http://www.redicals.com The spectrophotometer technique is to measures light intensity as a function of wavelength. • Measures the light that passes through a liquid sample • Spectrophotometer gives readings in Percent Transmittance (%T) and in Absorbance (A)

1. UV- Visible
Spectrophotometry

2. • The spectrophotometer technique is to
measures light intensity as a function of
wavelength.
• Measures the light that passes through a
liquid sample
• Spectrophotometer gives readings in Percent
Transmittance (%T) and in Absorbance (A)

3. The electromagnetic
spectrum

5. Energy levels of a substance in solution
UV-Visible spectroscopy: Valance electronic
excitation

7. The human eyes sees the complementary to which that is
absorbed

8. The absorption spectrum is complementary to the
transmission of light. Chlorophyll is green because it
absorbs strongly in the blue (435 nm) and red (660 nm)
regions of the spectrum. Thus, the "transmission
window" is left around 550 nm, which corresponds to
green light.
The absorbance spectrum of chlorophyll

9. The visible region of the spectrum comprises photon energies of 36 to 72
kcal/mole, and the near ultraviolet region, out to 200 nm, extends this
energy range to 143 kcal/mole
The energies noted above are sufficient to promote or excite a
molecular electron to a higher energy orbital. Consequently,
absorption spectroscopy carried out in this region is sometimes
called "electronic spectroscopy". A diagram showing the various
kinds of electronic excitation that may occur in organic molecules
is shown on the left. Of the transitions outlined, only the two
lowest energy ones are achieved by the energies available in the

10. THE ABSORPTION SPECTRUM
When sample molecules are exposed to light having an energy that matches a
possible electronic transition within the molecule, some of the light energy will be
absorbed as the electron is promoted to a higher energy orbital. An optical
spectrometer records the wavelengths at which absorption occurs, together with the
degree of absorption at each wavelength. The resulting spectrum is presented as a
graph of absorbance (A) versus wavelength (λ) is known as a spectrum.
The significant features:
 λmax (wavelength at which there is a maximum absorption)
 єmax (The intensity of maximum absorption)
UV-visible
spectrum
of isoprene
showing
maximum
absorption
at 222 nm

11. Light is a form of electromagnetic radiation. When it
falls on a substance, three things can happen:
• the light can be reflected by the substance
• it can be absorbed by the substance
• certain wavelengths can be absorbed and the
remainder transmitted or reflected
Since reflection of light is of minimal interest in
spectrophotometry, we will ignore it and turn to the
absorbance and transmittance of light.

13.  I – intensity where Io is initial intensity
 T – transmittance or %T = 100 x T
(T = I/ Io)
 A – absorbance
A = log1/ T = log Io/ I
(absorption: Abs = 1 – T or %Abs = 100 - %T)
Some terminology

14. ABSORBANCE LAWS
BEER’S LAW The Effect of Concentration
“ The intensity of a beam of monochromatic light decrease
exponentially with the increase in concentration of the
absorbing substance” .
Arithmetically;
- dI/ dc ᾱ I
I= Io. eˉkc --------------------------eq (1)

15. Transmittance and concentration – Beer’s
Law

16. LAMBERT’S LAW The Effect of Cell Path Length
“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 the light”
mathematically;
-dI/ dt ᾱ I
I= Io. eˉkt --------------------------eq (2)

17. Transmittance and pathlength – the Bouguer Lambert’s
Law

18. The law states that the amount of light absorbed by a solution
(colored) is proportional to the concentration of the
absorbing substance and to the thickness of the absorbing
material (path length). Absorbance is also called optical
density
the combination of eq 1 & 2 we will get
A= Kct
A= ℇct (K=ℇ)
where ℇ – molar absorptivity, t –
thickness/pathlength, and c – molar
concentration

19. Molar Absorptivity, ε = A / c l (where A= absorbance, c =
sample concentration in
moles/liter & l = length of light
path through the sample in
cm.)
Because the absorbance of a sample will be proportional to
the number of absorbing molecules in the spectrometer light
beam (e.g. their molar concentration in the sample tube), it is
necessary to correct the absorbance value for this and other
operational factors if the spectra of different compounds are
to be compared in a meaningful way. The corrected
absorption value is called "molar absorptivity", and is
particularly useful when comparing the spectra of different
compounds and determining the relative strength of light
absorbing functions (chromophores). Molar absorptivity (ε)
is defined as:

20. Deviation from Beer-Lamberts
law
• High concentration of analyte
• Scattering of light due to particulates in
sample
• Fluoresecence or phosphorescence of the
sample
• Non-monochromatic radiation

21.  The blank contains all substances
except the analyte.
 Is used to set the absorbance to zero:
Ablank = 0
 This removes any absorption of light
due to these substances and the cell.
 All measured absorbance is due to
analyte.

22. Nature of Shift Descriptive Term
To Longer Wavelength Bathochromic
To Shorter Wavelength Hypsochromic
To Greater Absorbance Hyperchromic
To Lower Absorbance Hypochromic
Terminology for Absorption Shifts

27.  The spectrophotometer technique is to measures
light intensity as a function of wavelength.
 It does this by:
1. diffracting the light beam into a spectrum of
wavelengths
2. direct it to an object
3. receiving the light returned from the object
4. detecting the intensities with a charge-coupled
device
5. displaying the results as a graph on the detector
and then the display device
Spectrophotometer

28. block diagrammatic representation of UV-spectrophotometer

29. Spectrophotometer:
a) Single-beam
b) Double-beam
[4]

30. Components of spectrophotometer
 Source
 Monochromator
 Sample compartment
 Detector
 Recorder
Instrumentation

31. LIGHT SOURCES
Characteristics of good light source:
1. High intensity but small surface area
2. Wide spectral range
3. Stable output
4. Long life at reasonable cost
UV Spectrophotometer
1.Hydrogen or Deuterium Lamp (Wavelength Range :190~420nm)
2.Mercury Lamp
3.Xenon Lamp (Wavelength Range :190~800nm)
Visible Spectrophotometer
1.Tungsten Lamp (Part of the UV and the whole of the Visible; Wavelength
Range :350~2500nm)
UV-Vis spectrophotometer have both deuterium & tungsten lamps.

