIE L*a*b* color scale, coordinates for the red of the pictured rose would be:
L* = 29.00, a* = 52.48, b* = 22.23
To learn more about the basics of color measurement, download the guide.
Providing an overall successful visual experience for consumers can influence the decision to buy. Therefore, color is important in the definition of brand identity and product consistency.
Different color scales are established to uniquely define a product according to industrial standards. These scales include:
Scale
Standard
Applications
Saybolt
ASTM D156, ASTM D6045
To determine if fuel (kerosene, gasoline, diesel, naphtha, etc.) is contaminated or has degraded in storage
APHA/Pt-Co/Hazen
ASTM D1209
Yellowness index used as a metric for purity checks in the water, chemical, oil, and plastics industries
Gardner
ASTM D1544/D6166, DIN EN ISO 4630-2
For testing products such as resins, fatty acids, varnishes and drying oils that have attained color through heating
CIELAB
DIN EN 11664-4, DIN 5033-3, 4630, ASTM Z 58.7.1 DIN 6174
Quality control for the flavor & fragrance and food & beverage industries
To measure color intensity and turbidity (haze) in EBC units of beer, malts, caramel, etc.
USP/EUP
USP-24 Monograph 631, EP method 2.2.2
Quality control of drugs
Hess-Ives
DGK test method F 050.2
Used to test chemicals and surfactant liquids (mainly in the cosmetics industry)
4. Microvolume Analysis Using a UV Vis Spectrophotometer
How to Perform Microvolume Analysis in UV Vis Spectroscopy?
A micro-volume spectrophotometer measures sample volumes as low as 1 µl. The concentration of nucleic acids in a sample is usually of the order of nano or microgram per milliliter.
Diluting such micro-volumes and getting accurate results is challenging. Therefore, microanalysis without dilution becomes important for downstream analysis of nucleic acids.
During analysis of nucleic acids the micro-volume sample is pipetted into the fine compartment on the pedestal surface. The light beam from the lamp source is guided by the fiber optics to the micro-volume platform. As the path length is reduced to the order of a millimeter, higher concentration of analyte can be analyzed precisely without multiple dilutions.
A micro-volume system uses fiber optic technology along with the inherent properties of the sample (such as surface tension) to retain the sample on the pedestal platform and determine the real-time absorbance of the samples at low volumes.
With these advantages, micro-volume analysis becomes an ideal choice for biomolecular analysis.
Attend the on-demand webinar “Maximize Workflow Accuracy through Good UV/VIS Practice in Nucleic Acid Analyses” for tips and tricks on microvolume analysis.
Quality Control of Nucleic Acids Nucleic acid quantification is an essential pre-analytical method for obtaining accurate and reliable results in many molecular biology assays such as Next-Generation Sequencing (NGS), Polymerase Chain Reaction (PCR), Real-Time PCR (quantitative PCR; qPCR), cloning and transfection.
Qualitative and quantitative control of nucleic acids can be performed by determining the purity and the concentration of nucleic acids.
DNA Analysis Methods A260 gives the correlation of the concentration of nucleotides and A280 gives that of the residual proteins. The amino acids tyrosine and tryptophan absorb at 280 nm and phenylalanine absorbs well at 260 nm. This allows calculation of the ratio A260/A280 for DNA purity using direct absorbance measurements. Good-quality DNA will have an A260/A280 ratio of 1.7–2.0
Protein Quantification Assays Different methods of total protein quantitation include A280, Bicinchoninic acid (BCA), Bradford, Lowry, Pierce and other novel assays. Proteins in solutions have maxima at 280 nm due to amino acids with aromatic rings and minima at around 220 nm due to the presence of peptide bonds.
The concentration is calculated by using the Warburg formula:
Protein concentration (mg/ml) = 1.55 X (A280 reading) – 0.76 X (A260 reading)
To learn more about DNA analysis with UV Vis spectroscopy, download the application editorial “260/280 Ratio: Indicator of Protein Contamination”.
What Are the Common Errors in UV Vis Measurements and Calibration?
