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Reliance Industries Ltd. Hz Basics of Spectrophotometry Page No.
1 of 24
Module No. : RELQQ009 Rev. : 00 Date: 01.01.2005
TRAINING MODULE
ON

BASICS OF
SPECTROPHOTOMETRY
PeIiunce Industries Limited
{Huziru Munufucturing Division}

Prepured by: , , PATL Approved by:
Reliance Industries Ltd. Hz Basics of Spectrophotometry Page No. 2 of 24
2.0 Content
Sr. No. Contents Page no.
1.0 Cover page. 1
2.0 Content 2
3.0 Introduction 3
4.0 Brief Theory of UV-VIS 4
4.1 Lambers Law 7
4.2 Beers Law 8
5.0
Principal of UV-VIS spectrophotometer
9
5.1
Wavelength
9
5.2
Absorptivity
11
5.3
Photometric accuracy
12
6.0
Quantitative Analysis
13
6.1
Scope
13
6.2
Light Scattering
13
6.3
Solvent Evaporation
13
6.4 Sample decomposition 13
6.5
Fluorescence
14
6.6 Temperature 14
6.7 Sample cell 14
6.8
Complementary Color
14
6.9
UV VIS Spectrum
15
6.10 Transmittance and Absorbance
16
6.11 Solvent properties
17
6.12 Solvent Transmittance
18
7.0 Instrumentation
19
7.1 Light Source
19
7.2 Monochromator
20
7.3
Z0
7.4 Type of UV Spectrophotometer
Z1
7.5 Calibration of UV VIS Spectrophotometer
Z1
7.6 Lamp Optimization
Z1
7.7 Alignment of Cell holder
ZZ
7.8 Minimum volume application
ZZ
7,9
Daily care of Instrument
Z3
7,10
8VH DQGFDUHRIFHOO
Z3
8,0
5HIHUHQFH
Z4
3.0 INTRODUCTION
Before invention of spectrophotometer, visual color comparator was used for color solution.

The color is usually due to the formation of a colored compound by the addition of an

appropriate reagent or it may be inherent in the desired constituent itself. The intensity of

the color may then be compared with that obtained by known amount of the substance in the

Same manner. Colorimetric is mainly concerned with determination of concentration of a

Substance by measurement of relative absorbance of light with respect to a known

Concentration of the substance. In visual colorimetry, natural or artificial white light is

generally used as a light source & determination are made with colorimeter or color

comparator. Colorimetric analysis is a special branch of photometric analysis. The

instrument used is known as colorimeter or color comparator. When the eye is replaced by a

photo-electric cell, the instrument termed a photoelectric colorimeter. It has increase the

accuracy and man to man variation was omitted. With the invention of Spectrophotometer

new field added. When white light passes through filter, i.e. passes through colored plates of

glass or gelatin, which transmitting only a specific spectral region & radiation are chosen

possessing a band with of less then 1nm. It is known as Spectrophotometer and the detector

known as photometer. The combination of photometer & spectrometer known as

Spectrophotometer. When the amount of light of a specific wave length absorbed by a

colored solution is made the basis of analysis, it is called Spectrophotometer, is really two

instrument in one cabinet, a spectrometer and photometer.

The colorimeter & spectrometer are based on optical principles and mainly divided in five

Type of optical instruments.

4.0 BRIEF THEORY OF UV-VIS
The UV region is sub divided in two,

a. 200 400nm is known as near UV region.

b. Below 200nm is called vacuum UV region.

The wavelength range of UV region start at the blue end of visible light(400nm). UV region

is normally expressed in nm. Molecular absorption in the ultraviolet and visible region of the

spectrum is dependent on the electronic structure of the molecules. Absorption of energy is

resulting in deviation of electrons from orbital in the ground state of higher energy orbital

in an excited state. We are discussing here only near ultraviolet(quartz) region extending

from 200 300nm.

