Saturday, August 19, 2017

UV-Vis Spectrophotometry

SPECTROSCOPY
Spectroscopy was originally the study of the interaction between radiation and matter as a function of wavelength (λ).
Spectrometry is the spectroscopic technique used to assess the concentration or amount of a given species. In those cases, the instrument that performs such measurements is a spectrometer or spectrograph.
Spectroscopy/spectrometry is often used in physical and analytical chemistry for the identification of substances through the spectrum emitted from or absorbed by them.
Spectroscopy/spectrometry is also heavily used in astronomy and remote sensing. Most large telescopes have spectrometers, which are used either to measure the chemical composition and physical properties of astronomical objects or to measure their velocities from the Doppler shift of their spectral lines.
Practical Significance: - Monochromatic wave
X-rays                                                 Medicinal Procedures                                  
UV-rays                                              Sun burns, Spectrophotometry
Visible                                                 Spectrophotometry, Colorimetry
Radio/Radar                                        Communication
            

UV/Visible Spectrophotometry and Colorimetry

·   Absorption spectrophotometry in the ultraviolet and visible region is considered to be one of the oldest physical methods used for quantitative analysis and structural elucidation.

Absorption spectroscopy
         Light is only absorbed if its energy (i.e. frequency) corresponds to the energy difference between two quantum levels in the sample
         Described by the Bohr frequency condition:

Δ E = E1− E2 = h g
where; h = Planck’s constant = 6.63×10-34 J·s
g = frequency of light (in Hz)
Wavelength:                λ = g / c           (c:        speed of light)
Wave number:            n = 1 / λ

Absorption of radiation
·         Light that is incident on a color sample is partially absorbed by it.
·         i.e.: at a certain wavelength there is less intensity coming out on the other side.
·         There is no breakdown in the law of conservation of energy, however. The result of the absorption may appear as:
·         Heat producing a temperature rise in the sample,
·         Luminescence in which a photon of the same or lower energy is emitted,
·         Chemical processes that incorporate energy into altered bonding structures.

·   The main types of instruments in use for measuring the emission or absorption of radiant energy are:    a) photometer.              b) Spectrophotometer.            c) Colorimeter

                                                   
Principle:

·         The study of interaction of light with a matter.
·         Widely used in the investigation of components of the matter (including living matter) to measure their concentration.
·         Let us consider a light beam passing through a sample of thickness d with absorbing species of concentration c.
·         The energy carried by the beam light per unit area per unit time, is called the intensity I and has initial value equal to I0.
·         The beam passes through the sample and emerges on the right with decreased, as the result of absorption, intensity It.
·         The “amount” of absorption depends on the sample thickness d (the path length), the sample concentration c and on the wavelength λ the light passing through the sample.
    Figure 1: Intensity of light passing through sample of thickness d is decreased due to adsorption


Lambert’s law

“When a beam of light is allowed to pass through a transparent medium, the rate of decrease of intensity with the thickness of medium is directly proportional to the intensity of light”
                                                              It = I0 e-e c d
                                                                             
                                                         Where e stands for absorption coefficient
%T = Io / It *100

·                     The ratio of the intensity of transmitted light (passing through a substance) It  to the intensity of the incident light Io is called the transmittance T or, when multiplied by 100%, percentage transmittance:
                                 
·                     The term absorbance, A, by definition, is the negative logarithm with base 10 of the transmittance T, that is:
A = - log10 (T)
                                                     
Beer’s law

The intensity of a beam of monochromatic light decreases exponentially with the increase in concentration of the absorbing substance arithmetically.

·                     The absorbance is proportional to the concentration, c, of the absorbing species and to the length of the path, d, of electromagnetic radiation through the sample containing the absorbing species:

                                                 A = e c d

Where the symbol ε stands for the absorptivity (the former name was the extinction coefficient).
·         The absorptivity ε depends on the wavelength, λ of light and plays the role of proportionality coefficient.

·         For a given path length and given wavelength the absorbance, A, is directly proportional to the concentration of a solution and is a suitable measure of the light absorption phenomenon.

·         If we plot the absorbance A versus concentration c, we obtain a straight line passing through origin (0, 0)
          



Combining the above two statement gives the Lambert-beer law ad states that the rate of decrease of intensity of light depends on the concentration and thickness of the medium and can be express by the equation:
                                             A = e c d

            Where A         = absorbance
ε              = molar absorptivity (L mol-1 cm-1)
d          = path length of the sample (cm)
c          = concentration of the sample in solution (mol L-1)
Absorbance is directly proportional to the other parameters, as long as the law is obeyed. After certain limitation the law is not obeyed and the straight line deviates from the normal in extreme cases of the concentration of samples and is called deviation of the law.
·   It explains why, for measurements made with samples of the same thickness d, the transmittance T of a sample decreases exponentially with increasing concentration c of the absorbing substance. (Fig. 4b).

