Optics

and
Instrumentation

Name : Shobhit Jain

Roll no. : 101503211
Group : 1COE8
 Preface
This project is about the concepts of optics used in the instruments based upon
Beer-Lambert law, especially the spectrophotometer. This project helps in
understanding the proper use of optics in spectrophotometer.
First of all this project tells us briefly about the Beer-Lambert Law i.e. its
basic idea, instruments based on it, conditions for its validity, limitations and its
applications. This project helps the readers to get a very basic and brief idea of what
Beer-Lambert Law is and what is its importance in various spectroscopic
techniques.
This project also helps the reader to understand the design, mechanism and
working of the spectrophotometer and spectrometer. This project proves to be very
useful in understanding the concepts of optics used in the functioning of these
instruments.
 Acknowledgements
First of all I would like to thank my Applied Chemistry teacher Dr.Deeptiman
for helping me in preparing this project and providing me great support regarding
the material used in making this project report. I would also like to thank my
friends in helping me to create this project.

I would also like to thank the various sources, as mentioned in the

Bibliography section, which provided me with the relevant material required for
making this report.
 Contents
Topics Page No.
1. Beer-Lambert Law 1-3
(i) Introduction 1
(ii) Instrumentation 1
(iii) Applications 2
(iv) Validity 2
(v) Limitations 3

2. Spectrophotometry 4-6
(i) Design and mechanism 4
(ii) Spectrophotometer 4
(a) Design 5
(iii) Spectrometer 6
(a) Mechanism 6

3. HPLC 7

4. Conclusion 8

5. Bibliography 9

Beer-Lambert Law
Introduction:
Beer-Lambert Law (Beers law) or Beer-Lambert-Bouguer Law relates the
attenuation of light to the properties of material through which the light is
travelling. The law is commonly applied to chemical analysis.[1]
The Beer-Lambert Law is a linear relationship between absorbance and
concentration of the absorbing species. The Beer-Lambert Law is written as:
A = *b*c
Where A is measured absorbance, is the wavelength-dependent molar
absorptivity coefficient with units of M-1cm-1, b is the path length and c is the
analyte concentration.
Data are frequently reported in percent transmission (I/I o * 100) or in
absorbance [A = log(I/Io)]. [2]

Instrumentation:
Experimental measurements are usually made in terms of transmittance (T),
which is defined as:

T = I / Io

where I is the light intensity after it passes through the sample and Io is the initial
light intensity. The relation between A and T is:

A = -log T = - log (I / Io).

Absorption of light by a sample:

Modern absorption instruments can usually display the data as either transmittance,
%transmittance, or absorbance. An unknown concentration of an analyte can be
determined by measuring the amount of light that a sample absorbs and applying
Beer's law. If the absorptivity coefficient is not known, the unknown concentration
can be determined using a working curve of absorbance versus concentration
derived from standards. [2]

Applications:
The law is used widely in infra-red spectroscopy and near-infrared
spectroscopy for analysis of polymer degradation and oxidation (also in biological
tissue). The carbonyl group attenuation at about 6 micrometres can be detected
quite easily, and degree of oxidation of the polymer calculated.[1]

This is highly useful in many fields of medical testing, where it is applied to

determine, among other things, the concentration of a chemical or substance in
blood by analyzing how the blood sample absorbs light.[3]

Beer-Lambert law is also applied to the analysis of a mixture by

spectrophotometry, without the need for extensive pre-processing of the sample.
Examples include the determination of bilirubin in blood plasma samples. The
spectrum of pure bilirubin is known thus the molar absorbance is known. Generally,
it can be used to determine concentrations of a particular substance, or determine
the molar absorptivity of a substance.[4]

Validity:
Under certain conditions BeerLambert law fails to maintain a linear relationship
between attenuation and concentration of analyte. There are at least six conditions
that need to be fulfilled in order for BeerLambert law to be valid. These are:

1. The attenuators must act independently of each other.

2. The attenuating medium must be homogeneous in the interaction volume.

3. The attenuating medium must not scatter the radiationno turbidityunless

this is accounted for as in DOAS.

4. The incident radiation must consist of parallel rays, each traversing the same
length in the absorbing medium.

5. The incident radiation should preferably be monochromatic, or have at least a

width that is narrower than that of the attenuating transition. Otherwise a
 spectrometer as detector for the power is needed instead of a photodiode
which has not a selective wavelength dependence.