The atmosphere is transparent in this region and quartz optic may be used for scan, from

200 300nm. Atmospheric absorption start near 200nm and extended in to shorter

wavelength, which known as vacuum ultraviolet region.

By increasing the frequency such that the energy of the incident quanta becomes equal to the

Difference in energy between the two electronic states of the molecules, the molecules jump

From one electronic states to another and gives rise the electronic spectrum in the UV

region.

Electronic transition occurs between different molecular orbits, which are , &
(nonbonding) type. The first two is also differentiated as bonding and antibonding. The

transitions of * and * occurs in vacuum UV only, because it require very

high energy. Therefore only * and * transitions are observed in normal

UV region.

a. * ------------------------------------------------------ *
>C = C: >C = C: ---------------------------------------------- *
---------------------------------------------------- (nonbonding)

b. * ----------------------------------------------------- (antibonding)

>C = C: >C = C:---------------------------------------------- (bonding)

The UV spectroscopy is only useful to determine characteristic of molecules containing and
type of molecular orbital. I.e. Olefins, Aromatic and those molecules containing N, O & S atoms.

& * transitions in case of Olefins absorb in vacuum UV and shift to UV Region on

conjugation. By measuring the wavelength of the radiation absorbed, the presence of certain

molecules can be inferred. This is the basis of all absorption spectroscopic methods of analysis.

For quantitative purposes, the relative reduction in the intensity of the incident light after it Passes

through the sample, is usually measured.

If I0 & I will be the intensity of incident and transmitted light, then according to Beer-Lambert Law,

I = I0.e-abc

OR

Log10 I0/I = A =abs log10 e

Where, A : Absorbance
a : absoptivity = E x 2.3026.
e : extinction coefficient
b : thickness of the sample
c : concentration

* the electron of unshared pair goes to an unstable (antibonding) * orbital.

* in which an electron goes from a stable (bonding) orbital to an unstable * orbital.

When a monochromatic or heterogeneous light falls upon a homogeneous medium, a

portion of the incident light is reflected, a portion is absorbed within the medium and the

reminder is transmitted. If the intensity of the incident light is represented by I0, that of

reflected light by Ir, that of The absorbed light by Ia and that of transmitted light by

It then,

I0 = Ia + It + Ir

For air glass interfaces, only 4% of the incident light is reflected, their for Ir is usually

eliminated by the use of a control, hence such as comparison cell, hence:

I0 = Ia + It. -------------------------------------(1)

Lambert in 1760 established the relation between I0 & It.

Beer in 1852 extended the experiments to solution.

Reflection Reflection

Incident beam Emergent beam

Scattering

Incident radiation falling on a system
4.1 LAMBERTS LAW
When monochromatic light passes through a transparent medium, the rate of decrease in

Intensity, with the thickness of the medium is proportionate to the intensity of the light.

i.e. for any layer given thickness of the medium, absorbs the same fraction of the incident

upon it, or intensity of emitted decrease exponentially as the thick ness of the absorbing

medium, increase arithmetically. Thus in the differential equation, we have,
dI
--------- = kI (2)
dl
Integrating (2) and putting I = I0, when I = 0, we obtain:
Io
In ------------- = kl or stated in other terms,
It

It = Io.e kl (3)

Where, I0 : Intensity of incident rays,
l : Thickness of the medium
It : Intensity of transmitted light.
K : is proportionality factor, depending upon the nature of medium.

By changing from natural to logarithm we obtain:
Io
Log e ---------------- = Kl or
It

It : Io10 -0.4343kl = Io. 10-kl (4)

Where K= k/2.3026 and is usually termed the absorption coefficient. It is generally defined

As the reciprocal of the thickness (1cm) required to reduce the light to 1/10 of its intensity.

We put this in our equation (4) since:

It/Io = 0.1 = 10-kl or Kl = 1 and K = 1/l.

The ratio It/ Io is the fraction of the incident light transmitted by a thickness 1cm of the

medium and is termed the Transmittance T.