Limitations of the Beer-Lambert law

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 equilibrium 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
Concept of λmax
·   For a given substance at a specified wavelength λ, the absorptivity ελ is a constant characteristic of the absorbing sample and is independent of both the concentration c of the solution and the thickness d of the absorbing layer.
·   Absorptivity and, the absorption itself depends strongly on the wavelength for nearly all compounds,
·   So, we must specify the wavelength at which the measurement of the absorbance versus concentration is made.
·   The way in which absorbance depends on wavelength, A= f(c) , defines the spectrum of the substance being studied (Fig. 5).



  
·   During spectrophotometric measurements the highest accuracy is achieved when these measurements are made at the wavelength at which the absorbance A takes the highest value.
Question: Why do we prefer to express the Beer-Lambert law using absorbance as a measure of the absorption rather than %T ?
Equation                 A = e c d
                              % T = e -e c d
Now, suppose we have a solution of copper sulphate (which appears blue because it has an absorption maximum at 600 nm). We look at the way in which the intensity of the light (radiant power) changes as it passes through the solution in a 1 cm cuvette. We will look at the reduction every 0.2 cm as shown in the diagram below.
The Law says that the fraction of the light absorbed by each layer of solution is the same.
For our illustration, we will suppose that this fraction is 0.5 for each 0.2 cm "layer" and calculate the following data:
Path length / cm
0
0.2
0.4
0.6
0.8
1.0
%T
100
50
25
12.5
6.25
3.125
Absorbance
0
0.3
0.6
0.9
1.2
1.5


The linear relationship between concentration and absorbance is both simple and straightforward, which is why we prefer to express the Beer-Lambert law using absorbance as a measure of the absorption rather than %T.

Note: that the Law is not obeyed at high concentrations. This deviation from the Law is not dealt with here.
Question: What is the significance of the molar absorbtivity, e ?
To begin we will rearrange the equation A = ε c d
e = A / d c
In words, this relationship can be stated as "e is a measure of the amount of light absorbed per unit concentration".
Molar absorptivity is a constant for a particular substance, so if the concentration of the solution is halved so is the absorbance, which is exactly what you would expect.
Let us take a compound with a very high value of molar absorptivity, say 100,000 L mol-1 cm-1, which is in a solution in a 1 cm pathlength cuvette and gives an absorbance of 1.
  e = 1 / 1 ´ c                             Therefore, c = 1 / 100,000 = 1 ´ 10-5 mol L-1
Now let us take a compound with a very low value of e, say 20 L mol-1 cm-1 which is in solution in a 1 cm pathlength cuvette and gives an absorbance of 1.
e = 1 / 1 ´ c
Therefore, c = 1 / 20 = 0.05 mol L-1
The answer is now obvious - a compound with a high molar absorptivity is very effective at absorbing light (of the appropriate wavelength), and hence low concentrations of a compound with a high molar absorptivity can be easily detected.

Spectrophotometry:

 Instrumentation




All photometers, colorimeters and spectrophotometers have the following basic components:
a)      Source: continuous source of radiant energy covering the region of spectrum in which the instrument is designed to work.
                                                              i.      Visible spectrum (320-700nm)-                      Tungsten lamp
                                                            ii.      UV-range (220-300 nm)-                                H2/2H2 lamp (Deuterium lamp)
·         Heating the tungsten lamp at 1725 °C- 1% of radiation is in between UV-visible range.
·         Heating the tungsten lamp at 2700 °C- 15% of radiation is in between UV-visible range.
·         Should be heated in control environment.
b)      Filter or monochromator: allow the light of the required wavelength to pass through but absorbs the light of other wavelength.
                                                              i.      Colorimetry-filters                 
1.      Transmit some λ absorbing other
2.      band width-30-250nm
3.      colored glasses/dyes sandwitched in glasses
4.      selection based on absorption and transmission spectra
5.      complementary color (N.B. color-when λ discrimination)



Figure: Complementary hue cycle



Table: Complementary hue (filter selection) for various solutions.

