Instrumental Analytical Techniques

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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- Atomic Absorption Spectroscopy(AAS)

- UV-Visible Spectroscopy (UV-Vis)

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

Gas Liquid Chromatography (GLC)

High Performance Liquid Chromatography (HPLC)

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Thin Layer (and Paper) Chromatography

TLC plates are inert supports (glass, plastic, aluminium) with a thin veneer of chromatographic media (silica,etc)
Apply a concentrated drop of sample () with a capillary or dropping tube to bottom of plate (origin pencil line) Stand plate in a sealed vessel. carefully add solvent (keep solvent level below sample).

Allow solvent to adsorb up the plate, drawing the sample with it.

Thin Layer (and Paper) Chromatography

The ratio of distance travelled by the component (from origin) compared with the distance travelled by the solvent front (from origin) is called the Rf value.

x
Solvent front

a
b c

Rf of Rf of

= a/x = b/x

Rf of

= c/x

Thin Layer and Paper Chromatography

A solution of a mixture is applied as a spot/band at the bottom of the plate and allowed to travel with the solvent up the plate.
Mixed standards Unknown + standards

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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Gas Liquid Chromatography

Gas Liquid Chromatography

A mixture is injected into a very thinsteel-jacketed chromatography column. Inject sample as gas or liquid. A solid component can be dissolved in solvent but a solvent peak will also be seen. Inject sample dense liquid (on solid) SP Gas mobile phase

Column in oven up to approx. 300 C. Substance must be able to vaporise and not decompose

Extremely sensitive FID detector

Elute with inert gas

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Gas Chromatogram of High Grade Petrol

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Qualitative
mixture of alcohols
air

known Rf values under standard conditions

mixture of hydrocarbons eg petrol

But

Must be able to be vaporised up to about 300oC

Must not decompose

Quantitative

Calibration graph of a series of standards of known concentration plotting area under peak vs concentration

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Eg. How much ethanol is in the blood?

A r e a u n d e r p e a k
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Use a series of standards of ethanol to determine area under peak.

Construct calibration graph

Area (or height at first approx.) is proportional to concentration. Find area of unknown and read off concentration

0 0 0.10 0.20 0.30 0.40 0.50 Concentration of alcohol in grams/Litre

High Performance Liquid Chromatography

(HPLC)

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.

Elute with solvent

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.

Normal Polarity Reverse Polarity Ion Exchange Size Exclusion

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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

Components elute in increasing order of polarity. Most polar.Least polar

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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

Components elute in decreasing order of polarity. Most non-polar.Least non-polar

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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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Regions of the Electromagnetic Spectrum

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 and Emission

of electromagnetic radiation by atoms

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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Emission Spectra of Elements

Continuous

Sodium

Hydrogen

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Calcium

Absorption Spectra

Sodium

For other Spectra, click on the hyperlink below: http://www.achilles.net/~jtalbot/data/elements/index.html

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The Spectroscopic Techniques are based on the fact that

Light absorbed (Absorption)

is directly proportional to the

Concentration

of the absorbing component.

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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

Detector measures red light absorbed

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

0.00 0.20 0.40 0.59 0.78 0.35

0.380
0.50 unknown

concentration of a species in solution is proportional to the light absorbed

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Absorbance 1.00 0.80 0.60 0,40 0.20 0

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Note that graphs may not be linear over a wide range of concentrations

0.10 0.20 0.30 0.40 Concentration in mol/Litre

0.50

1.00 0.80 0.60 0.40 0.20 0 0

Note that graphs may not be linear over a wide range of concentrations

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0.10 0.20 0.30 0.40 Concentration in mol / Litre

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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Atomic Absorption Spectroscopy

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Absorption Wavelengths of Iron

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Atomic Absorption Spectrophotometer (AAS)

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AAS Operation

Hollow Cathode Lamp

Gas Mixture Adjustment 39 Controls

Flame

Display

Monochromator

Atomic Absorption Spectrometer

Atomised sample in flame Lens Hollow Cathode Lamp

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Detector Monochromator

Lens

Flame
Solution Display Amplifier

Close-up view of AAS

Electrons return to ground Less energy is Ions absorb energy, state,and photons emitted in transmitted to detector jumpall to directions excited state

Hollow Cathode Lamp emits several unique wavelengths of light

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Ions in Flame

Transmittance

Atomic Absorption Spectrometry

measures small concentrations of metal ions in solution
Al, As, Au, B, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ge, K, Li, Mg, Mn, Mo, Na, Ni, Pb, Si, Sr, Ti, V, W and Zn

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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Advantages of using AAS

very sensitive:
can detect concentrations as small as a few parts to g / Litre (parts per billion) generally very specific: set wavelength is strongly absorbed by the particular metal ion being analysed (and not by other components)
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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.

Molecular formula: C40H56

Molar Mass Melting point
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537 178 - 179 C

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.

What can be analysed?

In its quantitative form, UV-visible spectroscopy can be used to detect coloured species in solution eg. bromine , iodine and organic compounds or metal ions that are coloured, or can be converted into a coloured compound.
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