Basics of UV Vis Spectroscopy: How It Works, Definition and Applications

UV Vis Spectroscopy: Essential Knowledge

Fundamentals, Instrumentation, Calibration, Color Scales and More

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UV Vis Spectroscopy
UV VIS spectrum
What is UV Vis Spectroscopy?

Absorbance/Transmittance Converter

=

Absorption of light according to Beer-Lambert Law
Scanning Spectrophotometer
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
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.

Array versus Scanning UV Vis Spectroscopy

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

Basics of UV Vis Color Measurement
Color Number
What is the color of this rose?

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

CIELab Color Measurement - UV Vis Spectroscopy

EBC

MEBAK Method 2.13.2, EBC Method 8.5, EBC Method 9.6

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)

 

Quality Control of Nucleic Acids
Cuvettes for UV Vis analysis

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

 

UV Vis Spectroscopy Cuvette

 

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.

UV Vis Spectroscopy in Food Industry
UV Vis Spectroscopy in Pharmaceutical Industry
UV Vis Spectroscopy in Cosmetics Industry
UV Vis Spectroscopy in Petrochemical Industry
UV Vis Spectroscopy in Chemical Industry
UV Vis Spectroscopy in Biotechnology

What Are the Different Types of Spectroscopy?

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?

Types of Transition in UV Region

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:

  1. statically, with partially transmitting mirrors or a similar device
  2. attenuating the beams using moving optical and mechanical devices

How to Analyze Solid Polymer Film Using UV Vis?

How to Analyze Solid Polymer Film Using UV Vis?

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?

What is 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.

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