C19 - Modern Analytical Techniques II

Paper chromatography key elements

• Mobile phase: liquid or gas that carries the mixture through the system

• Stationary phase: solid that does not move with the mobile phase

Separation process of paper chromatography

• Mobile phase moves mixture components based on solubility

  • More soluble ones move faster

• Stationary phase holds onto components differently through absorption

  • More absorptive ones move slower

Rf

• The retardation factor is the ratio of distance travelled to distance travelled by component

• 0 to 1

• Distance by spot/distance by solvent

HPLC key elements

• Stationary phase consists of small particles of solid (e.g. silica bonded to hydrocarbons) tightly packed into a column

• Mobile phase is polar mixture such as methanol and water

HPLC process

• Mixture is injected into solvent stream

• Carried through the column

• Components are attracted to the solid by varying degrees

  • Results in different travel times through the column

• As liquid exits the column, passes through a UV detector that measures UV light absorbance

• Chromatogram is produced

  • Shows retention time

Gas chromatography key elements

• Mobile phase

  • Sample injected into a stream of gas

• Stationary phase

  • A non-polar boiling liquid absorbed into solid support

Gas chromatography process

• Liquid sample is vaporised

• An inert carrier gas then transports the vaporised sample through a chromatographic column containing the stationary phase

• Components of the mixture are attracted to the solid by varying degrees

  • Results in different travel times  through the column

• As they exit a detector records the separation

• Like HPLC, components are identified by their retention times

Chromatography and mass spectrometry

Why

• Mass spectrometry is used to identify substances with m/z ratio

• Can give confusing results when analysing mixtures

• GC and HPLC can separate the mixtures and the mass spectrometry can identify the components

 

How GC-MS

• Sample is first separated using gas chromatography

• Separated components are introduced into a mass spectrometer

  • Are ionised

• A distinct mass spectrum for each component is generated

• Each substance is then identified by comparing mass spectrum to reference spectra

Combined techniques

Combustion analysis

• Can determine the empirical and molecular formula of organic compounds

 

Sequence of techniques for structure determination

• Mass spectrometry - molecular mass found

• IR spectrometry - finds functional groups

• NMR - chemical environments

NMR

• Determines molecular structure

  • Analyses the changes in magnetic properties of atomic nuclei

• Powerful analytical technique that is used to understand the structure of molecules

• Two types

  • 13C provides information about where carbons are

  • 1H is where the hydrogen atoms are

How NMR works

• When a molecule is placed in an external magnetic field

  • Nuclei within molecule experiences varying degrees of shielding from external magnetic field

  • Due to local chemical environment

• Electron density surrounding each nucleus acts as a magnetic shield

  • Protects it from the full strength of the external magnetic field

  • Nuclei in different environments experiencing slightly different resonance frequencies

• More shielded a nucleus is, the lower its resonance frequency will be

  • Requires less energy to flip its spin state

Chemical shift

• In NMR, chemical shift is a measure of the difference in resonant frequency

• TMS is used as the standard in NMR spectrometry

  • Has a single absorption peak (all carbons and hydrogens in same chemical environments)

  • Appears at a lower frequency which is to the right of more analytes

• TMS is assigned chemical shift value of 0 ppm

Number of peaks

Shows distinct carbon environment

Differing bonding of chlorine and hydrogen atoms to each carbon changes the electron density

Determining molecular structure

• Count number of distinct peaks to understand the variety of carbon environments

• Use chemical shift data to hypothesis the types of carbon present

• Combine all available evidence to propose molecular structure

Proton NMR graph

• Graph

  • Each peak represents a hydrogen environment

  • Peak area is proportional to number of hydrogens

  • Position of peak on scale can help identify the type of hydrogen atom

Spin-spin coupling

• Occurs among adjacent hydrogen atoms and results in the splitting of NMR peaks

• Occurs only between hydrogens in neighbouring carbons

• Reveal the number of non-equivalent protons on adjacent carbon atoms