1.4 – Chromatography Aaron Tieu 2 LEARNING OBJECTIVES • You should be able to:
qUnderstand and explain how components in a mixture are separated
based on polarities between stationary and mobile phase
qPredict the movement of components based on the polarity and charge
of molecules
qCalculate Rf values and retention times to identify components in a
mixture
qUnderstand how equilibrium can be used to separate ions in mixtures
3 SCIENCE UNDERSTANDING Chromatography techniques, including thin layer chromatography (TLC), gas chromatography (GC), high-performance liquid chromatography (HPLC), and ion chromatography (IC), involve the use of a stationary phase and a mobile phase to separate the components of a mixture. 4 ADSORPTION CHROMATOGRAPHY Do not get confused • Technique based on the principle of adsorption. with absorption!
• Adsorption: The attachment of particles to the surface of a
material (stationary phase).
• Results from the formation of interactions (primary and/or
secondary). • Stronger formation of secondary bonds results in a stronger adsorption to the surface. 5 ADSORPTION CHROMATOGRAPHY 6 ABSORPTION VS. ADSORPTION
ABsorption ADsorption Particle enters a material Particles attaches to the surface of a material 7 ADSORPTION CHROMATOGRAPHY • Affinity: the tendency of a molecule to associate with another molecule via interaction of intermolecular forces.
• All chromatography techniques involves a:
• Stationary phase: Solid material onto which components of mixture
are initially adsorbed. Ø Does not move. 8 ADSORPTION CHROMATOGRAPHY • Mobile phase: Liquid or gas that carries the components of a mixture over the stationary phase as it flows through it. Ø Components dissolve in the mobile phase (are desorbed).
Ø Different components travel at different rates.
• Stationary and mobile phases are chosen so that they have very different polarities 9 ADSORPTION CHROMATOGRAPHY 10 ADSORPTION CHROMATOGRAPHY • Components in a mixture adsorb to the stationary phase and dissolve in the mobile phase (desorb) to varying degrees.
• Components separate according to the strength of their interactions
with the stationary phase and mobile phase.
• Factors influencing interactions:
Ø Size
Ø Polarity/charge 11 ADSORPTION CHROMATOGRAPHY • REVIEW: Ø Ion-dipole interactions Ø Hydrogen bonding Increasing in strength Ø Dipole-dipole forces Ø Dispersion forces 12 SCIENCE UNDERSTANDING The rate of movement of the components is caused by the differences between the strengths of the interactions between atoms, molecules, or ions in the mobile and stationary phases. q Predict the relative rates of movement of components along a stationary phase on the basis of their polarities and charge, given the structural formulae or relative polarities of the two phases. 13 CHROMATOGRAPHY TECHNIQUES • Thin-layer chromatography (TLC)
• Gas chromatography (GC)
• High-performance liquid chromatography (HPLC)
• Ion chromatography (IC)
• Column chromatography
• Paper chromatography 14 THIN-LAYER CHROMATOGRAPHY • Separates components over thin surface of stationary phase. Ø Made of a thin layer of STATIONARY PHASE adsorbent material (silica or alumina) bonded to the surface of an inert sheet MOBILE (glass, aluminium, or polymer) PHASE 15 THIN-LAYER CHROMATOGRAPHY • Stationary phase – usually hydrated silica (SiO2) or alumina (Al2O3) Ø Polar -OH groups on surface 16 THIN-LAYER CHROMATOGRAPHY • STEPS:
• Pencil drawn ~1cm from the
bottom of the TLC plate – origin STATIONARY PHASE • Mixture to be separated prepared in solution
MOBILE PHASE 17 THIN-LAYER CHROMATOGRAPHY • Mixture spotted using a fine pipette along origin. Ø Multiple mixture can be STATIONARY PHASE spotted on the TLC plate leaving ~1cm distance.
• Solvent (mobile phase) placed in
MOBILE PHASE a beaker/jar. 18 THIN-LAYER CHROMATOGRAPHY • Lid placed on to allow atmosphere to become saturated with solvent. STATIONARY PHASE Ø Must sit below origin. Ø Must have different polarity to stationary phase MOBILE PHASE • TLC carefully placed in beaker 19 THIN-LAYER CHROMATOGRAPHY
STATIONARY PHASE
MOBILE PHASE 20 THIN-LAYER CHROMATOGRAPHY • Once mobile phase has almost travelled to the top, plate is removed. Ø Solvent front marked with a pencil STATIONARY PHASE Ø Allowed to dry
• Relative distance each component
travelled determined by calculation MOBILE PHASE of RF values 21 THIN-LAYER CHROMATOGRAPHY • TLC may contain a fluorescing agent.