Its reciprocal Io / It is the Opacity and the Absorbance A and it is termed as:

A = log Io / It. (5)
4.2 BEERS LAW
Beers studied the effect of concentration of the colored constituents in solution upon the

Light transmission or absorption. He found the same relation between transmission and

concentration as Lambert had discovered between transmission and thickness of the

medium.

i.e. the extinction is proportional to the concentration C of the absorbing species,

or

the intensity of a monochromatic light decrease expontialy as the concentration of the

absorbing substance increases arithmetically.

Thus, It = Io.e-kc = Io.10-0.4343kc = Io.10-kc (6)

Where c = Concentration of the solution and k and K are constants.

By combination of (4) and (5)

We have reference (6):

It = Io. 10-acl or log Io / It = acl (7)

This is a fundamental equation of colorimetric and spectrophotometer and it is known as

Beer Lambert Law.

Mainly spectrophotometers are divided in three categories as under with respect to wave

length and spectral band.

Out of this three, one and two are available in combined in one instrument.

1. Ultraviolet spectrophotometer.

2. Visible Spectrophotometer

3. Infrared Spectrophotometer
5.0 PRINCIPAL OF UV-VIS SPECTROPHOTOMETER:
Ultraviolet and Visible radiation are small segment of the vast electromagnetic spectrum

which can be divided in as under,

5.1 WAVE LENGTH(O)
------------------------------------------------------- ------------------------------------------------------
12.5 x 10 50 x 10
4 x 10-2 25 400 4000 25x 10 10 10
-------------------------------------------------------------------------------------------------------------
Electron spin Molecular Molecular Valence Electronic Inner Nuclear
orientation rotation vibration transmittance shell trans.
e-trans.

Radio Microwaves Far Fundamental Overton Near Vacuum Gama
Waves (radar) IR IR region UV UV x-ray rays
25cm 0.04cm 25m 2.5m 0.8m 400 200 1 0.1
400m 800nm nm nm nm nm
Fig.-1

Wavelength Frequency
(meters)(10-12-14 -22
-13 -21________________
Picometre -12 -20 Gama rays
-11- -19____________________
1 Angstrom -10- -18
1 Nanometre -9- -17___________ X rays
-8- -16 Ultraviolet
-7- -15
1 Micrometre -6- -14 Visible
-5- -13
-4- -12
1 Milimeter -3- -11-------------------------------------
-2- -10
-1- -9 Hertzian waves
1 Meter 0- -8--------------------------------------
1- -7
2- -6 1 Megahertz Radiowaves
1 Kilometre 3- -5---------------------------------------
4- -4
5- -3 1 kilohertz Audible
6- -2 frequencies
7- -1(10)
8-

Fig.-2

Ultraviolet Visible Infrared

200 250 300 400 500 600 750 1500 Wavelength
1600 1400 1200 1000 800 600 400 200 Frequency
50000 40000 30000 20000 10000 Wave number

Fig.-3

Electromagnetic radiation can be considered to be an oscillating electric field with an

associated magnetic which travels with a wave motion in discrete units called photons. Since

electromagnetic radiation acts as a wave it can be classified according to its wavelength,

which is the distance between the crests of two adjacent waves measured along the line of

propagation.
5.2 ABSORPTIVITY:
Absorptivity is an intensive property of a substance, where as absorbance is its extensive

property. With the change of concentration and thickness of the container, the absorbance will

very significantly but the absorptivity remain constant within the range of Law. When

molecular weight of the substance is known, absoptivity can be represented by the symbol ,
the molar absoptivity ( = A M/bc).The absoptivity will very in a particular system if the

absorbance fluctuate with wavelength.

2400-
2000-
1600-
1200 -

800 -

400 -

400 440 480 520 560 600 640 680

The above figure it is evident that molar absoptivity will be different at different wavelengths.

This asserts why a monochromatic radiation is preferable i.e. spectrophotometer is preferred to

a filter photometer.

