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An Overview
of Chromatography and Spectroscopy
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Chromatographic Techniques
-Thin layer and column chromatography
-Gas Chromatography (GC) -High Performance Liquid Chromatography (HPLC)
Spectroscopic Techniques
- Colorimetry
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Chromatography
A technique exploiting the interaction of the components of a mixture with a stationary phase and a mobile phase (solvent) in order to separate the components.
Components are separated by different levels of adsorption to the stationary phase and solubility in the the mobile phase.
Types of Chromatography
Paper Chromatography and Thin Layer Chromatography (TLC)
Column Chromatography
Allow solvent to adsorb up the plate, drawing the sample with it.
x
Solvent front
a
b c
Rf of Rf of
= a/x = b/x
Rf of
= c/x
standards
A+B+C
A+B+C ?
Column Chromatography
A mixture is applied to a solid support in a chromatography column, and eluted by a solvent.
Elute with solvent
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Absorbent medium tap Cotton wool plug
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Column in oven up to approx. 300 C. Substance must be able to vaporise and not decompose
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Qualitative
mixture of alcohols
air
But
Quantitative
Calibration graph of a series of standards of known concentration plotting area under peak vs concentration
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A r e a u n d e r p e a k
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A mixture is injected into a steel-jacketed chromatography column and eluted with solvent at high pressure (4000psi or approx 130 atm).
Inject sample as gas or liquid. A solid component can be dissolved in solvent but a solvent peak will also be seen.
UV detector
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STATIONARY PHASES
The surface of the stationary phase can be altered to create a surface wirh different bonding properties in TLC, column chromatography, GLC and HPLC.
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STATIONARY PHASES
(NORMAL POLARITY)
Silica or alumina possess polar sites that interact with polar molecules.
silica
O HO Si O
Polar Group
STATIONARY PHASES
(REVERSE POLARITY)
If the polar sites on silica or alumina are capped with non-polar groups, they interact strongly with non-polar molecules.
silica C18 phase
O Si O Si Me O Me
STATIONARY PHASES
(CATION EXCHANGE)
Silica is substituted with anionic residues that interact strongly with cationic species (+ve charged)
Cations exchange Na+
Na O O S O
silica
+ve charged species adhere to the support and are later eluted with acid (H+) Most +ve.Least +ve
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STATIONARY PHASES
(ANION EXCHANGE)
Silica is substituted with cationic residues that interact strongly with anionic species (-ve charged)
Anions exchange ClMe Cl Me N CH2 Me
silica
-ve charged species adhere to the support and are later eluted with acid (H+) Most -ve.Least -ve
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STATIONARY PHASES
(SIZE EXCLUSION)
Size exclusion gels separate on the basis of molecular size. Individual gel beads have pores of set size, that restrict entry to molecules of a minium size.
Large molecules elute fast (restricted path), while small molecules elute slowly (long path length) Larger molecules.Smaller molecules
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Light waves all travel at the same speed through a vacuum but differ in frequency and, therefore, in wavelength.
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Spectroscopy
Utilises the
Absorption:
Low energy electrons absorb energy to move to higher energy level
Emission:
Excited electrons return to lower energy states
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Absorption v. Emission
Energy is emitted by electrons returningto lower energy levels 3rd Excited 2nd States 1st Energy is absorbed as electrons jump to higher energy levels Ground State
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Sodium
Hydrogen
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Calcium
Absorption Spectra
Sodium
Concentration
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An introduction to Colorimetry
Colorimetry is a quantitative technique which makes use of the intensity in colour of a solution is directly related to the concentration of the coloured species in it. Colorimetry can be used if the substance to be analysed is coloured, or if it can be made coloured by a chemical reaction. The concentration of the unknown solution can be estimated by the naked eye by comparing its colour to those of a series of standard solutions prepared by successive dilution. However at low concentrations, colour may not be detected.
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A more accurate quantitative analysis can be made using an instrument called a Colourimeter. The light
source of a kind that will be absorbed by the solution, ie if the solution is blue then light of a colour other than blue will be absorbed by it. Simple colourimeters allow a choice of three wavelengths using blue, green and red Light Emitting 28 D iodes (LEDs)
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Red LED
Green LED
Blue LED
In this example, the blue solution would absorb red (or green) and reflect blue. The chosen red LED is passed through the a transparent plastic or glass cell (cuvet) of fixed pathlength (1cm) containing the blue solution to be investigated and a Detector measures the amount of light absorbed measured.
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Collect data
A set zero adjustment enables the instrument to factor out any absorbance of the solvent and the material the cuvet is made from.
Absorbance
Concentration
0.0 0.125 0.250
0.380
0.50 unknown
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Note that graphs may not be linear over a wide range of concentrations
0.50
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0.50
The concentration of an unknown solution of a food colouring can be determined by measuring its absorbance and reading the concentration from the calibration graph. Using the data in the graph above, if a sample of this food colouring was found to have an absorbance of 0.35, then its concentration would be ______ M.
Questions What would happen to absorbance if the path length of the cuvet was doubled? What would happen if the cuvet was handled on the transparent outer surface?
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AAS Operation
Flame
Display
Monochromator
Detector Monochromator
Lens
Flame
Solution Display Amplifier
Ions in Flame
Transmittance
used by industry analysis of ores for metal content quality control of metals in steel testing water for metals ions analysing food and pharmaceuticals for metal ions
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A Source of Error
Another species may be absorbing at the same wavelength.
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UV-Visible Spectroscopy
A UV-visible spectrophotometer measures the amount of energy absorbed by a sample.
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The optics of the light source in UV-visible spectroscopy allow either visible [approx. 400nm (blue end) to 750nm (red end) ] or ultraviolet (below 400nm) to be directed at the sample under analysis.
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Why are carrots orange? Carrots contain the pigment carotene which absorbs blue light strongly and reflects orange red and so the carrot appears orange.
400nm
500nm
600nm
700nm
BLUE
GREEN
Y E L LO W
O R A N G E
RED
420nm
520 nm
600nm
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Carotene beta-Carotene forms orange to red crystals and occurs in the chromoplasts of plants and in the fatty tissues of plant-eating animals.
Absorbance is set to 0% or light transmitted using a solvent blank in a cuvet. This compensates for absorbance by the cell container and solvent and ensures that any absorbance registered is solely due to the component under analysis.
The sample to be analysed is placed in a cuvet (as for colorimetry).
Qualitative analysis is achieved by determining the radiation absorbed by a sample over a range of wavelengths. The results are plotted as a graph of absorbance/transmittance against wavelength, which is called a UV/visible spectrum.
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The UV- Visible absorption spectrum for carotene in the non-polar solvent, hexane
I N T E N S I T Y O F
400nm
700 nm
A B S O R P T I O N
ultra- violet
visible
infrared
320nm
460nm
540nm
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Although the light absorbed is dependent on pathlength through the cell, a usual standard 1cm pathlength is used so that pathlength can effectively be ignored.
Quantitative analysis is achieved in a manner similar to colorimetry. The absorption of a sample at a particular wavelength (chosen by adjusting a monochromator) is measured and compared to a calibration graph of the absorptions of a series of standard solutions.