Chapter 6 UV VIS SPECTROMETER

Spectroscopy UV-VIS Spectrophotometer Application Case Study

Spectroscopy
The procedures that uses light to measure chemical concentration

Spectroscopy

Light is described as photon because of its ability to carry energy, E, that is given by E = hv where : h is a Plank constant (6.626x10-34 J.s) v is frequency of light in Hz

Spectroscopic analytical method are based on measuring the amount of radiation produced or absorbed by molecular or atomic species of interest We can classify spectroscopic methods according to the region of the electromagnetic spectrum involved in the measurement. The region that have include such as X-ray, Ultra violet, Visible, Infrared(IR) and etc

The Electromagnetic Spectrum

Gamma Rays X Rays nm 1 UV IR Radar TV Radio meters 10 100 1000 cm 1000 0.001 0.01 0.1

0.001 0.01

0.1

10

100

10

visible
UVB UVA Near IR

200

300

400

500

600

700

800

900

Electromagnetic Spectrum
Regions which represent molecular processes that occur when light in each region is absorbed. Electromagnetic radiation in the UV-VIS portion of the spectrum ranges in wavelength from approx. 200 to 700 nm. The UV range runs from 200 to 350 nm and the VIS range from 350 to 700 nm.

Absorption
The process by which energy is transferred from an electronic wave to an atom or molecule and causes it to move to an excited state. Absorption can only occur when an atom or molecule absorbs a photon of light that has an energy which exactly corresponds to the difference between two energy levels, that is it must be quantized. If an atom or molecule is subjected to electromagnetic radiation of different wavelengths (energies) it will only absorb photons at those wavelengths which correspond to exact differences between two different energy levels within the material.

Chromophore
Absorption of UV VIS radiation results from the excitation of electrons from ground to excited states. Nuclei inside the molecules play an important role in:
Determining wavelength of radiation absorbed Determine the strength with which the electrons are bound and thus influence the energy spacing between ground state and excited state.

Chromophore
Hence the energy of transition and the wavelength of radiation absorbed are properties of a group of atoms rather than the electron themselves called chromophores.

Beers Lambert Law

Beers Lambert law states the relationship between the absorbance of a solution and the concentration of the absorbing species. (1) A=abc Where a = Proportional equation; absorptivity (Lg-1cm-1), b = optical pathlength (cm) and c = Concentration (g L-1).

This Law tell us quantitatively how the amount of attenuation depends on the concentration of the absorbing molecules and the pathlength over which absorption occur. Absorption may be presented as: Transmittance ( T = I / Io ) or Absorbance ( A = log Io / I )
b

(2) A=bc Where A= absorbance, = molar absorptivity (L mol-1cm-1), b = optical pathlength (cm) and c = Concentration (mol L-1).

Note: Transmittance is often

Io

expressed as percentage and called percent transmittance (%T= T x 100) 100)

Beer law also known as absorption law or beer-Lambert law

Absorbing solution of concentration c

Absorbance VS Transmittance
The absorbance is a logaritma function of transmittance, A = - log T Note: Since transmittance is often expressed as percentage and called percent transmittance (%T= T x 100 %), A also can calculate as, A = 2 log %T Where 2 = log 100

Note: 1. Absorbance is zero when transmittance is 100%, ie. No light absorbed by the sample I = Io b=1 cm T = 1.0 or 100 % and A = 0

I0
Transmitted light = I Incident light = Io

2. Absorbance is infinity when transmittance is 0%, ie. all light absorbed by the sample

If all the light passes through a solution without any absorption, then absorbance is zero, and percent transmittance is 100%. If all the light is absorbed, then percent transmittance is zero, and absorption is infinite.

Absorption
The process by which energy is transferred from an electronic wave to an atom or molecule and causes it to move to an excited state. Absorption can only occur when an atom or molecule absorbs a photon of light that has an energy which exactly corresponds to the difference between two energy levels, that is it must be quantized.