6. The incident flux must not influence the atoms or molecules; it should only
act as a non-invasive probe of the species under study. In particular, this
implies that the light should not cause optical saturation or optical pumping,
since such effects will deplete the lower level and possibly give rise to
stimulated emission.
If any of these conditions are not fulfilled, there will be deviations from Beer
Lambert law.[1]

Limitations:
The linearity of the Beer-Lambert law is limited by chemical and instrumental
factors. Causes of nonlinearity include:

Deviations in absorptivity coefficients at high concentrations (>0.01M) due to

electrostatic interactions between molecules in close proximity.

Scattering of light due to particulates in the sample.

Fluorescence or phosphorescence of the sample.

Changes in refractive index at high analyte concentration.

Shifts in chemical equilibria as a function of concentration.

Non-monochromatic radiation, deviations can be minimized by using a

relatively flat part of the absorption spectrum such as the maximum of an
absorption band.

Stray light.[2]
Spectrophotometry
Spectrophotometry is a method to measure how much a chemical substance
absorbs light by measuring the intensity of light as a beam of light passes through
sample solution. The basic principle is that each compound absorbs or transmits
light over a certain range of wavelength. This measurement can also be used to
measure the amount of known chemical substances.

Devices and mechanism:

Figure 1 illustrates the basic structure of spectrophotometers. It consists of a
light source, a collimator, a monochromator, a wavelength selector, a cuvette for
sample solution, a photoelectric detector, and a digital display or a meter. Detailed
mechanism is described below.

Figure 1: Basic structure of spectrophotometers[5]

Spectrophotometer:
A spectrophotometer measures either the amount of light reflected from a
sample object or the amount of light that is absorbed by the sample object.
Design:

Single beam spectrophotometer

In short, the sequence of events in a modern spectrophotometer is as follows:

1. The light source shines on the sample.

A fraction of the light is transmitted or reflected from the sample

2. The light from the sample is directed to the entrance slit of the
monochromator

3. The monochromator separates the wavelengths of light and focuses each of

them onto the photodetector sequentially.
There are two kinds of spectrophotometers: single beam and double beam. A double
beam spectrophotometer compares the light intensity between two light paths. One
path containing a reference sample. The other the test sample. A single beam
spectrophotometer measures the relative light intensity of the beam before and after
a test sample is inserted. A double beam machine makes comparison readings easier
and more stable. But a single beam machine can have measure a wider range of
light frequencies. Single beam machines have simple optical systems and are more
compact. When the spectrophotometer is built into another device (like microscopes
or telescopes) only single beam machines will work.
Many older spectrophotometers must be calibrated by a procedure known as
"zeroing." The absorbancy of a reference substance is set as a baseline value, so the
absorbancies of all other substances are recorded relative to the initial "zeroed"
substance. The spectrophotometer then displays % absorbancy (the amount of light
absorbed relative to the initial substance).
Spectrophotometers can also measure luminescence. For example, the machine can
shine ultraviolet light of one frequency on the sample. This will excite the sample
and make it glow. The detectors can then measure the light glowing from the
sample at a different frequency.[6]

Spectrometer:
It produces a desired range of wavelength of light. First a collimator (lens)
transmits a straight beam of light (photons) that passes through a monochromator
(prism) to split it into several component wavelengths (spectrum). Then a
wavelength selector (slit) transmits only the desired wavelengths.[5]

Mechanism:

The first step in this process is to direct light through a fiber optic cable into
the spectrometer through a narrow aperture known as an entrance slit. The slit
vignettes the light as it enters the spectrometer. In most spectrometers, the divergent
light is then collimated by a concave mirror and directed onto a grating. The grating
then disperses the spectral components of the light at slightly varying angles, which
is then focused by a second concave mirror and imaged onto the detector.
Alternatively, a concave holographic grating can be used to perform all three of
these functions simultaneously. This alternative has various advantages and
disadvantages, which will be discussed in more detail later on.
 Once the light is imaged onto the detector the photons are then converted into
electrons which are digitized and read out through a USB (or serial port) to a
computer. The software then interpolates the signal based on the number of pixels
in the detector and the linear dispersion of the diffraction grating to create a
calibration that enables the data to be plotted as a function of wavelength over the
given spectral range. This data can then be used and manipulated for countless
spectroscopic applications, some of which will be discussed here later on.[7]

HPLC
High-performance liquid chromatography (HPLC; formerly referred to as high-pressure liquid
chromatography), is a technique in analytical chemistry used to separate, identify, and quantify each
component in a mixture.

