![Selectivity factor=α
o It’s the ratio of distribution constant strongly retained species to
less strongly retained (rapidly eluted species “A”) in
chromatographic column.
α = KB/KA, α >1
Or α = kB/kA [kB =(tR)B-tM/tM & kA =(tR)A-tM/tM ]
o Putting kA and kB
α = (tR)B-tM/(tR)A-tM](https://image.slidesharecdn.com/lec3separationbychromatography-231119102146-7c87c642/85/Lec-3_Separation-by-Chromatography-ppt-4-320.jpg)
Lec#3_Separation by Chromatography.ppt
- 1. Analytical Chemistry-II BS-V, CH-311 Department of Chemistry, SNS, NUST By Dr. Musammir Khan Selectivity factor, column efficiency
- 2. Migration rates of solute_ Derivation of equation • The effective chromatographic separation of two solutes A & B (mixture) depends on the relative rates at which the two species are eluted. • Distribution constant (Kc): The ratio of the molar concentration of solute in the stationary phase to mobile phase i.e. • A (mobile phase_A (stationary) • Kc = (aA)S/(aA)M or CS/CM = Ideally constant over a wide range C • Retention time (tR): The time required for the solute molecules to reach the detector (R = retained by st. phase). • Dead/void time tM: The time taken by an un-retained solute specie to pass through. It provide an average time taken by the mobile phase. • This can be achieved by adding an un-retained specie into the sample if not already present e.g. LHS peak Figure---next page
- 3. • LHS peak represent un-retained specie or average time taken by mobile phase, while peak at RHS represent time taken by sample through the stationary phase: tR = tM + tS • Average linear velocity of solute migration: (L =length of column) ν = L/tR • Average linear velocity of mobile phase = µ = L/tM
- 4. Selectivity factor=α o It’s the ratio of distribution constant strongly retained species to less strongly retained (rapidly eluted species “A”) in chromatographic column. α = KB/KA, α >1 Or α = kB/kA [kB =(tR)B-tM/tM & kA =(tR)A-tM/tM ] o Putting kA and kB α = (tR)B-tM/(tR)A-tM
- 5. Band broadening: Efficiency (Rate theory + Plate theory) • Why the band broaden as they move through the column? • Described by Rate Theory=random walk mechanism: • Consider a single solute undergoing thousand of transfer between the stationary and mobile phase i.e. residence time in either phase is quite irregular. • Transfer from one phase to another phase require energy and molecule acquire this energy from the surrounding. • Movement through the column occur only when the molecules are in the mobile phase. • The chromatogram behave much like the Gaussian shape i.e. • Random uncertainty to be +ve or –ve, each of which has equal probability of occurrence.
- 6. Gaussian shape curve/chromatogram is usually obtained, which is described by standard deviation δ and variance δ2 standard deviation δ: How individual values are far from each other. variance δ2: how actual value are far from mean.
- 7. Tailing (favor phase) vs. Fronting (favor stationary phase)
- 8. Column efficiency: o Quantitative measure of band broadening or the degree to which a column and other components effect the separation process. o Plate theory: Column consists of large number of theoretic plates (N), where components of a mixture gain equilibrium (between stationary and mobile phase). o Column efficiency is usually expressed in term of theoretical plate number: N = L/H [L =length of column, H = height of plate The efficiency of a column increases with plate count increase or with smaller plate height (H).
- 9. Theory of peak broadening: Column efficiency Longitudinal diffusion coefficient B: Diffusion of solute species on either side of flow from region of high conc to low conc. Stationary and mobile phase mass transfer coefficient (Cs and CM) *In case of liquid immobilized as stationary phase: Mass transfer coefficient (CS) α sq of film thickness (df)2 *In case of solid stationary phase: Mass transfer coefficient (CS) α time required for adsorption/desorption For mobile phase: CM α 1/DM For packed column: CM α square of particle diameter (dp)2 For capillary column: CM α square column inner diameter (dc)2 Van Deemter Equation
- 10. Formula for number of theoretical plates Packed column=particle diameter Dp
- 11. For packed column where flow dominate diffusion Height equivalent of theoretical plate
- 12. Variable effecting column efficiency Mobile phase flow rate: Gas vs. Liquid • Capillary vs. packed column For liquid 10 time slower than for gaseous phase, because of lower flow rate and lower plate height
- 13. Gas Chromatography (GC) • The components of a vaporized mixture are separated/distributed between mobile gaseous phase and liquid/solid mobile phase. • Used for qualitative and quantitative analysis of components in a mixture ( important industries, biomedical and forensic areas). • Unlike other chromatographic techniques, gaseous mobile phase doing only elution. • Two types: Gas-solid (GS) and gas-liquid (GL) chromatography; – GSC (solid stationary phase): Retention occur only by physical adsorption – Limited due to SEMIPERMANENT retention/severe tailing occur. – Used for smaller molecules eg gas – GLC (liquid stationary phase): partition occur between liquid immobilized on stationary phase and gaseous mobile phase – Liquid immobilized on inert solid (packed or coated on inner walls of capillary tubing) (useful = 1941 by Martin).
- 14. Instrumentation/components: • Carrier Gas System, • Sample Injection system, • Column Configurations and Column Ovens • Detectors Carrier Gas System: • Mobile phase inert carrier gas available in tank e.g. (He, Ar, N2, H2) • Flow rate can be controlled by two-stage regulators i.e. gas inlet pressure and some commercially available regulators. • A gas inlet pressure of 10-50 psi (1atm=14.6 psi) • *Volumetric flow rate (F) =25-150 mL/min packed column • *1-25 mL/min - Capillary column (low diameter~0.2-5 mm)
- 15. Instruments for Gas- Liquid Chromatography (GLC)
- 16. • Sample injection: Suitably sized sample is injected through calibrated micro-syringes. • For packed analytical column = 0.1-20 µL sample required • For capillary column- 0.1-20 µL/100 i.e. smaller sample needed • Currently auto-injector and autosamplers are available • For injection volume = 0.1 µL -10-200 µL micro-syringes • Column Configurations and Column Ovens • Two types of column • *Packed column- Used in old time • *Capillary column currently used • L = 2-60 m, composition = stainless steel/silica/glass/Teflon • Inner diameter = 0.2-5 mm, coiled for fitting into oven = 10-30 cm coiled diameter • Column Temperature: Depend on B.P. of sample (T=B.P. =2-3 min eluted)
- 17. • Temperature programming improve separation as compared with ISOTHERMAL chromatogram (i.e. increasing the column temp either continuously or in steps. e.g. (a) Isothermal at 458C. (b) Isothermal at 1458C. (c) Programmed at 308 to 1808C
- 18. Detectors • Characteristics of ideal detectors: • Sensitivity~ 10-8 -10-15 g/s • Stability/reproducibility • Foolproof in hand of less skilled person • Temperature = up to 400 oC • Short response time • Universal • Non-destructive
Editor's Notes
- #10: C