5.3 PHOTMATRIC ACCURACY:

The concentration range over which Law is valid may not necessarily be the length over which

photometric analysis is practicable. Photometric analysis is limited at both high & low values.

When the concentration is too high, only a little of radiant energy will come out and then

photometer proves quite inadequate to monitor that. Again when the concentration is very low,

more power will come out making the galvanometer reading too large and therefore highly

erratic compared to the quantity being measured. The accuracy for measurement is. which
A/A, small change of absorbance with respect to total absorbance is minimum.

The value for absorbance A stands at 0.4343, corresponding to a transmittance T = 36.8%. It is

shown as under graphically.

10-

8-
6-
4-
2-
0 20 40 60 80 100

From above figure is apparent that although the error is minimum at 36.8% T,It will not be

very high over the range of transmittance between 15 and 65% ( absorbance 0.8 0.2)

6.0 QUANTITATIVE ANALYSIS

6.1 SCOPE:
To determine the concentration in an unknown sample, a calibration curve of absorbance

versus concentration at a relevant wave length is produced by using standard of known

sample is measured and the concentration of the analysis can be read off the

concentration versus absorbance curve. This appears straight forward but there are some

potentially large source of error.

6.2 LIGHT SCATTERING:
An apparent light absorption will be recorded by a spectrophotometer if a sample

contains a colloidal dispersion, dust or other particulate matter which can scatter the

incident light beam. If possible these type of samples should be filtered, centrifuged or

allowed to settle before measurement. Where turbid sample must be measured, a

correction can sometimes be applied by measuring the scattered light at a wavelength

where the sample does not absorb and subtracting this reading from the e\reading at the

centre of the absorption band.

6.3 SOLVENT EVAPORATION:
Where volatile solvent such as acetone, ether, chloroform etc. are used, care must be

taken to limit the evaporation of such solvents which can lead to a concentrating effect on

the absorbing species. In such cases stopper cells should be used when using volatile

solvents.

6.4 SAMPLE DECOMPOSITION:
Some samples are very sensitive to UV light. These samples can under go photo-chemical

Reactions in the cells it self, if it exposed to UV light.
6.5 FLUORESCENCE:
Fluorescence is the phenomenon where by a molecule absorbs incident light of one

wavelength and emits light of a longer wavelength. If the fluorescent substance is the

molecular species being analyzed there is no problem. However if the fluorescent

material is not the analyzed species, the fluorescence can introduced significant error.

This can be reduced by placing a wavelength filter in the optical beam of the

spectrophotometer between the sample and the detector. The filter is selected on its

ability to be transparent to the desired wavelength but opaque to the longer emitted

fluorescent wave length.

6.6 TEMPERATURE:
A change in temperature can change the volume of a solvent, due to thermal expansion

and it can also change the absorption coefficient. These both effect create error in

concentration.

6.7 SAMPLE CELL:
Dirty sample cells will absorb more light then cells. Care should be taken to avoid finger

print etc. on the optical surface of the sample cells.

6.8 COMPLEMENTARY COLORS:
Electromagnetic radiation which the human eye can detect is called visible light. Which

found in the region 400 to 700nm of the spectrum. Ordinary visible light is a mixture of

various wave length between these two limits. The relationship between the colors, their

complementary colors and associated wavelengths is as under.

Absorbed Transmitted Wavelength
color color nm
<400
400

Ultraviolet

<400
450
Violet

Yellow green

400-435
500
_______
_______
Blue

Green blue
Blue green
Yellow

Orange
Red
435-480

480-490
490-500
550 Green

Red violet

500-560
600
Yellow green

Yellow

Orange

Violet

Blue

Green blue

560-580

580-595

595-610
650

Red

Blue green

610-750
700
Red violet

Green

Near Infrared

6.9 UV VIS SPECTRUM
When light falls upon a substance, a part of the light may be selectively absorbed depending

on the energy levels of the constituents molecules in the substance. When light having an

energy equal to the difference between one energy level and another impinges on the

substance, some of the light of this specific energy is absorbed by the substance.