High-performance liquid chromatography (HPLC; formerly referred to as high-pressure liquid

chromatography), is a technique in analytical chemistry used to separate, identify, and quantify each
component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture
through a column filled with a solid adsorbent material. Each component in the sample interacts slightly
differently with the adsorbent material, causing different flow rates for the different components and leading to
the separation of the components as they flow out the column.

HPLC has been used for medical (e.g. detecting vitamin D levels in blood serum), legal (e.g. detecting
performance enhancement drugs in urine), research (e.g. separating the components of a complex biological
sample, or of similar synthetic chemicals from each other), and manufacturing (e.g. during the production
process of pharmaceutical and biological products) purposes.[1]

Chromatography can be described as a mass transfer process involving adsorption. HPLC relies on pumps to
pass a pressurized liquid and a sample mixture through a column filled with a sorbent, leading to the
separation of the sample components. The active component of the column, the sorbent, is typically a granular
material made of solid particles (e.g. silica, polymers, etc.), 250 micrometers in size. The pressurized liquid is
typically a mixture of solvents (e.g. water, acetonitrile and/or methanol) and is referred to as a "mobile phase".
Its composition and temperature play a major role in the separation process by influencing the interactions
taking place between sample components and sorbent. These interactions are physical in nature, such as
hydrophobic (dispersive), dipoledipole and ionic, most often a combination.

HPLC is distinguished from traditional ("low pressure") liquid chromatography because operational pressures
are significantly higher (50350 bar), while ordinary liquid chromatography typically relies on the force of
gravity to pass the mobile phase through the column. Due to the small sample amount separated in analytical
HPLC, typical column dimensions are 2.14.6 mm diameter, and 30250 mm length. Also HPLC columns are
made with smaller sorbent particles (250 micrometer in average particle size). This gives HPLC superior
resolving power (the ability to distinguish between compounds) when separating mixtures, which makes it a
popular chromatographic technique.

The schematic of an HPLC instrument typically includes a sampler, pumps, and a detector. The sampler brings
the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the
desired flow and composition of the mobile phase through the column. The detector generates a signal
proportional to the amount of sample component emerging from the column, hence allowing
for quantitative analysis of the sample components. A digital microprocessor and user software control the
HPLC instrument and provide data analysis. Some models of mechanical pumps in a HPLC instrument can
mix multiple solvents together in ratios changing in time, generating a composition gradient in the mobile
phase. Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or based on mass
spectrometry. Most HPLC instruments also have a column oven that allows for adjusting the temperature the
separation is performed at.[8]

. Conclusion
Thus I would like to conclude that the Beer-Lambert law is a very useful law
to understand the linear relationship between absorbance and concentration of the
absorbing species. In this project we also learn about various applications of Beer-
Lambert Law and also the instruments based on it such as Spectrophotometer and
Spectrometer. We also come to know various conditions and limitations of this law
such as:
 1. Deviations in absorptivity coefficients at high concentrations.
2. Scattering of light due to particulates in the sample.

Spectrophotometry is the modern method to measure how much a chemical

substance absorbs light by measuring the intensity of light as a beam of light passes
through sample solution. Spectrophotometer is the instrument which measures the
amount of light absorbed or gets reflected.

Spectrometer is the device which produces a desired range of wavelength of

light.

Thus this project helps us in understanding the mechanism of

Spectrophotometer and Spectrometer.

Bibliography
1. https://en.wikipedia.org/wiki/Beer%E2%80%93Lambert_law

2. http://life.nthu.edu.tw/~labcjw/BioPhyChem/Spectroscopy/beerslaw.htm
3. http://www.answers.com/Q/What_are_the_applications_of_beer-
Lambert_law

4. http://www.wikilectures.eu/index.php/Lambert-Beer's_law

5. http://chemwiki.ucdavis.edu/Physical_Chemistry/Kinetics/Reaction_Rates/E
xperimental_Determination_of_Kinetcs/Spectrophotometry

6. https://simple.wikipedia.org/wiki/Spectrophotometer

7. http://bwtek.com/spectrometer-introduction/

8. https://en.wikipedia.org/wiki/High-performance_liquid_chromatography