Good accuracy and precision in UV Vis measurements can be attained by taking precautions to avoid errors. Typical error risks that should be accounted for when taking UV Vis measurements includes:
Spectral characteristics. Spectral characterization is performed during the calibration process. Major factors that may lead to erroneous results are wavelength accuracy, spectral bandwidth, stray light, and linearity.
Photometric characteristics. Photometric characteristics include the spectral sensitivity of the light source, the temperature-dependent sensitivity of the light source and detector, etc.
Optical interactions. The radiations of the lamp source may interact with the cuvette material, altering the intensity of sample absorbance. Such optical interactions can be avoided by selecting the right cuvette material.
Miscellaneous factors. Other factors such as temperature, line voltage fluctuations, vibrations, contamination, or heating of the sample by the photometer also affect measurements adversely.
GUVP™
Although not all errors can be avoided, errors can be minimized for better results.
'Download the Good UV/VIS Practice brochure “Trustworthy Results for UV/Vis Spectroscopy”.
Choosing the right cuvette involves selecting the right material and the correct size based on your sample and instrumentation.
The material of cuvette should have a sufficient transmission at a given wavelength. Light attenuation on the cuvette walls should not affect the outcome of an analysis. Glass cuvettes are not used in the UV region for analysis below 370 nm as they absorb the radiation. It is recommended to use them only in the visible region.
The chart that follows gives the usable transmission ranges of cuvettes:
Material
Theoretical transmission range (nm)
Far UV quartz
170-2700
Optical glass
320-2500
Near IR quartz
220-3800
UV silica
220-2500
UV plastic
220-900
Disposable PS cell
340-750
Disposable PMMA cell
285-750
The size of the cuvettes also affect measurement capabilities. The nominal radiation path length of the cuvette is 10 mm. Depending upon the samples, the length can be varied from 1 mm to 100 mm.
Standard cuvettes can be used for most of samples under study. Common absorption and fluorescence cuvettes have an external base of 12.5 mm x 12.5 mm, a height of 45 mm, and internal dimensions 10 mm x 10 mm.
Long path cuvettes (cuvettes having a pathlength more than 10 mm) are used when the sample is too dilute or the sample vaporizes or undergoes a chemical change during the measurement process.
Short path cuvettes (cuvettes having a pathlength less than 10 mm) are used when absorbance is high and dilution is difficult.
How to handle cuvettes correctly?
When handling cuvettes, always carry the cuvette using the frosted sides. Avoid touching the transparent optical surfaces with your fingers, as fingerprints can cause significant absorbance and thus impact accuracy.
Avoid using glass pasteur pipettes to fill the cuvette, as they could scratch the optical surface causing further interference. Pipettes with disposable plastic tips are recommended.
The cleanliness of cuvettes has a major effect on results, so we must consider this as a very important factor.
The following steps are recommended for deep cleaning of quartz cuvettes:
Soak the cuvettes in the cleaning solution.
Remove the cleaning solution and rinse the cuvettes with deionized water.
Wash the cuvettes with ethanol or acetone, except in cases of proteins (see table below).
Dry the cuvettes by wiping with a lint-free tissue followed by air drying or oven drying.
The chart that follows gives the usable transmission ranges of cuvettes.
Aqueous solutions
Organic molecules
Difficult to remove particles
Proteins
Heavy metals
Fatty acids
Cleaning solutions
Equal parts by volume of 3 M HCl and ethanol
Wash with 50% nitric acid
Concentrated HNO3 or 2 M HCl
Equal parts by volume of ethanol and 3 M HCl
Incubate at room temperature with trypsin
(Ethanol and acetone are not recommended for cleaning.)
Equal parts by volume of sulfuric acid 2 M and 50% deionized water
Aqua regia
Equal parts by volume of IPA and Deionized water
Soaking time*
10 minutes
10 minutes
30 seconds
Overnight
20 minutes
Wipe
*The soaking time stated in the table is rough estimation; however, it is only recommended that you soak cuvettes until stains/contaminants are removed.
Glass cuvettes can be cleaned by rinsing the cuvettes with acetone or ethanol, followed by rinsing with water. Air-drying is recommended.
Plastic cuvettes can be washed with deionized water several times. Washing plastic cuvettes with chemicals is not recommended.