33. Accepts polychromatic input light from a lamp and outputs
monochromatic light
Characteristics desired of a monochromator
1. minimum absorption of light as it passes through
the system
2. High degree of accuracy in wavelength selection
3. High spectral purity over a broad spectral range
Components : Entrance slit, Dispersion device, Exit slit.
The resolving element are of two kinds namely,
Prisms Simple glass prisms are used for visible range. For
UV region silica, fused silica or quartz prism is used.
Fluorite is used in vaccum UV range.
Gratings are often used in the monochromators of
spectrophotometers operating in UV, visible and infra red
regions. Their resolving power is far superior to that of
prisms & they yield a linear resolution of spectrum.
MONOCHROMATOR

35. UV Spectrophotometer
Quartz (crystalline silica)
Visible Spectrophotometer
Glass
SAMPLE CONTAINERS / SAMPLE
CELLS / CUVETTES

36. Three common types of detectors are used
I. Barrier layer cell
II. Photo cell detector
III. Photomultiplier , Photo voltaic cells
DETECTORS
Convert radiant energy (photons) into an electrical signal
The photocell and phototube are the simplest
photodetectors, producing current proportional to the
intensity of the light striking them

37. RECORDERS
Display devices:
The data from a detector are displayed by a readout
device, such as an analog meter, a light beam
reflected on a scale, or a digital display , or LCD
The output can also be transmitted to a computer or
printer

38. 1. Concentration measurement
 Prepare samples
 Make series of standard solutions of known
concentrations
APPLICATIONS

39. APPLICATIONS
 Set spectrophotometer to the λ of maximum light
absorption
 Measure the absorption of the unknown, and
from the standard plot, read the related
concentration

40. APPLICATIONS
2. Detection of Impurities
 UV absorption spectroscopy is one of the
best methods for determination of impurities in
organic molecules.
Additional peaks can
be observed due to
impurities in the
sample and it can be
compared with that of
standard raw material.

41. APPLICATIONS
3. Structure elucidation of organic compounds.
 From the location of peaks and combination of
peaks UV spectroscopy elucidate structure of
organic molecules:
o the presence or absence of unsaturation,
o the presence of aromatic ring

42. APPLICATIONS
4. Chemical kinetics
 Kinetics of reaction can also be studied using
UV spectroscopy. The UV radiation is passed
through the reaction cell and the absorbance
changes can be observed.

43. Limitations of UV-visible spectrosco

44. 1. Sample
UV-vis spectroscopy works well
on liquids and solutions, but if
the sample is more of a
suspension of solid particles in
liquid, the sample will scatter the
light more than absorb the light
and the data will be very
skewed. Most UV-vis
instruments can analyze solid
samples or suspensions with a
diffraction apparatus (Figure),
but this is not common. UV-vis
instruments generally analyze
liquids and solutions most
efficiently.Schematic representation of the
apparatus for collecting UV-vis
spectra from solid materials

45. 2. Calibration and reference
• A blank reference will be needed at the very beginning
of the analysis of the solvent to be used (water,
hexanes, etc), and if concentration analysis needs to be
performed, calibration solutions need to be made
accurately .
• If the solutions are not made accurately enough, the
actual concentration of the sample in question will not
be accurately determined.

46. 3. Choice of solvent
Every solvent has a UV-vis absorbance cutoff wavelength. The
solvent cutoff is the wavelength below which the solvent itself
absorbs all of the light. So when choosing a solvent be aware of
its absorbance cutoff and where the compound under
investigation is thought to absorb. If they are close, chose a
different solvent. Table below provides an example of solvent
cutoffs.
UV absorbance cutoffs of various common
solvents
solvents UV absorbance cutoff
(nm)
Acetone 329
Benzene 278
Dimethylformamide (DMF) 267
Ethanol 205
Toluene 285
water 180

47. 4. Choice of container
The material the cuvette (the sample holder) is made from will
also have a UV-vis absorbance cutoff. Glass will absorb all of
the light higher in energy starting at about 300 nm, so if the
sample absorbs in the UV, a quartz cuvette will be more
practical as the absorbance cutoff is around 160 nm for quartz
Three different types of cuvettes commonly used, with different usable
wavelengths.
Material Wavelength range (nm)
Glass 380-780
Plastic 380-780
Fused quartz Below 380

48. 5. Concentration of solution
To obtain reliable data, the peak of absorbance of a given
compound needs to be at least three times higher in
intensity than the background noise of the instrument.
Obviously using higher concentrations of the compound in
solution can combat this. Also, if the sample is very small
and diluting it would not give an acceptable signal, there
are cuvettes that hold smaller sample sizes than the 2.5 mL
of a standard cuvettes. Some cuvettes are made to hold
only 100 μL, which would allow for a small sample to be
analyzed without having to dilute it to a larger volume,
lowering the signal to noise ratio.

49. JOB EFFECT
If we continue to take measurements beyond the
colour reagent limit, it is observed that the linearity
of the Beer- Lambert calibration does not continue
indefinitely but forms a plateau at a point which
indicates that there is insufficient reagent to
produce any more colour. This phenomenon is
known as job effect.
To extrapolate beyond the linear portion of the
curve, therefore, would potentially introduce
enormous errors.