Analytical methods

Chromatography – general principles

Mobile phaseStationary phase Compound for analysis - solute

Principles • The sample to be analyzed is introduced in

small volume to the stream of mobile phase. The solute’s motion through the column is slowed by specific chemical or physical interactions with the stationary phase as it traverses (passes through) the length of the column. The amount of retardation depends on the nature of the solute, stationary phase and mobile phase composition.

HPLC – High Performance Liquid Chromatography

Mobile phase – liquid solvent Stationary phase - small silica beads (typical size = 5 micro metres) (1) Solvent reservoirs, (2) Solvent degasser, (3) Gradient valve, (4) Mixing vessel for delivery of the mobile phase, (5) High-pressure pump, (6) Switching valve in "inject position", (6') Switching valve in "load position", (7) Sample injection loop, (8) Pre-column, (9) Analytical column, (10) Detector (i.e. IR, UV), (11) Data acquisition, (12) Waste or fraction collector.

GLC or GC – Gas Chromatography

GC in more detail• Stationary phase = 1.5 - 10 m in length and have

an internal diameter of 2 - 4 mm• Mobile phase – a gas - typical carrier gases

include helium, nitrogen, argon, hydrogen and air. Which gas to use is usually determined by the detector being used

• Gas-liquid chromatography (GLC), or simply gas chromatography (GC), is a common type of chromatography used in organic chemistry for separating and analyzing compounds that can be vaporized without decomposition

Fluorescence spectroscopy

• Fluorescence spectroscopy aka fluorometry or spectrofluorometry, is a type of electromagnetic spectroscopy which analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light of a lower energy, typically, but not necessarily, visible light. A complementary technique is absorption spectroscopy.

Atomic absorption spectroscopy

atomic absorption spectroscopy is a technique for determining the concentration of a particular metal element in a sample. The technique can be used to analyze the concentration of over 70 different metals in a solution.

IR or Infrared Spectroscopy• Symmetrical and antisymmetrical stretching,

scissoring, rocking, wagging and twisting

Infrared spectroscopy (IR spectroscopy) is the subset of spectroscopy that deals with the infrared region of the electromagnetic spectrum. It covers a range of techniques, the most common being a form of absorption spectroscopy. As with all spectroscopic techniques, it can be used to identify compounds or investigate sample composition

Bond Compound Type Frequency range, cm-1

C-HAlkanes

2960-2850(s) stretch1470-1350(v) scissoring and bending

CH3 Umbrella Deformation1380(m-w) - Doublet - isopropyl, t-butyl

C-H Alkenes3080-3020(m) stretch1000-675(s) bend

C-HAromatic Rings 3100-3000(m) stretchPhenyl Ring Substitution Bands 870-675(s) bendPhenyl Ring Substitution Overtones 2000-1600(w) - fingerprint region

C-H Alkynes3333-3267(s) stretch700-610(b) bend

C=C Alkenes 1680-1640(m,w)) stretchCºC Alkynes 2260-2100(w,sh) stretchC=C Aromatic Rings 1600, 1500(w) stretch

C-O Alcohols, Ethers, Carboxylic acids, Esters 1260-1000(s) stretch

C=O Aldehydes, Ketones, Carboxylic acids, Esters 1760-1670(s) stretch

O-H

Monomeric -- Alcohols, Phenols 3640-3160(s,br) stretchHydrogen-bonded -- Alcohols, Phenols 3600-3200(b) stretch

Carboxylic acids 3000-2500(b) stretch

N-H Amines 3500-3300(m) stretch1650-1580 (m) bend

C-N Amines 1340-1020(m) stretchCºN Nitriles 2260-2220(v) stretch

NO2 Nitro Compounds1660-1500(s) asymmetrical stretch1390-1260(s) symmetrical stretch

NMR - Nuclear Magnetic

Resonance