Download Our Collection of Posters with Tips and Tricks to Keep Your Lab Instruments Clean
Good Practice for Using Micro Volume Spectrophotometers
Prepare the sample Instrument sensitivity may be low for diluted concentrations of biological samples. To increase the sensitivity of such samples, consider taking higher concentration of the sample. Micro volume measurements typically need 1-2 µl of sample volume. Use calibrated pipettes for taking the sample. Care must be taken that a homogeneous sample is prepared and taken for analysis.
Clean the micro volume platform Ensure that the micro volume pedestal surface and the the mirror are cleaned properly. Simply wipe off the surfaces with a lint-free tissue using deionized water. If using a buffer solution, detergents or a sticky sample, clean the surface multiple times before proceeding for the next sample.
Place the sample on the platform Slowly and steadily, place the sample onto the surface to avoid bubble formation.
Work within detection limits Know the instrument's lowest and highest detection limits and work within that range.
General Practices for Accurate UV Vis Measurements
Be careful while preparing the sample and pipetting it into a cuvette or onto a microvolume platform. The sample should be homogeneous.
If any suspended solid particles are present in the sample, the light may scatter. In such cases, filter the sample using a syringe filter.
Fill the sample in a cuvette considering the z dimension of the sample holder. This will ensure that the light is passing through the sample. z-dimension is the distance from the bottom of a cuvette to the height at which the light beam passes through the sample.
Before every measurement, clean the cuvette with a lint-free tissue. Use new tissue every time.
Use the same solvent/solution buffer that was used in the sample preparation as a blank.
6. What Are the Applications of UV Vis Spectroscopy in Various Industries?
UV Vis spectroscopy is a versatile analytical technique with a wide range of applications in various industries. Some of the significant applications of UV Vis spectroscopy in different industries are:
Food & Beverage
UV Vis spectroscopy determines the quality and composition of food and beverage products. It can be used to analyze the color (e.g., wine), flavor, and aroma of food products, as well as to detect the presence of contaminants or adulterants.
Pharmaceutical
UV Vis spectroscopy analyzes the purity, concentration, and identity of drugs and other pharmaceutical products. It is also used to monitor the stability of pharmaceuticals over time.
Cosmetics
Evaluation of photostability of agents for formulations, particle characterization of UV blocking agent, assessing the color index, detecting adulteration (perfume industry), study of optical properties, quantification of dyes, antioxidants, etc.
Petrochemical
Characterization of crude oil, calculation of asphaltene fractions, formulation of indices for aromatic content, quality of crude oil gravity, sulfur content, calculating Hildebrand solubility factor.(Extended to bitumen, heavy & shale oils and oils from fluid catalytic cracking, coking, or coal liquefaction)
Chemical
Determining chemical properties, final quality assessment of finished product, study of polymer composition, qualification of waste water, determination of purity & dyeing efficiency, photocatalytic degradation of polymers/dyes, pesticides residues in soil or water
Biotechnology
Concentration and purity of nucleic acid, proteins (A280, BCA, Biuret, Bradford Lowry, OD 600), microbial cell culture measurements, denaturation of protein, kinetic studies (enzymatic activity), biological samples such as blood, plasma, serum, etc.
METTLER TOLEDO offers a wide range of validated application methods. Find the application that best suits your needs through our online search engine.
The different spectroscopic techniques are mainly differentiated by the radiation they use, the interaction between the energy and the material, and the type of material and applications they are used for. The spectroscopic techniques commonly used for chemical analysis are atomic spectroscopy, ultraviolet and visible spectroscopy (UV Vis spectroscopy), infrared spectroscopy, Raman spectroscopy and nuclear magnetic resonance.