• Components may be colourless.
• Treatment with UV light may
show components as darker patches 22 THIN-LAYER CHROMATOGRAPHY • Distance a substance moves along a particular stationary phase, using a particular mobile phase, is a characteristic of that substance. Ø Results often tabulated so a comparison can be made and components can be identified
Ø Applicable to all chromatography techniques.
Video: https://www.youtube.com/watch?v=CmHFVxTxkGs 23 ADSORPTION CHROMATOGRAPHY • Rate of movement of a component along a stationary phase depends on: Ø Size and polarity of component
Ø Relative polarities of stationary and mobile phase
• Follows “likes dissolve likes” principle.
• Better rationalized by discussion of the types of secondary
• Determined by: 1. Number of polar/charged groups in molecule/ion Ø More polar groups = more polar
2. Size of molecule/ion Ø Larger molecules = less polar 25 SCIENCE UNDERSTANDING Data from chromatography techniques can be used to determine the composition and purity of substances. q Calculate and apply RF values and retention times in the identification of components in a mixture. 26 RETENTION FACTOR • Distance component travels as a fraction of the distance travelled by solvent front (from origin).
• Formula:
RF = distance travelled from the origin to the centre of the component
distance travelled from the origin to the solvent front Note: RF values do not have units * Can be compared to known standard values, allowing identification of component 27 EXAMPLE Calculate the RF value of the red component
1.7 RF = 5.0 RF = 0.34
Solvent front = 5.0 cm
Red component = 1.7 cm 28 POLARITIES 29 EXAMPLE Consider the three amino acids: glycine, isoleucine and aspartic acid.
Q: Sort the amino acids from
most polar to least polar 30 EXAMPLE A: Aspartic acid is most polar, with 3 polar groups and medium length. Glycine second most polar, with 2 polar groups and a short length.
Isoleucine will be the least
polar, with 2 polar groups but the greatest length 31 EXAMPLE Q: Which of these molecules will adsorb most strongly to a polar stationary phase?
A: Aspartic Acid 32 EXAMPLE Q: Which of these molecules will be most soluble in a non- polar mobile phase?
A: Isoleucine 33 EXAMPLE Now consider these 3 amino acids as part of a mixture placed on a polar stationary phase, in combination with a non-polar mobile phase. 34 EXAMPLE Q: Which one will have a greater rate of movement along the stationary phase? (remember the mobile phase is non-polar)
A: Isoleucine 35 EXAMPLE Q: Which one will have the smallest rate of movement along the stationary phase?
A: Aspartic Acid 36 EXAMPLE Q: Which one will have the largest RF value?
A: Isoleucine 37 EXAMPLE Q: Which one will have the smallest RF value?
A: Aspartic Acid 38 EXAMPLE In a different case scenario, consider a mixture of lactic acid and tartaric acid. Their individual structures are given: 39 EXAMPLE Q: Which molecule will be more polar?
A: Tartaric acid, as it is a small
molecule with four polar groups (two carboxyl and two hydroxyl groups) 40 EXAMPLE Hydrated silica is commonly used as a stationary phase in TLC. 41 EXAMPLE Q: Would this be regarded as a polar or non-polar stationary phase?
A: Polar 42 EXAMPLE Q: Would lactic acid adsorb more or less strongly compared to tartaric acid? (remember it is less polar)
A: Less 43 EXAMPLE Q: What would a TLC plate look like for a mixture of the two acids separated by TLC? 44 EXAMPLE Q: What would a TLC plate look A: like for a mixture of the two acids separated by TLC? 45 COLUMN CHROMATOGRAPHY • Used to isolate components of a mixture Ø Analysis or identification can then follow
• Uses a column similar to a burette
• Vertical column packed with a solid adsorbent (stationary phase)
• Eluent (mobile phase) constantly supplied
Ø Passes through by gravity
46 COLUMN CHROMATOGRAPHY 47 COLUMN CHROMATOGRAPHY 48 GAS CHROMATOGRAPHY • Can effectively separate: * Components Ø Gaseous mixtures transported through the Ø Volatile (low boiling point) mixtures column by an inert
• Very sensitive carrier gas (H, N, He)
• Components in mixtures must not decompose when vaporised
• Not suited for mixtures susceptible to degradation at high temperature
49 GAS CHROMATOGRAPHY 50 RETENTION TIME 51 RETENTION TIME • Time taken for a component to be eluted from (pass through) a column.
