Month: May 2021

Ultra Violet Visible spectroscopy Principles


 UV-Visible spectroscopy is a mature and well-established analytical technique used extensively in many industry sectors including Environmental Analysis, Pharmaceutical Testing, Food and Beverage Production etc.

Spectroscopy is the measurement and interpretation of electromagnetic radiation absorbed or emitted when the molecules or atoms or ions of a sample moves from one energy state to another energy state.

UV spectroscopy is type of absorption spectroscopy in which light of ultra-violet region (200-400 nm) is absorbed by the molecule which results in the excitation of the electrons from the ground state to higher energy state.


UV-Visible Spectroscopy is based on the Lambert-Beer principle which states that the absorbance of a solution (A) is directly proportional to its path length (l) and its concentration (c) when the wavelength of the incidence light remains fixed.

Basically, spectroscopy is related to the interaction of light with matter.

As light is absorbed by matter, the result is an increase in the energy content of the atoms or molecules.

When ultraviolet radiations are absorbed, this results in the excitation of the electrons from the ground state towards a higher energy state.

Molecules containing π-electrons or non-bonding electrons (n-electrons) can absorb energy in the form of ultraviolet light to excite these electrons to higher anti-bonding molecular orbitals.

The more easily excited the electrons, the longer the wavelength of light it can absorb.

The absorption of ultraviolet light by a chemical compound will produce a distinct spectrum which aids in the identification of the compound.

If there is no absorption of the light passing through the solution, the transmittance is 100%.


The UV-Visible Spectrophotometer is the analytical instrument used for the UV-Vis spectroscopic analysis. Spectrophotometers are available in different configurations however most can be categorized into either single beam, split beam or double beam types depending on the design of their optical system. Such types of instrument comprise the following components in their constructions:

1. Light Source :

  • Tungsten filament lamps and Hydrogen-Deuterium lamps are most widely used and suitable light source as they cover the whole UV region.
  • Tungsten filament lamps are rich in red radiations; more specifically they emit the radiations of 375 nm, while the intensity of Hydrogen-Deuterium lamps falls below 375 nm.

2. Monochromator :

  • Monochromators generally is composed of prisms and slits.
  • Most of the spectrophotometers are double beam spectrophotometers.
  • The radiation emitted from the primary source is dispersed with the help of rotating prisms.
  • The various wavelengths of the light source which are separated by the prism are then selected by the slits such the rotation of the prism results in a series of continuously increasing wavelength to pass through the slits for recording purpose.

3. Cell Compartment :

  • One of the two divided beams is passed through the sample solution and second beam is passé through the reference solution.
  • Both sample and reference solution are contained in the cells.
  • These cells are made of either silica or quartz. Glass can’t be used for the cells as it also absorbs light in the UV region.

4. Detector :

  • Generally two photocells serve the purpose of detector in UV spectroscopy.
  • One of the photocell receives the beam from sample cell and second detector receives the beam from the reference.
  • The intensity of the radiation from the reference cell is stronger than the beam of sample cell.

5. Signal Processing System :

  • Most of the time amplifier is coupled to a pen recorder which is connected to the computer.
  • Computer stores all the data generated and produces the spectrum of the desired compound.

This absorption spectroscopy uses electromagnetic radiation between 190 and 800 nm and is divided into two regions

1. UV (190–400 nm)- Deuterium lamp

2. Visible (400–900 nm) – Tungsten lamp

Because the absorption of UV or visible radiation by a molecule leads to transition among electronic energy levels of the molecule, it is also often called electronic spectroscopy.

UV-Visible Analysis is Suitable For,

  1. Analytes that can be dissolved in solvents like water, ethanol and hexane.

  2. The analyte need to absorb UV or Visible light.

  3. With UV /Vis we can do quantitative measurements a single analyte in solution(or more than one analytes om solution provided that do not interfere with each other).

Not Suitable For,

1. Analytes that have a photochemical reaction at the wavelength range of interest.

2. Partially dissolved, unclear or colloidal samples.

The UV-Visible spectrum shows the absorbance of one or more sample component in the cuvette when we scan through various wavelengths in the UV/Vis region of the electromagnetic spectrum.