Figure 3 : The relationship between absorbance and transmittance

absorption spectroscopy If an atom or molecule is subjected to electromagnetic radiation of different wavelengths (energies) it will only absorb photons at those wavelengths which correspond to exact differences between two different energy levels within the material.
The amount of light absorbed as a function of wavelength is measured, which give qualitative and quantitative information about sample The wavelength of maximum absorbance is a characteristic value, designated as max

0.10 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 250 300 350 400 450 500 550 NM 600 650 700 750 800 850

ABS

Coffee Sample

100 98 96 94

%T
%T

92 90 88 86 84 82 80 250 300 350 400 450 500 550 NM 600 650 700 750 800 850

Different compounds may have very different absorption maximum ( max) and absorbances. Intensely absorbing compounds must be examined in dilute solution, so that significant light energy is received by the detector, and this requires the use of completely transparent (non-absorbing) solvents. The most commonly used solvents are water, ethanol, hexane and cyclohexane. Solvents having double or triple bonds or heavy atoms (e.g. S, Br & I) are generally avoided.

Path length / cm

0 100 0

0.2 50 0.3

0.4 25 0.6

0.6 12.5 0.9

0.8 6.25 1.2

1.0 3.125 1.5

Most spectrophotometers display absorbance on the vertical axis, and the commonly observed range is from 0 (100% transmittance) to 2 (1% transmittance).
Why do we commonly obtain and utilize absorbance values rather than transmittance values to quantitative compounds by UV Vis Spectroscopy ?

%T Absorbance

Standard calibration graph

 A = bc tells us that absorbance depends on the total quantity of the absorbing compound in the light path through the cuvette. If we plot absorbance against concentration, we get a straight line passing through the origin (0,0). The linear relationship between concentration and absorbance is both simple and straightforward, which is why we prefer to express the BeerLambert law using absorbance as a measure of the absorption rather than %T.

ote that the Law is not obeyed at high concentrations.

Using the Beer Law

Some factors causing deviation from BeerLambert Law : Gravimetric errors eg. mood Incomplete spectral resolution due to wrong slit width selection Turbidity must filter the sample first to avoid cloudy Aggregation at high concentration the particles stick together Contamination of cuvettes must clean the cuvettes immediately after using
Beer law expressed in equation 1 and 2 , can be used in several different way: 1. Calculate molar absortivity of species if their concentration are known 2. Can use to measure value of absorbance to obtain concentration if absorptivity and pathlength are known. 3. More often , we use a series of standards solution of the analytes to concentration to construct a calibration curve, or working curve , of A versus c (see figure 3). Then it can be used to determine the concentration of unknown.

Example 1
A 7.50 x 10 M solution of potassium permanganate has a transmittance of 36.4% when measured in a 1.05 cm cell at wavelength of 525 nm. Calculate (a) The absorbance of this solution (b) the molar absortivity of KMnO4. Answer: A = - log T = - log 0.364= 0.439 Using equation 2, = A / bc = 0.4389 / 1.05 cm x 7.50 x 10-5 mol L-1 = 5.57 x 103 Lmol-1cm-1
-5

Example 2
At 580nm,The Wavelength of its maximum absorption, the complex Fe(SCN)2+ has a molar absorptivity of 7.00 x 103 Lmol-1.Calculate the absorbance of a 2.50 x 10-5 M solution of the complex at 580 nm in 1.00-cm cell Answer:

A=bc = (7.00 x 103 Lmol-1)(1.00-cm) (2.50 x 10-5 molL-1)

= 1.75 x 10-7

Example 3

UV VIS pectrophotometer Animation

Instrumentation and Basic Components

Double Beam Instrument

Schematic diagram of a double-beam UVVIS spectrophotometer

Perkin Elmer Lambda 25

Lambda 25 Optic diagram

Lambda 25/35/45

Sources (UV and Visible)

Types of Spectroscopy Source

A. Continuum Sources Provides a broad distribution of wavelengths within a particular spectral range. This distribution is known as a spectral continuum B. Line sources which emit a limited number of spectral lines, each of which spans a very limited wavelength range.

