ATOMIC ABSORPTION SPECTROSCOPY
much more sensitive method
requires the sample to first be atomised
The sample solution is injected into this and vaporises completely
Light of the right wavelength is shone through the sample – the
amount absorbed is related to
the
concentration of the metal in the sample
require calibration using standard solutions
ATOMIC SPECTROSCOPY
measures transitions between the electronic energy levels of
(metal) atoms
Spectroscopy normally implies that a spectrum of
absorption or emission versus
wavelength (or frequency) is obtained at a
fixed concentration.
Spectrometry usually measures absorption (or emission)
at a single wavelength (frequency) over a range of concentrations.
- turns the sample into atoms and measures photons emitted or absorbedas a result of
transition of an electron between energy levels
atomic emission - creates colours when metallic salts are burned in flames
flame photometry - samples dissolved in a solvent are volatilised and sprayed into a flame.
- depends on being able to create a significant population in the excited states
- difficult at flame temperatures.
- widely used in the analysis of blood found coupled with more complicated excitation sources such as plasmas
Electrochemical method
measure pH
HA(aq) + H2O(l) ⇋ H3O﹢ (aq) + A﹣ (aq)
pH = −log10 [H3O﹢]
pH is measured using an
electrode, using the
potential (voltage) between an
Ag/AgCl and the solution for which the pH is to be measured
The meter measures the
effect of the H3O+ ions in the solution on the
pH-sensitive glass
membrane on the electrode potential
between buffer and reference electrode
electrodes which are sensitive to other species :
- ion-selective electrodes
- dissolved-oxygen sensors
Quantifying absorption strength
Spectriscopic experiments tent to involve passing light of some particular range of wavelength through a sample and recording what gets absorbed
Beer-Lambert law , the absorbance is given by
A = −log10(I/Io) = ε × c × ℓ
I0 is the incident intensity (at a given wavelength),
I is the intensity after passing through the sample,
ε is the molar absorptivity (extinction coefficient) (usually m2 mol-1),
c is the concentration (mol m-3) and
ℓ is the path length through the sample (m)
ε =A / c × ℓ
UV-VISIBLE SPECTROSCOPY
Electromagnetic Radiation & Spectroscopic Transition
↳ consists of orthogonal oscillation electric and magnetic fields
Main descriptive parameters :
• The wavelength (λ, m)
• The frequency (ν, s−1 or Hz)
ν = c/λ
• The wavenumber (⊽ , cm-1)
⊽ = 1/λ
E = hν = hc/λ= hc⊽
When the frequency ,ν of the radiation matches the energy gap between two quantum states in an atom or molecules , the absorption may occur
There are three types of absorption that can occur :
- stimulated absorption ( used for spectroscopy )
- stimulated emission ( used for laser )
- spontaneous emission ( fluorescence / phosphorescence )
UV-VISIBLE (electronic) SPECTROSCOPY
↳ measures transition between electronic energy level
- The most common transition are from the Highest Occupied Molecular Orbital (HOMO) to Lowest Unoccupied Molecular Orbital (LUMO)
If you know the actual values of the energy levels , you can calculate the energy gap and thus the frequency or even wavelength
Colored compound means that it has absorption bands in the visible region
- the UV-visible spectrum will shows the exact positions and intensities of these absorption
HOMO-LUMO band gap will have the highest peak (wavelength) as can be seen in the spectrum
Other transition to higher energy level can also occur at shorter wavelength
These are due to combined electronic and vibration transition
R M I V U X G
VISIBLE (700-350nm)
UV (350-200nm)
So typical range for UV-visible spectrum is between 200-700nm
Electric dipole transition mainly occur between orbitals of the same symmetry
When the HOMO is a σ orbital, we get σ→σ* transitions.
• These are generally high energy – see, for example, ethane
• They would be quite strong if we could measure them
• This is a region called the ‘vacuum ultraviolet’ because air absorbs in these regions.
Detection in HPLC
REFRACTIVE INDEX DETECTOR
The solvent has refractive index
the presence of analyte will change the refractive index
NON-SPECIFIC because it doesn't specify the species
UV ABSORPTION
Most compound have some absorption in the UV
- Use solvent with no absorption
Some detector is single wavelength ( 254nm)
NON-SPECIFIC because no spectral resolution
But spectra of analytes can be recorded (UV- visible spectroscopy) with very specific kind of spectrometer (diode array)
Notes to UV-visible spectroscopy
Choosing solvent system
- A constant composition of solvent - ( isocratic elution )
- A variable composition of solvent - ( gradient elution )
Isocratic elution
1 solvent or 1 mixture of solvent of constant composition during elution of compound
- Commonly used for routine analysis of 1 or 2 compound in a single run
➤ This method employs 1 mobile phase (1 solvent)
- Polar analyte might elutes (keluar) very quickly while the non polar compound will left the
phase very slow
- This will takes longer time to run
➤A mixture of 2 mobile phases with same composition
- 90% polar 10% non polar
- better run time
Gradient elution
2 or more solvents programmed to change composition of POLARITY during elutions of compound
- This method employs 2 or more solvent system that differ significantly in polarity
- The mobile phase is programmed to change in composition during elution
a bit like temperature programming in GC
- used to shorten the retention time of strongly-retained species
➤ When polar compound has left the stationary phase . The mobile phase is programmed to non polarity
NURBALQIS BINTI ZAILAN
Allah S.W.T.
you 
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