Color of Visible Light
Color                                                  Wavelength, nm                                Filter color
Voilet                                                              400-435                                   Yellow-Green
Blue                                                                 435-480                                   Yellow
Green-Blue                                                      480-490                                   Orange
Blue-Green                                                      490-500                                   Red
Green                                                              500-560                                   Purple
Yellow-Green                                                 560-580                                   Voilet
Yellow                                                            580-595                                   Blue
Orange                                                            595-610                                   Blue-green
Red                                                                 610-750                                   Green-blue
(NOTE: blue absorbing solution appears yellow or green absorbing solution appears purple)
c)      Sample cells:
·         A container for the sample.
·         Cuvettes- transparent for λ, reproducible path length
·         Should transmit the maximum wavelength
·         Should be economic
·         Different cuvette for different transmission capacity
a.       Glass cuvettes-            λ=400-2500nm
b.      Quartz                         λ=200nm
c.       Silica                           λ<180 nm="" o:p="">
d.      Pyrex                           λ=300-2500nm
e.        
d)      Detector: for measuring the radiant energy transmitted through the sample.
a.       Broad λ, sensitive, rapid responding, amplifiable signal
b.      Two types =   
1.       Heat detector- IR, heat detection
2.       Photodetector-          
a.       photon amplification
b.      radiant energy to electrical
c.       three different types of photodetector:
                                                              i.      photovoltaic cell
                                                            ii.      phototubes
                                                          iii.      photo amplifiers
I. Photovoltaic cell:
There are different designs of photovoltaic cells. It operates without the use of a battery. A typical photovoltaic cell consists of a metal base plate (irons or aluminium) acts as one electrode. On the surface of the base plate, semiconductor thin metal layer of selenium is coated. Further it is covered by a thin layer of silver or gold on the outer most surface. It acts as a second collector electrode. When incident radiation strikes at surface it generates electrons in Se-Ag interface. Then the electrons are collected by silver and created electric voltage. It is limited to visible region (450-650 nm) and sensitive to the whole visible range. However output depends upon the wavelength of the incident light. Current can’t be amplified readily in this design and so there are fatigue effects after long time operation.

Figure: Photovoltaic cells
II. Phototubes:
It is also known as photo-emissive cells. It consists of an evacuated glass bulb, inside which a light sensitive cathode in form of a half cylinder of metal is fitted. The cathode is coated with light sensitive layer, cesium, potassium oxide or silver oxide. A metal ring is inserted near the center acts as an anode for capturing of electrons. The incident beam when falls on cathode it emits photoelectrons which are attracted by an anode. The electrons return via the external circuit which is amplified to read out readily. It measures the amount of the light striking the photo-sensitive surface and expressing it in absorbance or transmittance or concentration forms. For the wavelength of 350-450 nm the cathode should be coated with sodium.
Figure: Phototubes
III. Photo amplifiers:
It is one of the highly sensitive devices used today.  It consists of an electrode covered with a photoemissive material. A large number of plates known as dynodes are used. A dynode is cover with a material which emits several electrons. Once electrons are ejected from the cathode and accelerated to the sensitive surface of the dynode, secondary electrons are emitted in greater amount than the striking the plate by 4 x to 5 x factor Each dynode is maintained at 75-100 V more positive than the preceding dynode. Over all amplification by about 10 dynodes will be 106. So it can be used to measure the intensities about 200 x weaker than those by conventional methods. It also prevents from stray light.
Figure: Photo amplifier

How to use?              
·         Warm up an instrument for 10-30 minutes
·         Set a suitable wavelength and switch on respective lamp
·         Use of optically matched cuvettes
·         Blank- all expect the sample under test
·         Zeroing- with blank
·         Absorbance start from dilute sample
§  Use of standard calibration curve
§  Interpolation is critical-Job’s Phenomenon
·         Accuracy of instrument may not be uniform for all λ or T-range
§  20-80 % transmission (sd=±2)
·         Error at low or high absorbance values so best to try middle of T-range
·         Select complementary hue.


Applications:-

  Qualitative Analysis:
  To identify compound in pure / biological mixture
  To confirm the presence of particular compound in the mixture

  Quantitative Analysis
  For the estimation of any organic and inorganic compounds such as
  Chromophores = absorbs specific wavelength of light
  Protein = 280 nm
  Nucleic acids = 260 nm
  Amino acids estimation by Ninhydrin = 570 nm
  Bradford for protein estimation = 595 nm
  Total carbohydrate estimation

  Binding Spectra
  Can be used in the study of enzyme kinetics e.g. ALT and AST profile
  To know whether the reaction is complete or not
  Estimation of reactants and products

  Structural studies
  SS DNA, DS DNA, RNA
  Binding to DNA by ethidium bromide

  Addition of functional group shift the wavelength maxima

2 comments:

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