Sources of UV radiation
1. Deuterium ( and also Hydrogen ) lamps Electrical excitation of deuterium or hydrogen at low pressure produces a continuous UV spectrum. The mechanism involves formation of an excited molecular species, which breaks up to give atomic species and an ultraviolet photon.

Sources of UV radiation
Both deuterium and hydrogen lamps emit radiation in the range 160 375 nm. Quartz windows must be used in these lamps, and quartz cuvettes must be used, because glass absorbs radiation of wavelengths less than 350 nm.

Sources of visible radiation

2. Tungsten filament lamp
commonly employed as a source of visible light. This type of lamp is used in the wavelength range of 350 2500 nm. The energy emitted is proportional to the fourth power of operating voltage. For stable energy output, the voltage to the lamp must be very stable. Electronic voltage regulators or constant-voltage transformers used to ensure this stability.

Sources of UV- ViS radiation

3. Tungsten/halogen lamps
Very efficient, and their output extends well into the ultra-violet. They are used in many modern spectrophotometers. The lifetime of a tungsten/halogen lamp is approximately double that of an ordinary tungsten filament lamp.

2. Wavelength selector (monochromator)

Wavelength Selector (monochromator)

The aim of a monochromator is to isolate narrow band of wavelengths centered on selected . We want to have high intensity at the selected and minimum light of other s. All monochromators contain the following component parts:

An entrance slit A collimating lens A dispersing device (usually a prism or a grating) A focusing lens An exit slit

Polychromatic radiation (radiation of more than one wavelength) enters the monochromator through the entrance slit. The beam is collimated, and then strikes the dispersing element at an angle. The beam is split into its component wavelengths by the grating or prism. By moving the dispersing element or the exit slit, radiation of only a particular wavelength leaves the monochromator through the exit slit.

3. Cuvettes (sample containers)

Czerney-Turner Grating Monochromator

sample containers
Sample Container, usually called cells or Cuvettes, must have windows that are transparent in spectral region of interest. Types of material normally used are: 1. Quartz or fused silica required for the UV region ( wavelength less than 350 nm) and may used in the visible region and out to about 3000 nm in the IR region. 2. Silicate Glass ordinary used for the region of 375 to 2000nm because of its low cost compare quartz 3. Plastic cells also used in visible region Sample The most common cell pathlength for studies in the UV VIS region is 1 cm.

The containers for the sample and reference solution must be transparent to the radiation which will pass through them. Quartz or fused silica cuvettes are required for spectroscopy in the UV region. These cells are also transparent in the visible region. Silicate glasses can be used for the manufacture of cuvettes for use between 350 and 2000 nm.

Different Types of Cuvettes

Cell and Cell Holders

standard

cylindrical

Long path length

Handling of Cell Clean and Dry Immediately after used Avoid scratches on polished cell windows Cleaning of Cell Use the solvent in which the substance was dissolved After cleaning rinse with distilled water Never use hydrofluoric acid for cleaning will etch the surface Never use ultrasonic cleaning may cause cavitation

Detector
Usually uses photo-electric devices. It converts the radiant energy (hv) into an electrical signal. Examples of detectors are:

Silicon photodiode array Vacuum phototube Photomultiplier tube

Type of Instrument
a) Single -Beam UV VIS Spectrophotometer

Single Beam VS Double Beam In single-beam uv-vis absorption spectroscopy, obtaining a spectrum requires manually measuring the transmittance (see the Beer Lambert Law) of the sample and solvent at each wavelength. The double-beam design greatly simplifies this process by measuring the transmittance of the sample and solvent simultaneously. The detection electronics can then manipulate the measurements to give the absorbance

B) Double Beam UV VIS Spectrophotometer

Advantage of Single beam instrument

It is well suited for quantitative absorption measurement at single wavelength type. Here, simplicity of the instrumentation, low cost, and ease of maintenance offer distinct advantages.