Type of Spectroscopy
Type of Radiation
Interactions
Wavelength
ϒ-ray spectroscopy
ϒ-rays
Atomic nuclei
< 0.1 nm
X-ray fluorescence spectroscopy
X – rays
Inner shell electrons
0.01 – 2.0 nm
Vacuum UV spectroscopy
Ultraviolet (UV)
Ionization
2.0 – 200 nm
UV Vis spectroscopy
UV Vis
Valance electrons
200 – 800 nm
Infrared & Raman spectroscopy
Infrared
Molecular vibrations
0.8 – 300 mm
Microwave spectroscopy
Microwaves
Molecular rotations
1 mm to 30 cm
Electron spin resonance spectroscopy
Electron spin
Nuclear magnetic resonance spectroscopy
Radio waves
Nuclear spin
0.6 – 10 m
What Are the Different Molecular Interactions in the UV Region?
The absorption of UV light results in electronic transitions from lower energy levels to higher energy levels. Absorption of ultraviolet radiation in organic molecules is restricted to certain functional groups (chromophores) that contain valence electrons of low excitation energy. The molecular transitions/interactions that take place due to UV absorption are:
π- π* (pi to pi star transition) – bonding to anti-bonding orbital
n - π* (n to pi star transition) – non-bonding to anti-bonding orbital
These transitions need an unsaturated group in the molecule to provide the π electrons.
σ (bonding) to σ* (anti-bonding) transitions require higher energy and therefore cannot be detected using UV Vis spectroscopy.
How Do Functional Groups Affect the Spectra?
Consider a functional group containing atoms with one or more lone pairs of electrons that do not absorb ultraviolet/visible radiation. However, when this functional group is attached to a chromophore, it alters the intensity and wavelength of absorption. This phenomena is called an auxochrome or a color-enhancing group.
The presence of an auxochrome causes the position shift of a peak or signal to a longer wavelength, which is called a bathochromic or red shift. The functional groups contributing to bathochromic groups are substituents such as methyl, hydroxyl, alkoxy, halogen and amino groups.
The auxochrome that causes position shift of a peak or signal to shorter wavelength is called a hypsochromic or blue shift. Actually, the combination of chromophore and auxochrome behaves like a new chromophore having a different absorption maxima (λmax). For example, benzene shows λmax at 256 nm, whereas aniline shows λmax at 280 nm. Hence, the NH2 group acts as an auxochrome and causes the shift of λmax to a larger value.
What Is the Difference between Spectral Bandwidth and Resolution in UV Vis Spectroscopy?
The spectral bandwidth (SBW) of a spectrophotometer is related to the physical slit-width and optical dispersion of the monochromator system. Resolution is the ability of an instrument to separate light into finite, distinct wavelength regions and to distinguish each finite region. Spectral bandwidth is typically used for scanning instruments, whereas resolution is typically used for array instruments.
For most pharmacopeia quantitative purposes, a spectral bandwidth of less than 2 nm is sufficient and the acceptance criteria for the ratio is 1.3. Spectral resolution can be used for comparison with spectral bandwidth.
The table shows the resolution of METTLER TOLEDO's UV/VIS Excellence spectrophotometers, which is measured using toluene in hexane, and the equivalent SBW.
Instrument
Spectral resolution
Equivalent SBW (nm)
UV5
> 1.5
< 2.0
UV5Bio
> 1.5
< 2.0
UV5Nano
> 1.7
< 1.5
UV7
> 1.9
≤ 1.0
What Are the Different Light Sources Used in a UV Vis Spectrophotometer?
The best light source would be one that provides good intensity with low noise across all ultraviolet and visible wavelengths and offers stability over a long period. There is a range of light sources which are commonly employed as mentioned below.
Light Source
Wavelength Range
(nm)
Region
Lifetime
Tungsten filament lamp
350 – 2500
VIS + IR
3,000 hr
Deuterium arc lamp
190 – 400
UV
1,000 hr
Hydrogen lamp
190 – 400
UV
1,000 hr
Xenon flash lamp
190 – 1100
UV + VIS + NIR
5,500 hr*
* Corresponds to 50 Hz flashes at constant operation
How Is Diffraction Grating Better Than a Prism?
Prisms and diffraction grating are typical dispersive elements. A prism achieves dispersion due to the difference in the material refractive index according to the wavelength. However, a diffraction grating uses the difference in diffraction direction for each wavelength due to interference. Both prisms and diffraction gratings can spread light spectra into many colors for analysis. However, a diffraction grating is less sensitive to the color of the light and can be made to spread colors over a larger angle than a prism. The glass in a prism is clear to visible light, but it absorbs and blocks light in the infrared and ultraviolet part of the spectrum. A diffraction grating with a few hundred lines per inch can deflect light in the middle of the visible spectrum by at least 20 degrees. The deflection angle of a glass prism is generally much smaller than this.
