Spectrophotometer

UV-Vis Spectrophotometer is widely used instrument in Lab analysis. This instrument works on a principle of light detection in UV and Visible range. Most of HPLC Detectors are UV and Visible light detector. To understand UV and Visible light let’s understand Electromagnetic spectrum.

Concept of light
One of major difference between various compound is, its color. Such as Tree leaf has chlorophyll, which is green and Apple is Red. Here one question arises, what causes object to be appearing colored?

Sunlight comprises a broad range of light, which includes UV, Visible, and Infrared etc. It forms an Electromagnetic spectrum. Electromagnetic spectrum interacts with matter. Visible light is a part of Electromagnetic spectrum, that’s why matter appearing colored. Human eye can identify visible light colors and their combination colors. So we can say that our Eye is working as a spectrometer. Some part of Electromagnetic spectrum is shown in below (Image A) with some parameter like Frequency, wavelength.

Light is measured by Wavelength or Frequency. In terms of light, Frequency can be defined as number of wave passes through a given point per unit time (Generally number of cycle per second). Its unit is Hertz (Hz). 

Wavelength can be defined as distance between two adjacent Peaks in one cycle. See below Image B. For illustration purpose, sinusoidal waves are shown.

Wavelength and frequency can be related by equation: c = f λ

Where c = speed of Light in vacuum (3 x 10⁸ m/s)

F=frequency and λ = wavelength

Also λ = c/f

If light falls on Object, then what will happen? 

• All light will be transmitted through Object. It results colorless object. For example: Glass.

• All light will be reflected from Object. Result will appear as white light.

• From all light, some part of light will be absorbed and some will reflected. It will result detection of reflected color by our eyes.

• All light will be absorbed by object. As a Result, object will appear as black.

Visible Color detection is based on complementary colors. Why Apple is Red color? Apple does not contain Red color, but it reflects Red color and absorbs blue-green color. So our eye identifies it as Red color. 

If some objects absorbs all color then they appears as Black, if object reflects all color then appear as White. Complementary color wheel (Means Two colors, when mixed in proper combination with each other will produce white light) is shown below.

As per color wheel, if sample absorbs wavelength of 625 nm to 800 nm, then it will appear as green color. As Green is the complementary color for Red.

Spectroscopy has two types: Atomic and Molecular.

• To know only Atom properties like nature and amount, Atomic spectroscopy is used.

• To know structure of matter, its chemical function, molecular spectroscopy is used. It is all about electromagnetic waves interaction with matter. 

Here, we will discuss widely used Molecular spectroscopy.

Spectrophotometer:
In Spectrophotometer light falls on sample, sample molecules absorbs light and unabsorbed light is reaches to Detector and get detected.

Object is made from molecules. A molecule is made from an Atom. For example: CH₄ is methane. It is molecule, where as C is Carbon atom and H is Hydrogen Atom. 

When light falls on object, some or all portion of light is absorbed by object molecules. Some portion is transmitted through object. Light absorption by object depends on molecule structure. If compound has conjugated more multiple bonds then it will appear as visible. Conjugated bond means single bond, double bond, single bond, double bond continuously changing structure. See below figure.

When light absorbed by compound, its ground state electron moves to higher level or excited state. It depends on energy level of excited state. 

If energy level of excited state is higher than ground state, then more energy is required to move electron from ground state to excited state and if energy level of excited state near to ground state, then less energy is required to move electron from ground state to excited state. 

For Conjugated system, lower energy is required to excite electron, as ground state and excited state is closer in conjugated system compared to non conjugated system. It means lower energy light is absorbed by conjugated system. For less conjugated system, high energy is required to excite Electron. In visible spectrum all different light has different energy. Visible light spectrum has lower energy as per equation “E= hv”. Description is given below.

Here energy and Frequency is also related to each other by equation: E=hv

Where h = Planck constant

E = Energy and v = frequency

Wavelength as per Electromagnetic spectrum give above.

We have seen that c = f λ;  So f = c / λ

So E = hv = h x (c/ λ) = 1 / λ; as h and c is constant So, E α 1 / λ

As Wavelength decreases, Energy increases. This energy can cause to move electron into excited state.

There is a group of molecule which has a structure to give absorption in ultra violet region which is called as Chromophoric Group. Sample molecule has electron of certain energy level. Photons of light interact with sample molecule electron. Photon with right wavelength (energy) will excite electron and moves electron into excited state. Means sample molecule is absorbing energy. Unabsorbed (remaining) energy (Light) is transmitted to Detector. UV light range has low wavelength so Energy is more, this more energy cause Electron to move into excited state.

Basic principle of spectrophotometer: 

Spectrophotometer consists of two main parts, one is spectrometer which produces light of selected wavelength, and another one is photometer, which measures the intensity of light. Light generated from Light source (UV or Visible or both) passes to Grating and is diffracted to different colors. This is same fundamental as white light diffraction done in Prism. Visible spectrum can be separated using Prism from white light. Visible spectrum is shown in below Image C.

Grating is connected with stepper motor type mechanism. (This mechanism is called as monochromator, which convert source light into selected wavelength light and passes to slit.) Grating motor moves in such a way that selected wavelength light goes to entrance Slit. This light passes from sample. Sample is filled in Cuvette. Cuvette is rectangle small tube made of good quality glass or quartz. Sample absorbs some light and remaining light passes to Detector. This is very basic idea to understand Spectrophotometer. There are some additional optical component is used in Optical unit. To increase performance various different configuration is used in Spectrophotometer. Basic Spectrophotometer diagram is shown below Image D. Spectrophotometer diagram shown in below image is not exact design. It is to understand basic only. Original design is more complex.

 

Detector converts light intensity into electrical signal. This signal is very small, so it is amplified and given to recorder. Recorder is usually a computer which has Data acquisition software installed. Finally software gives recorded chart output as absorbance versus wavelength. This chart is called Spectra. It looks like below.

Here as per above Image E, selected wavelength light passes from sample. Every solvent, used in chromatography has a cut off wavelength. For selected solvent, below cutoff wavelength, solvent itself absorbs all light. So it is required to select proper wavelength.

Let’s say Incident light (Light signal before sample) = Io

Light signal after passing sample= I

So Transmittance = I / Io, and percentage (%) T = (I/Io) x 100

When incident light passes from cuvette, molecule of sample will absorb energy of incident light and moves to excited state. Transmitted Light Wavelength versus absorbed Energy is plotted on a graph. This graph is called Spectra. If the sample compound does not absorb light, for given particular wavelength, then transmitted light from sample becomes equal to Incident Light. 

That is I = Io. So no absorbance of energy by sample compound. 

So Transmittance (T) = 1

Beer-Lambert law is used to know absorbed light. This law states that, absorbed light depends on Path length (length of light path through sample), Sample concentration and extinction coefficient.

Absorbance or O.D = ecl

Transmittance and absorbance can be related as:

A = -log T = log(1/T) =ecl

e= concentration, co-efficient, l = path length