• Retention times are characteristics of the component, with
respect to the particular stationary and mobile phase used. 52 RETENTION TIME • Shorter retention times: Ø Less strongly adsorbed to stationary phase. Ø More readily dissolved (desorbed) in mobile phase.
• Longer retention times:
Ø More strongly adsorbed to stationary phase.
Ø Less readily dissolved (desorbed) in mobile phase. 53 EXAMPLE Refer to the following chromatogram.
Q: State the retention time of
component E.
A: ~11.4 min 54 EXAMPLE Q: State the component with the shortest retention time.
A: Component A 55 EXAMPLE Q: If the stationary phase was polar, state the most polar component. Explain your answer. 56 EXAMPLE A: Component F is more polar. Component F formed the strongest interactions with the polar stationary phase, therefore taking the longest time to elute from the column and therefore having the longest retention time. HIGH-PERFORMANCE LIQUID 57
CHROMATOGRAPHY (HPLC) • Similar principles to gas chromatography.
• Difference: Ø Column is much shorter
Ø Utilises smaller adsorbent particles as stationary phase
Ø Uses liquid as the mobile phase
Ø High pressure required to force solvent (mobile phase) through column à
otherwise very slow movement
Ø Can be used for mixtures that aren’t suitable for gas chromatography HIGH-PERFORMANCE LIQUID 58
CHROMATOGRAPHY (HPLC) HIGH-PERFORMANCE LIQUID 59
CHROMATOGRAPHY (HPLC) 60 EFFECTS OF PHOTOCHEMICAL SMOG 61 SCIENCE UNDERSTANDING Ion chromatography (also known as ion exchange chromatography) is used to remove either cations or anions from a mixture by replacing them with ions of another type. q Explain, using equilibrium principles, how ions attached to the surface of a resin can be exchanged with ions in aqueous solution. 62 ION CHROMATOGRAPHY
• Similar principles to column chromatography, HPLC.
• Utilises the electrostatic attraction (or repulsion) between ions (or
polar molecules).
• Examples: • Two types:
Ø Protein analysis - Anion-exchange Ø Ion concentration in water - Cation-exchange 63 ION CHROMATOGRAPHY 64 ION CHROMATOGRAPHY 65 ION CHROMATOGRAPHY
• Column contains porous beads --> composed of charged polymer
resin with cations or anions attached to the surface
• Ions with strong ion-interactions will remain on surface of the bead
after the solvent has eluted.
• Strong ions can only be removed by altering the ion concentration
or pH à weakens electrostatic interactions 66 ION CHROMATOGRAPHY 67 ION CHROMATOGRAPHY
• EXAMPLE:
Na+ (resin) + NH4+(aq) ⇌ Na+(aq) + NH4+ (resin)
• Cations adsorbed in resin are in equilibrium with cations in solution.
• Le Châtelier’s Principle explains the ability for ion exchange.
68 ION CHROMATOGRAPHY
• Le Châtelier’s Principle (Overview)
Ø System must be in a state of dynamic equilibrium (balance). Ø When a stress is placed on the system, equilibrium will shift to offset the stress. Ø Equilibrium will shift to the left (reverse reaction) or right (forward reaction) until equilibrium is re-established.
• Will cover in more detail - subtopic 2.2
69 ION CHROMATOGRAPHY
• EXAMPLE 1:
Na+ (resin) + NH4+(aq) ⇌ Na+(aq) + NH4+ (resin)
• Prior to sample injection, sodium ions adsorbed to resin surface.
• Sample is injected Ø [NH4+(aq)] is increased 70 ION CHROMATOGRAPHY
Na+ (resin) + NH4+(aq) ⇌ Na+(aq) + NH4+ (resin)
Ø System in equilibrium acts to offset stress by favouring the
reaction that decreases [NH4+(aq)]
Ø Equilibrium shifts to the right (favours forward reaction).
Ø NH4+ adsorbs to surface of resin, Na+ goes into solution. 71 ION CHROMATOGRAPHY
• EXAMPLE 2:
Na+ (resin) + NH4+(aq) ⇌ Na+(aq) + NH4+ (resin)
• Flooding resin column with a concentrated sodium solution will allow
for sample to be eluted.
Ø [Na+(aq)]is increased. 72 ION CHROMATOGRAPHY
Na+ (resin) + NH4+(aq) ⇌ Na+(aq) + NH4+ (resin)
Ø System in equilibrium acts to offset stress by favouring the
reaction that decreases [Na+(aq)].
Ø Equilibrium shifts to the left (favours reverse reaction).
Ø Na+ adsorbs to surface of resin, NH4+ goes into solution. Ø Process continues until sample is eluted from column. 73 Your Turn! • Complete practice questions worksheet