An absorbance spectrum is obtained by successively allowing light of different energy to

Impinge on a substance and measuring the degree of absorption. The total potential energy

of a molecules can be considered to be the sum of its electronics, vibration and rotational

energies. Energy differences between rotational states of a molecule are very small

compared to the energy difference between electronic states, while vibration transitions are

intermediates between the two. Thus rotational transitions will absorb in the far infrared

region of the spectrum. Vibration transitions will absorb in the near infra-red region, whilst

electronic transition Will absorb in the X-ray, Ultraviolet & visible region of the spectrum.

The inner shell Electronic transition which are unaffected by the physical environment of the

atom, have higher energy transitions and thus absorb in the X-ray region. And because they

are un affected by their environment, they have sharp quantum absorption bands.

However the other shell electrons which are involved in molecular bonding are affected by

their physical environment, have lower energy transitions and thus absorb in the UV-Visible

region of the spectrum, and because they are affected by their environment, they have broad

absorption bands. They physical environment of the atom may include the proximity and

type of adjacent atom and the degree of vibration and rotation of the molecule itself.

6.10 TRANSMITTANCE AND ABSORBANCE:
If light of intensity I0 is incident on a substance and light of intensity It passes

through the substance,

i.e. is transmitted, then the Transmittance (T) is expressed by,
It
T : -----------
I0
Hence, percentage transmittance (%T) is expressed by:

%T : (It/I0) x 100

Incident light Transmitted light

Now if the substance contains a molecular species that absorbs at the wavelength of the

incident light, then the following equation holds, as per Beer Lambers Law.

It : I0 . 10 -cl (9)

Where, : absorption coefficient
c : Concentration
l : path length

(Molar absorption coefficient in c is mol./L & l in cm.)

This equation illustrates that concentration C is not a liner function with transmittance(T).

Therefore the concept of absorbance (A) is introduced & given as under.

A = -log 10.It/I0 (10)

This equation combined with equation (9), leads to,

A = - cl

i.e. absorbance is directly proportional to the concentration of the absorbing species.

6.11 SOLVENT PROPERTIES:
The solvent should meet the following requirement.

It should dissolve the sample without reacting with it.

The absorption of radiation in the scanning region should be low.

Evaporation should be low at ambient temperature.

Aromatic compounds are not suitable as solvent in UV region, as it has
high absorption in this region.

Water is almost ideal solvent in visible, UV & below 195nm also, but it
should free from oxygen.
6.12 SOLVENT TRANSMITTANCE:
Lower wavelength limits of commonly used solvent.(10mm pure solvent &
transmittance 10% min.)
Acetone
m- Xylene
Toluene
Chloroform
Glycerol
Dioxane
Hexane
i- octane
Acetonitrile
Cyclohexane
Methanol
Ethanol
Methyl cyclo hexane
IPA
water
190 210 230 250 270 290 310 330 350 370
nm
7.0 INSTUMENTATION
OPTICAL INSTRUMENT
Colorimeter Flurometry Nephelometry Emission Flame
Spectrometer Turbidimetry Spectrograph Spectrometry
-------------------
UV-VIS IR
The essential parts of spectrophotometer are as under:

a. a source of radiation energy.

b. a monochromator to separate the radiation according to its frequency.

c. a detector and indicating systems.

d. sampling systems.

e. recorder.

7.1 LIGHT SOURSE:
An ideal source of radiation energy should be of uniform intensity throughout the entire

region of its application or preferably of greater intensity.

Any source can be used, but it should be fulfill the following requirement,

It must be stable.

It should have sufficient intensity.

It must supply continuous radiation over to the entire region.

The common source in ultra violet region, are Hydrogen discharge lamp or Deutorium lamp,

which covers entire interested UV range (above 185nm).

