Rigorous analysis is vital for verifying the purity, concentration, and identity of drugs. Monitoring the stability of medications is also essential to ensure efficacy over time under varying environmental conditions.

Evaluates product safety and effectiveness by analyzing the photostability of UV filters, characterizing particles, and measuring color indices. It also detects adulteration and quantifies dyes and antioxidants to meet consumer expectations.

Characterizes crude oil, calculates asphaltene fractions, formulates aromatic content indices, determines sulfur content, and calculates solubility factors.

Determines chemical properties, assesses final product quality, studies polymer composition, qualifies water, determines purity and dyeing efficiency, analyzes photocatalytic degradation and pesticide residues.

Determines concentration and purity of nucleic acids and proteins, monitors microbial cell cultures, studies protein denaturation and kinetics, and analyzes biological samples like blood plasma and serum.

To read a UV/Vis spectrum, you analyze the plot of absorbance (or sometimes transmittance) versus wavelength. Key points to focus on include:

• Absorption Peaks: Identify the wavelengths where the absorbance is highest; these correspond to electronic transitions in the molecules.
• Peak Wavelengths (λmax): The wavelength(s) at which maximum absorbance occurs, characteristic of specific molecular structures or functional groups.
• Peak Intensity: The height of the absorption peaks indicates how strongly the substance absorbs at that wavelength, related to concentration and molar absorptivity.
• Baseline: Check the baseline absorbance in regions without absorption to assess instrument or sample issues.
• Shape of Spectrum: The shape and number of peaks can provide information on the types of electronic transitions and the environment of the molecules.

By interpreting these features, you can identify compounds, determine concentration, and study molecular properties.

UV/Vis spectroscopy typically covers the wavelength range from 190 nm to 780 nm. 

More specifically: 

• The ultraviolet (UV) region generally ranges from 190 nm to 390 nm.
• The visible (Vis) region generally ranges from 390 nm to 780 nm.

METTLER TOLEDO’s UV/VIS Excellence spectrophotometers extend further into the near-infrared region, reaching 1100 nm.

UV/Vis spectroscopy is important because it enables both qualitative and quantitative analysis of substances by measuring their absorption of ultraviolet and visible light.

This method helps determine the concentration of analytes, study chemical kinetics, assess purity, perform objective color measurements, and analyze molecular structures. Its applications cover a wide range of fields, including chemistry, biology, environmental science, and materials science, making it a versatile and essential tool for analysis.

UV/Vis spectroscopy measures the light absorbed by a sample to determine its concentration and identify compounds. This process involves the removal of light.

In contrast, fluorescence spectroscopy measures the light emitted by a sample after it absorbs light, usually at a longer wavelength. This technique focuses on the re-emission of light, providing much higher sensitivity for specific fluorescent molecules.

To determine the concentration of an unknown solution using UV/Vis spectroscopy, follow these steps:

1. Prepare a calibration curve: Measure the absorbance of a series of standard solutions with known concentrations at the wavelength of maximum absorbance (λmax) for the analyte.
2. Plot absorbance vs. concentration: Create a calibration curve by plotting the absorbance values against the known concentrations. According to Beer's Law, absorbance is directly proportional to concentration.
3. Measure the unknown sample: Record the absorbance of the unknown solution at the same λmax.
4. Determine the concentration: Use the calibration curve to find the concentration corresponding to the measured absorbance of the unknown sample.

This method relies on Beer-Lambert Law, which states that absorbance A=εbc, where ε is the molar absorptivity, b is the path length, and c is the concentration.

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 (λ_max). For example, benzene shows λ_max at 256 nm, whereas aniline shows λ_max at 280 nm. Hence, the NH₂ group acts as an auxochrome and causes the shift of λ_max to a larger value.

Diffraction gratings are generally better than prisms for splitting different wavelengths because:

Higher Spectral Resolution: Gratings can provide much higher spectral resolution due to their ability to produce multiple sharp diffraction orders, allowing finer separation of closely spaced wavelengths.

Linear Dispersion: The angular separation between wavelengths in a grating is more linearly related to wavelength, making it easier to analyze and calibrate spectra compared to the nonlinear dispersion of prisms.

No Material Dispersion Limitations: Prisms rely on material dispersion (variation of refractive index with wavelength), which can limit performance, especially in certain wavelength ranges. Gratings use interference effects, which are not limited by material properties.

Broad Wavelength Range: Gratings can work efficiently over a broader range of wavelengths, including ultraviolet and infrared, whereas prisms have absorption and dispersion limitations.

Overall, diffraction gratings provide more precise and versatile wavelength separation than prisms, which is why they are commonly used in spectrometers and optical instruments.

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.

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 device

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.

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.