Which Inorganic Compounds Can Be Measured by UV Vis Spectroscopy?
Molecules can be analyzed using UV Vis spectroscopy if they possess any functional group or conjugation, or if they produce a color complex. As inorganic compounds do not contain any functional group or conjugation, the common method for analyzing them is by reaction with a suitable compound. This produces a color complex whose absorbance can be photometrically measured in the visible region and correlated with its actual concentration. For example, iron is commonly analyzed by a reaction with 1, 10-phenthroline to produce a red color complex. The absorbance of the complex is measured at 570 nm to estimate iron concentration.
How Do Single Beam and Double Beam Spectrophotometers Differ?
The main difference between a single beam and double beam spectrophotometer follows.
Single beam spectrophotometer: A single beam from the light source passes through the sample
Double beam spectrophotometer: The light beam from the light source is split into two parts: one part goes through the sample, and the other part passes through the reference
Beam splitting in a double beam spectrophotometer is achieved in two ways:
statically, with partially transmitting mirrors or a similar device
attenuating the beams using moving optical and mechanical devices
How to Analyze Solid Polymer Film Using UV Vis?
The analysis of a solid sample is performed mainly by estimating its absorbance, transmittance and reflectance. Common parameters determined for solid polymers include % transmittance, cutoff wavelength, and yellowness index. The sample is mounted on a holder specifically designed for solid samples and readings are taken in the same manner as they are for liquid samples. A solid sample holder enables measuring of solid samples such as films or glass.
Does Temperature Affect UV Vis Analysis?
Temperature affects absorbance values. Different solvents undergo different interactions at different temperatures. Solution parameters that change due to temperature changes are:
Rate of reaction. The rate changes when temperature is elevated. This can cause a change in the activity of the sample. Enzymatic/biomolecular reactions are very sensitive to temperature.
Solubility of a solute. Solubility is affected with variations in temperature. Poor solubility may result in imprecise absorption.
Expansion or contraction of the solvent. This may lead to a change in the concentration of the solution and affect the absorbance, as absorbance is linearly related to concentration.
Schlieren effect. This effect may occur with temperature changes, leading to a series of convective currents which may change the true absorbance.
Optical performance parameters such as photometric noise, wavelength accuracy/repeatability, photometric repeatability and stray light are not influenced by temperature within a range of 10 – 40 °C.
Whereas, optical parameters like photometric resolution (toluene/hexane ratio) and photometric accuracy wavelengths (K2Cr2O7 in HClO4) show a temperature dependency ranging from 0.014 to -0.034/unit within 10 – 40 °C.
Temperature control for UV Vis spectrophotometry can be achieved using high-performance thermostating systems like CuveT and CuvetteChanger. Learn more here.
What Is Stray Light?
Stray light is defined as light that reaches the detector which is not from the instrument's light source and does not follow the optical path, causing a deviation at the correponding wavelength. Therefore, the light intensity measured by the detector is higher than it actually should be. Conversely, this also means that the measured absorbance is lower than the true absorbance because it is reduced by the contribution of stray light. This effect is more prominent at higher absorbance values (high sample concentrations).
Download the whitepaper to learn more about the origin and accurate measurement of stray light:
Why Is the Sample Compartment in UV Vis Array Spectrophotometers Open?
The sample compartment in UV Vis array spectrophotometers is open due to the fact that array instruments use reverse optics and the simultaneous detection of all wavelengths of the spectrum.
Reverse optics: The light is diffracted after it has gone through the sample. Due to this, only a small fraction of the external ambient light contributes to the signal in a given wavelength region.
Simultaneous detection: Using an array detector which provides 2048 light intensity signals at the same time, full spectrum is recorded within one second. Because the measurement is very fast, the effect of ambient light is significantly reduced.
Applications
Download the specific application guides for UV Vis spectroscopy below
