Section 3: Separation Methods (Chromatography)

Topics covered:
 
 

1.	Non-chromatographic methods - precipitation, masking, distillation, extraction      - in a 2-phase (organic/aqueous) extraction, the amount of analyte A remaining in the aqueous layer of Vaq after i extractions with Vorg is: Example: 24-1
2.	Chromatographic experiments   (a) Normal-phase - stationary phase is polar (b) Reverse-phase - stationary phase is non-polar (c) Ion-Exchange - stationary phase a resin capable of exchanging ions (d) Size-exclusion (SEC) - stationary phase a gel with pores, separate by molecular volume      - biomolecules a hydrophilic gel      - gel-permeation chromatography (GPC) uses hydrophobic gel, used to separate synthetic polymers
3.	Column Chromatography - can use any of the above types of chromatographic experiments (column chromatography normally refers to gravity-fed, preparatory scale)      - chromatographic peaks suffer broadening      - several formulae to describe the quality of separation (resolution):   distribution coefficient K (or Kc) for analyte A undergoing a (pseudo)equilibrium between the mobile and stationary phases, A (mobile) <===> A (stationary) retention factor k, where tR is retention time, tM is retention time of solvent, VS, VM are volumes of stationary and mobile phases selectivity factor a for two analytes A and B number of theoretical plates N, where H is average plate height, L is the column length, W is the width at the base of a peak, and W1/2 is the width of the peak at the half-way point of its height resolution RS between two peaks A and B length of time for second analyte B to reach the detector (centre of peak), where u is the speed of the mobile phase (L/tM) Figures: 24-4, 24-5, 24-6, 24-7, 24-10, 24-11, 24-12 Example: 24-2
3.	Gas (and/or Gas-Liquid) Chromatography (GC) - several parts to the instrument:      (a)  injector assembly                - mix carrier gas and sample, heated to volatilise sample Figure: 25-4      (b) column assembly                - oven to keep column at elevated temperature to keep sample in gas phase                - mobile phase: inert gas (N2, Ar, Ne)                - column - open capillary with stationary phase on walls or packed with stationary phase on packing material                              - column made from: steel or other metal, plastic/polymer, glass                              - packing material: silica gel, beads of metals, etc.                              - stationary phase: normal or reverse phase                                                          - liquid polymer attached to support (packing material or capillary wall)                                                         - siloxanes, poly(ethylene glycol), etc.      (c) detector                - spectroscopic (mass spec, UV-visible absorption, IR, fluorescence)                - electron-capture (ECD)                - flame-ionisation (FID) Figure: 25-5      - both quantitative (by peak area) and qualitative analysis possible, depending on which type of detector is used Figures: 25-1, 25-9
4.	High-Performance Liquid Chromatography (HPLC) - instrument run at high pressure (up to 400 atm), all components designed to withstand pressure:      (a) injection assembly                - sample loop - to protect syringe from pressure                - solvent reservoirs - need ultra-pure solvent                - pump - to deliver solvent at high pressure Figure: 25-15      (b) column - steel, 10-30 cm long, could be any type (reverse, normal, size-exclusion, or ion-exchange)                - packed with silica, alumina, polymer beads, etc.                          - adsorption chromatography - no coating on packing material                          - partition chromatography - packing material coated with stationary phase                - guard column before actual column - to protect, filter out particles                - no temperature ramping - may be oven to maintain constant T, usually <50oC      (c) detector                - spectroscopic (UV-vis absorption, IR, fluorescence, MS (expensive!))                - refractive index (RI)                - light scattering Figures: 25-13, 25-17, 25-19, 25-25-20, 25-21, 25-22
5.	Comparison between GC and HPLC      - HPLC more general, more expensive, can be preparatory scale, sharper flow rate/resolution profile      - GC has more general detectors (including MS), cheaper Figure: 24-11
6.	Electrophoresis - place voltage across an LC column, separates by "charge density;" we will focus on capillary methods for this course      (a) use of capillary:                - can use higher voltage (efficient heat disapation), therefore faster                - migration rate (v), plate count (N) directly proportional to voltage, V:   migration rate: (me = mobility, relates to size, shape, and viscosity) plate count: (D = diffusion constant)      (b) electroosmotic flow:                - glass has surface Si-O- groups, positive buffer ions (in mobile phase) attracted to wall and cathode                - this drags solvent and solutes along                - neutral solutes dragged at same rate as solvent front                - positively charged ions move faster (solvent flow + voltage)                - negatively charged ions move slower (solvent flow - voltage)                - to stop electroosmotic flow, attach trimethylsilane (TMS) groups to capillary wall                - to reverse, coat wall with Si-NH4+ groups      (c) capillary zone electrophoresis (CZE)                - buffer constant through capillary                - used for: proteins, pesticides/pharmaceuticals, carbohydrates      (d) capillary gel electrophoresis                - buffer contained in pores, better size selectivity than CZE                - used for DNA fragments      (e) micellar electrokinetic capillary chromatography                - add surfactant, forms micelles, micelles move more slowly (larger)                - polar molecules stay in buffer (mobile phase), non-polar go into micelles Figures: 26-4, 26-5, 26-6, 26-7, 26-8, 26-9, and from Zumdahl (1st-year book) 23.39
7.	Chirality      - if stationary phase chiral, can separate optical isomers      - used in HPLC, electrophoresis, prep scale or analytical scale

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