Xenon discharge lamp has greater intensity than Hydrogen lamp, but due to stray light problem

it is not used.
7.2 MONOCHROMATOR:
Monochromator used for to disperse the radiation according to the wave length. The main

part of monochromator is entrance slit. It controls the unwanted radiation and allows only

fixed wave length radiation.

Dispersing elements are prism or grating.

In visible glass prism used.

In ultra violet silica or quartz prism are used. Now a days it is replaced by gratings.

In vacuum fluorite ( CaF2 or LiF) used.

Prism monochromators have the advantage of being simple and cheep, its intensity is higher.

The main disadvantage of prism is that,
it damages easily by abrasion and by Mishandling.

It has limited resolution.

Grating monochromators are superior to prism, because,
it has improved.

Linear dispersion.

The higher resolution gives greater qualitative and quantitative accuracy. The typical grating

for the UV & VIS region may have 10000 to 6000 rulings/cm. The disadvantage of gratings

are more scattered light and a limited wavelength range for single grating. So it is over come

by using double monochromator, i.e. prism followed by gratings or different filters. Usually

many filters and two or more gratings are used to cover complete spectrum.

7.3 DETECTOR:
Three detectors are commonly used in UV determination.
Barrier layer cell.

Photocell.
-
Photomultiplier tube.

Photomultiplier tube, as detector are generally used in UV. It is extremely sensitive and

fast response detector. A photomultiplier may have as many as 12 dynodes, with total

voltage difference of 500-2000V.

7.4 TYPE OF UV SPECTROPHOTOMETER
1.Singlr beam

2. Double beam

7.5 CALIBRATION OF UV VIS SPECTROPHOTOMETER:
By Holmium oxide standard calibration filter.

1. By 4% Holmium oxide solution in perchloric acid.

2. Potassium chromate test solution.

4omgs. of Potassium chromate + 28gms. Of 85% KOHdilute to 1L with DM water.

Std. wave length Absorbance

250nm 0.460

273nm 0.760

350nm 0.550

370nm 1.000

400nm 0.400

7.6 LAMP OPTIMIIZATION AGAINST:
Lamp Wave length Acceptable absorbance

QI lamp 550nm 30-40

D2 lamp 240nm 12-40

7.7 ALIGNMENT OF CELL HOLDER:
Alignment is correct when the radiation beam is just above the floor of the cell(min 2mm)

7.8 MINIMUM VOLUME APPLICATION:
Minimum volume required is the cell height of liquid slightly more than height of beam. Cell

Window should be completely filled with liquid

7.9 DAILY CARE FOR INSTRUMENT:
Keep it cleaned and dust free.

To protect the optical system from dust and fumes, keep sample compartment cover
closed.

Immediately clean all spilled material by using lintless paper or cloth.

Be careful while cleaning sample windows, because it is part of optical system.

Do not leave sample, particularly those gives fuming, evaporating in the sample
compartment for longer then necessary.

7.10 USE AND CARE OF CELL:
Cell is a part of optical device of spectrophotometer.

Scratches, lint finger mark on the optical side of cell, can leads to substantial analytical
error.

To avoid such damage following care must be require.

Hold cell by non optical surface, means matt finished surface.

Protect cell from scratches and never permit to rub against any hard surface.

Avoid any abrasive, corrosion, or stain producing cleaning agent.

Make sure that exposed surface of cells are optically cleaned.

Always wipe optical surface of cell and make it free of finger marks by using soft tissue
paper just before placing in sample holder.

When measuring cold solution, always keep in mind that moisture condensation can
form on the optical surface.

Make sure that no bubbles cling to the inner surfaces of the cells, particularly when
handling cold solution.

For maximum precision and accuracy for calibration and test, use same cells and
orientation of holder.

8.0 REFERENCE:

1. A Textbook of quantitative Inorganic analysis by A I Vogel

2. Spectrometric Identification of organic compounds.

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