QCEChemistry

Analytical techniques

Chromatography (size)

explain how proteins can be analysed by chromatography and
electrophoresis

select and use data from analytical techniques, including mass spectrometry,
X-ray crystallography and infrared spectroscopy, to determine the structure of
organic molecules

analyse data from spectra, including mass spectrometry and infrared
spectroscopy, to communicate conceptual understanding, solve problems
and make predictions.

- Chromatography is a technique for the separation of a mixture by passing it in solution or suspension through a medium in which the components move at different rates.

• Mobile phase is the phase the flows in chromatography, moving the component of a sample at different rates over the stationary phase

▪ Usually a solvent

• Stationary phase is the phase to which the components of a chromatographic sample is adsorbed

▪ Either a solid or liquid coated onto a solid support

▪ Does not move

• Affinity - the interaction of a substance within a sample with the mobile or stationary phases

▪ High affinity means component interacts strongly with either the mobile or stationary phases

• Adsorption - the attraction of a substance within a sample to the stationary phases

▪ Adsorption is the adhesion of a substance to the surface

▪ Has affinity for the stationary phase

• Desorption - the release of a substance within a sample from the stationary phase into the mobile phase

▪ Has affinity for the mobile phase

Thin layer chromatography or paper chromatography

Column chromatography

- Stationary phase (resin or gel)is packed into glass column. Sample mixture is applied to top of packed solid. Solvent is dripped slowly and a tap at the bottom of the column allows the solvent (eluent) to leave the column at the same rate as it enters it at the other end

• Components of the mixture are carried down the column, and are repeatedly adsorbed to the stationary phase, and desorbed back to mobile phase
• Largest proteins elute first because proteins that are too large to fit into the pores of the resin pass through the column with no separation

• Small proteins spend more time inside the pores of the resin and take longer to elute

- Chromatogram is connected to bottom of column

• As components pass through the detector, peaks are formed
• The larger the area under the peak, the higher the concentration

High-performance liquid chromatography (HPLC)

- Most common form is reversed-phase HPLC

• Phases

▪ Stationary phase - silica

□ Modified to make it non-polar by attached long hydrocarbon chains

▪ Mobile phase - polar solvent such as mixture of methanol and water

• Polar molecules in the sample form strong IMF with polar mobile phase and weakly adsorbed to non-polar stationary phase. Polar molecules elute first
• Non-polar molecules spend more time in the column
• Time taken to pass through column is called the retention time (Rt)

Key differences

Electrophoresis (molecular weight)

explain how proteins can be analysed by chromatography and
electrophoresis

select and use data from analytical techniques, including mass spectrometry,
X-ray crystallography and infrared spectroscopy, to determine the structure of
organic molecules

analyse data from spectra, including mass spectrometry and infrared
spectroscopy, to communicate conceptual understanding, solve problems
and make predictions.

Analysis of proteins

- Electrophoresis is a separation technique that involves the movement of charged particles along an electric field

- Native PAGE (polyacrylamide gel electrophoresis) is electrophoresis under conditions in which the protein retains its natural shape

• PAGE is the stationary phase (or agarose for larger proteins), water is the mobile phase
• Proteins are treated with sodium dodecyl sulfate (SDS) to denature and coat proteins with negative charge

▪ Boiling proteins with SDS breaks weak bonds in the protein, loosening secondary and tertiary structures

• Battery is used to force a current - attached to negative terminal of gel
• Proteins are attracted to electrode with an opposite charge of their own. Smallest proteins will travel furthest.

Analysis of amino acids

Why do cations forms as pH lowers and anions
form as pH increases?

As pH lowers, there are more H+ ions in solution,
therefore the amine group will accept a H+, i.e act
as a base and the cation will form.
As the pH lowers OH- concentration is increasing.
Therefore H+ ions will dissociate, and neutralise
the OH-, from the acid group (COOH), from
COO-, the anion

Mass Spectroscopy (molecular mass)

explain how proteins can be analysed by chromatography and
electrophoresis
select and use data from analytical techniques, including mass spectrometry,
X-ray crystallography and infrared spectroscopy, to determine the structure of
organic molecules
analyse data from spectra, including mass spectrometry and infrared
spectroscopy, to communicate conceptual understanding, solve problems
and make predictions.

Components of mass spectrometer

- Ions are formed in the ionisation chamber where the sample is exposed to high voltages. Bonds break causing molecule to fragment.

- Ions are separated in a magnetic field based on their mass-to-charge ratio (m/z)

• Most ions have a +1 charge, meaning the m/z ratio = molecular mass of the ion

- The number of ions with different m/z values are measured by a detector and the data is recorded as a mass spectrum

Key terms

- Molecular ion peak is the significant peak with the highest m/z value. This is the molar mass of the substance.

• Isotopes may cause a small peak after the significant one - this is not the ion peak (if the ratio of the second last and last time is 10:1)

- Base peak is the peak with the greatest abundance

X-ray Crystallography (shape)

explain how proteins can be analysed by chromatography and
electrophoresis
select and use data from analytical techniques, including mass spectrometry,
X-ray crystallography and infrared spectroscopy, to determine the structure of
organic molecules
analyse data from spectra, including mass spectrometry and infrared
spectroscopy, to communicate conceptual understanding, solve problems
and make predictions.

- X-ray crystallography is the study of crystal structures

• X-rays are used to bombard crystalline lattice and characteristic diffraction pattern is used to determine the molecules' structure
• Diffraction pattern obtained from X-ray scattering off the ordered structure of molecules or atoms in crystals is used to reconstruct the electron density
• Electron density map is used to represent the molecular structure.

- Observations

• Identity of atoms

▪ Atoms with higher atomic masses and therefore more electrons diffract X-rays more than lighter atoms

• Distance between atoms

▪ Single bonds will be further apart than double bonds

• Bond angles

Generalisation of analytical techniques and their purposes

Infrared Spectroscopy
explain how proteins can be analysed by chromatography and
electrophoresis
select and use data from analytical techniques, including mass spectrometry,
X-ray crystallography and infrared spectroscopy, to determine the structure of
organic molecules
analyse data from spectra, including mass spectrometry and infrared
spectroscopy, to communicate conceptual understanding, solve problems
and make predictions.

- Infrared spectroscopy looks at how infrared radiation interacts with organic molecules

• Absorbed energy changes the vibration of molecules

▪ Higher mass will absorb lower frequency radiation (higher wavelength)

▪ Stronger bonds will absorb higher frequency radiation (lower wavelength)

▪ Triple bonds require more energy to stretch than single bonds

• Is used to identify functional groups, but does not provide information about size or structure

- Transmittance is the ratio of the intensity of transmitted light (light that has passed through the sample) to incident light (light first put out by light source)

• Higher transmittance means more of the light has been absorbed

- Fingerprint region is from 1500 to 800 cm-1 (unique to each compound)

- Peaks are formed in graph when light has been absorbed

• Broad peak is usually if the properties of the bonds are different (e.g the terminal C-H bond is connected to 3 hydrogens and 1 carbon, while the middle C-H bond is connected to 2 hydrogens and 2 carbons, meaning the energy absorbed is slightly different)
• In general, the higher the different types of bonds, the broader the peak

- Acids have a typical hook shape

- Aldehydes form a doublet

- Ketone form single peak