Advantage of Double beam instrument

Double beam instrument offer the advantage that they are compensate for all but the most the most short term fluctuation in the radiant output of the sources. They also compensate for wide variation of source intensity with wavelength. Furthermore, the Double Beam design is well suited for continuous recording of absorption spectra

Single-Beam UV-Vis Spectrophotometer

Dual-Beam uv-vis Spectrophotometer

Applying UV VIS Molecular Absorption Methods

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Molecules absorbing UV VIS radiation

Molecules absorbing UV VIS radiation

1.Absorption by Organic Species Species with unsaturated bonds generally absorb in the UV. Unsaturated organic functional groups that absorb in the UV VIS region are known as Chromophores. Table list common chromophores and the approximate wavelength at which they are absorb (Table 1.0). For many non nonabsorbing analytes, reagents are available that react with organic functional group to produced species that absorb in the UV or Visible reagent. For example: carbonyl containing compound react with dinitrophenylhydrazine to form colored species that absorb at 480 nm.

Absorption measurement in the UV VIS region of the spectrum provide qualitative and quantitative information about organic, inorganic and biochemical molecules.

Table 1.0 : Absorption Characteristic of Some Common Organic Chromophores

2. Absorption by Inorganic Species

C hrom op ho re A lkene C o n ju g a te A lkene A lky ne E x a m p le C 6H 13= C H 2 C H 2= C H C H = C H 2 C 5H 1 1 C - C H 3 S o lv e n t n-h e xa n e n- heptane n- heptane
m ax,

nm 177 217 178 196 225

m ax 1 3 ,0 0 0 2 1 ,0 0 0 1 0 ,0 0 0 2 ,0 0 0 160

O C arbon yl C H 3C C H 3 O C H 3C C H O C H 3C H 2 C H 3C O O H C H 3= C H 3 C H 3= O 2 C 2H O O 2 C 9H 9 O B en ze n e W ate r E th an o l E th an o l I s o o c ta n e D io x a n e E th yl E th er n -H e x an e 214 204 339 280 270 300 665 204 256 60 41 5 22 12 100 100 7900 200 n- hexane 180 293 L arg e 12 n- hexane 186 280 1 ,0 0 0 16

C arbon yl A zo itr o itr a t e itr o s o A r o m a t ic

In, general the ions and complexes of elements in the first two transition series absorb broad band of visible region in at least one of their oxidation states and are, as consequence colored. Example : Ni2+, Cr2O72-, Cu2+, C02+. Many inorganic species absorb in the UV VIS region. Ions such as nitrate, nitrite, and chromate show characteristic UV VIS absorption

Analysis using UV VIS Spectrophotometer

3. Absorption by Biochemical Species Many species of biochemical importance also exhibit strong absorption in the UV Vis region. For example, NADH, the reduced form of the coenzyme nicotinamide adenine dinultide ( NAD+ ), absorb at 340 nm

Qualitative Analysis

Quantitative Analysis

Even though it may not provide the unambiquous identification of an organic compound nevertheless useful for detecting the presence of certain functional group that act as Chromophores Example: Group containing phenyl, conjugated double bond and the nitro group. Compilation of UV VIS absorption spectra are available to compare with the absorption spectrum of a pure unknown compound. Usually, UV VIS absorption spectroscopy is only used for conformation in conjunction with more useful qualitative technique, such as NMR, IR and Mass spectrometry

One of the most powerful and widely used tools for quantitative analysis. Important Characteristics : Wide applicability to organic, inorganic and biochemical system Typical sensitivities of 10-4 to 105M Moderate to high selectivity Good accuracy ( Typically , relative uncertainties of 1% to 3% encounters) Ease and Convenience data acquisition

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General UV VIS Methods

1.Scanning Methods
Obtain typical spectrum of sample (using full range scan, 190 1100 nm)

Simple measurement of absorbance and transmittance values A, % T Scanning % A Scan, % T Scan Concentration Measurement Kinetic ( time Drive ) Single wavelength

2.Concentration Measurement
Used for quantitative analysis

3.Kinetic Analysis
Time based method Graphical real time display of absorbance vs time ( study course of a chemical or biochemical reaction with time) Used for enzyme kinetic study