System suitability in HPLC Analysis

System suitability is to prove that system is working perfectly before the analysis on HPLC, GC, TOC analyser or any other system. It is required to done before every sample analysis. HPLC, short for High-performance liquid chromatography is a technique used for separating the components in a mixture.

HPLC chromatographic technique is used in pharmaceutical industries for analysis. System suitability testing limits are acceptance criteria that must met before starting the analysis.

There are some System suitability parameters which can be used to check the system before starting the sample analysis are listed below.

1.    Retention time

2.    Resolution

3.    Repeatability

4.    Plate Count

5.    Tailing Factor

6.    Signal-to-noise ratio

7.    Pressure

Retention Time:

In liquid chromatography and gas chromatography, the retention time, tR, is defined as the time elapsed between the injection of the sample and the appearance of the maximum peak response of the eluted sample zone. tR may be used as a parameter for identification. Chromatographic retention times are characteristic of the compounds they represent but are not unique. Coincidence of retention times of a sample and a reference substance can be used as a partial criterion in construction of an identity profile but may not be sufficient on its own to establish identity. Absolute retention times of a given compound may vary from one chromatogram to the next.

 Resolution (Rs):

Resolution is a measure for the ratio of the distance of two adjacent peak maxima and their widths. For complex sample mixtures Rs should be determined for the critical pairs of components to characterize their separation.

Resolution is calculated by following formula,

RS = 2(tR2 − tR1) / (W1 + W2)

Where, tR2 and tR1 are retention times of two compounds, W2 and W1 are the corresponding widths at the bases of the peaks obtained by extrapolating the relatively straight sides of the peaks to the baseline.


Replicate injections of a standard preparation are used to demonstrate the system performance when it gets exposed to some specified column usage, environment, and plumbing conditions. Data from five or six replicate injections are used if requirement of relative standard deviation is less than 2%.

Plate Count/ Column Efficiency:

The Column Efficiency is measured by following formula,

N = 16(tR/W)2

Where tR is the retention time of the substance, and W is the peak width at its base, obtained by extrapolating the relatively straight sides of the peak to the baseline. The value of N depends upon the substance being chromatographed as well as the operating conditions, such as the flow rate and temperature of the mobile phase or carrier gas, the quality of the packing, the uniformity of the packing within the column, and, for capillary columns, the thickness of the stationary phase film and the internal diameter and length of the column.

Tailing Factor/Symmetry Factor:

Tailing Factor is calculated by following formula,

AS = W0.05/2f

where W0.05 is the width of the peak at 5% height and f is the distance from the peak maximum to the leading edge of the peak, the distance being measured at a point 5% of the peak height from the baseline.

Signal-to-noise ratio:

This parameter is used for the lower-end calculation of the performance of the system. 

Noise: It is measured between two specific lines that bracket the baseline. 

Signal: It is measured starting from the baseline’s middle and ending to the peak’s top.

Once calculating both these factors, the ratio can be measured by dividing the signal value by the noise value. With this, generally, the noise value has to be reduced using one of the following methods:

  • Signal Averaging
  • Reagent and Solvent Purity
  • Column Flushing and Sample Clean-Up
  • Temperature Control
  • Additional Pulse Damping and Mixing

The signal-to-noise ratio (S/N) is a useful system suitability parameter. The S/N is calculated as follows:

S/N = 2H/h

where H is the height of the peak measured from the peak apex to a baseline extrapolated over a distance ≥5 times the peak width at its half-height; and h is the difference between the largest and smallest noise values observed over a distance ≥5 times the width at the half-height of the peak and, if possible, situated equally around the peak of interest

System Pressure:

System suitability tests must be performed under controlled pressure limit. Monitor the pressure variation throughout the analysis.

Conclusion: The above mentioned system suitability parameters are not must. These parameters and acceptance criteria are performed during the method validation and fixed based on the method development outcome results. But system suitability should meet the acceptance criteria before starting the sample analysis.