1 Instrumental Analytical Techniques An Overview of Chromatography and Spectroscopy

2  Chromatographic Techniques -Thin layer and column chromatography -Gas Chromatography (GC) -High Performance Liquid Chromatography (HPLC)  Spectroscopic Techniques - Atomic Absorption Spectroscopy(AAS) - Colorimetry - UV-Visible Spectroscopy (UV-Vis)

Chromatographic Chromatography is the term used to describe a separation technique in which a mobile phase carrying a mixture is caused to move in contact with a selectively absorbent stationary phase. 3

4 Types of Chromatography Column Chromatography Gas Liquid Chromatography (GLC) High Performance Liquid Chromatography (HPLC) Paper Chromatography and Thin Layer Chromatography (TLC)

1.the mobile phase is also a solvent, and the stationary phase is a thin of finely divided solid, such as silica gel or alumina, supported on glass or aluminium. 2.Thin layer chromatography is similar to paper chromatography

Thin Layer (and Paper) Chromatography  Thin layer chromatography in that it involves spotting the mixture on the plate and the solvent (mobile phase) rises up the plate in the chromatography tank 6  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.

7 Thin Layer and Paper Chromatography The components of the mixture move up the paper with the solvent at different rates due to their differing interactions with the stationary and mobile phases. ABCA+B+C standards Mixed standards Unknown + standards A+B+C ? ?

8 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 R f value. Solvent front x a b c R f of = a/x R f of = b/x R f of = c/x

R f = Distance the solute moves Distance the solvent front moves

Column Chromatography In column chromatography, the mobile phase is again a solvent, and the stationary phase is a finely divided solid, such as silica gel or alumina. Chromatography columns vary in size and polarity. if the sample was too soluble the mobile phase (solvent) would move the solutes too quickly, resulting in the non-separation of the different constituents

11 Column Chromatography A mixture is applied to a solid support in a chromatography column, and eluted by a solvent. Elute with solvent 2134 Absorbent medium Cotton wool plug tap

Gas Chromatography A gas is the mobile phase and the stationary phase can be either a solid or a non- volatile liquid.

13 Elute with inert gas Column in oven up to approx. 300 C. Substance must be able to vaporise and not decompose Gas Liquid Chromatography A mixture is injected into a very thin“steel-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. FID detector Gas mobile phase dense liquid (on solid) SP Inject sample Extremely sensitive

14 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). Elute with solvent UV detector Inject sample as gas or liquid.A solid component can be dissolved in solvent but a solvent peak will also be seen.

Spectroscopy  Spectroscopy may also be defined as the study of the interaction between light and matter. UV-Visible Spectroscopy (UV-Vis) UV-Visible Spectroscopy (UV-Vis)  Qualitative Analysis by UV-Visible Spectroscopy (UV-Vis) UV-VIS spectroscopy studies the electronic transitions of molecules as they absorb light in the UV and visible regions of the electromagnetic spectrum. The data is used to produce absorbance spectra.

1. prism used to analysis the light to seven light 2.The function of the monochromator is to select a single wavelength or frequency from the light source. 3.sample cells composed of basic salts, such sodium chloride or potassium bromide, are commonly used because they will not absorb light 4.The detector measures the quantity of radiation that passes thru the sample by converting it to an electrical signal

18 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.

20 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

21 Absorption v. Emission Ground State 1st 2nd 3rd Energy is absorbed as electrons jump to higher energy levels Energy is emitted by electrons returning to lower energy levels Excited States photo

Electronic Transitions: 1. Transitions involving π, σ, and n electrons 2. Transitions involving charge-transfer electrons 3. Transitions involving d and f electron s 1. Transitions involving π, σ, and n electrons

σ → σ* Transitions: * The energy required for an electron in a bonding σ orbital to get excited to the corresponding anti bonding orbital is large. For example, methane (which has only C-H bonds, and can only undergo (σ → σ* transitions) shows an absorbance maximum at 125 nm and are thus not seen in typical UV-VIS. spectra ( nm). σ σ* Methane (125nm)

n → σ* Transitions: * The energy required for an electron in a bonding n orbital to get excited to the corresponding anti bonding orbital is large. For example, alcohols, amines (which has only C-OH bonds and NH 2, and can only undergo (n → σ* transitions) compounds absorb light having wavelength in the range nm and are thus not seen in typical UV-VIS. spectra ( nm). n σ* Methane (125nm)

π → π* Transitions: * The energy required for an electron in a bonding π orbital to get excited to the corresponding anti bonding orbital is large. For example, alkenes absorb at nm π π*π* Methane (125nm)

n → π* Transitions The energy required for an electron in a bonding n orbital to get excited to the corresponding anti bonding orbital is large. For example, ketone(which has only c=o bonds aliphatic Ketones absorb at 280nm n π*

Quantitative UV/vis Analysis Quantitative UV/vis is used to determine the concentration of an analyte, which can absorb in this region, in a solution. Beer's Law, which gives a linear relationship between absorbance and concentration for dilute solutions, is used. A calibration plot is formed by measuring the absorbance of a series of analyte solutions with different known concentrations and the concentration of the target analyte in the sample under study is determined from the plot The Beer-Lambert Law A=  bc

 ) is the molar absorptivity with units of L mol -1 cm b is the path length of the sample We will express this measurement in centimetres. 3. c is the concentration of the compound in solution, expressed in mol L-1 A=  bc

30 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

31 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)

32 An introduction to Colorimetry Colorimetry can be used if the substance to be analysed is coloured, or if it can be made coloured by a chemical reaction. 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. 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.