Typical Industrial Application

Chemical analysis quality control, quality analysis Biochemical analysis proteins, DNA, enzymes Water and environment analysis EPA & DIN methods, cation/anion Kinetics enzymes kinetics, substrate kinetic Food analysis food colors (dyes), toxins, additives (BHA, BHT), contaminants (heavy metals) Pharmaceutical drugs (opium, heroin, cocaine)

Industrial Application

Determining the concentration of an unknown solution

Most dilute solutions obey the Beer-Lambert law and it can be used to determine the concentration of an unknown solution. Examples include the amount of iron in a sample of blood, the percentage of copper in brass (by first converting it into a copper (II) ion solution, or investigating the reaction kinetics of a reaction involving colored species. The method is essentially the same in each case and is illustrated by the following example.

A student was asked to plan an experiment to determine the concentration of Cu2+ in an unknown solution. She was provided with a separate solution of 1.00 mol dm-3 copper (II) sulphate. Using a spectrometer the student measured the absorbance of a diluted sample of the copper (II) sulphate solution at different wavelengths to obtain the value of 720 nm for max. She then carefully diluted the 1.00 mol dm-3 solution to give separate solutions with a range of known concentrations. The absorbance at 720 nm for each of these diluted solutions was then measured.

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Concentration of CuSO4 (aq) / mol dm-3

1.50 1.4 1.2 1.0 0.8 A 0.6 0.4 0.2 0.01 225.0 250 300 350 nm 400 450 500.0

Absorbance at 720 nm

0.100 0.150 0.200 0.250 0.300 0.500

0.362 0.498 0.798 0.901 1.002 1.751

The student used these results to plot a calibration curve using the line of best fit. The absorbance of the unknown solution at 720 nm was then measured.

Quantitatative analysis
Calculate the Relationship Between Absorbance and Concentration

Scans of Potassium Dichromate solutions of different Concentrations

1.50 1.4 1.2 1.0

Y = mx+C
4.00 3.5 3.0 2.5 2.0 A 1.5 1.0 0.5

2 1

0.8 A 0.6 0.4 0.2 0.01

0.00 0.00 0.5 1.0 1.5 mg/ml 2.0 2.5 3.00

225.0

250

300

350 nm

400

450

500.0

Case Study

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Transmittance Measurements

Transmission spectra of two different suntan lotion showing different levels of UVA and UVB light protection.

UVA=320-400nm(suntan)

UVB=290-320nm(damage skin)

What to Look for When Shopping for a Sunglasses? The standards for sunglasses place heavily in blocking UVB and UVA rays. UVC are blocked by our earth's atmosphere and does not reach the earth. According to the standards, to effectively protect your eye's from harmful UV rays it must block out at least 70% of UVB and 60% of UVA. However, we suggest that to best protect your eyes look for sunglasses that blocks out 98% of both UVB and UVA. All our sunglasses we provide are made with UV400 nm lenses. Which means it blocks out 100% of both UVB and UVA rays. It exceeds industry standards and suggested protection.

UVC
UVC is a type of magnetic waveform. It absorbs by the earth's atmosphere below 280mm. 'The "C" is considered a part of the UV family and has germicidal affects. When around the 260-nanometer frequency, it is known to have the highest affects. UVC is not harmful as compare to UVA and UVB. However, with long and sustained exposure it can make your skin read and your eyes feel like there is sand in them. With proper protection from our sunglasses, it can effectively block this type UV wave.

UVA UVB
UVB causes cancer and burning of the eye. It can be most damaging between 280 to 315 nanometer. It has a shorter wavelength; therefore, it is known to cause greater damage to the skin and eyes. The medical community suggests any person tanning indoor or outdoor to wear protective sunglasses. With long exposure to UVB and UVA, it is known to cause skin cancer and chronic eye diseases, such as cataract which is clouding or hazy of the eye's lens.

UVA is a longer waveform than UVB. It is the closest of the three waveforms to visible light. Most people get sunburns because of UVA because of its longer waveform it causes excitement in your skins cells within the epithelial melanocytes. UVA can also cause cataracts and Pterygium within 300 to 300 nanometer.

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