HPLC Background Chem 250 F 2008 Page 1 of 24

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1 HPLC Background Chem 250 F 2008 Page 1 of 24 Outline: General and descriptive aspects of chromatographic retention and separation: phenomenological k, efficiency, selectivity. Quantitative description of zone migration in partition chromatography: migration velocity, partition coefficient and theoretical k. Theoretical description of efficiency: zone broadening and the Van Deemter equation. Solving the general elution problem: Gradient vs. isocratic elutions. HPLC Instrumentation: pump, injector, column, detector, data collection. Chromatographic modes: HPLC (reverse and normal phase or flash ), gel permeation (GPC), gel filtration, ion exchange. Electrophoresis.

2 HPLC Background Chem 250 F 2008 Page 2 of 24 Chromatography: separation of chemicals in a mobile phase by differential flow rates through stationary phase. t M t R Skoog Holler and Nieman Principles of Instrumental Analysis 5 th ed.

3 HPLC Background Chem 250 F 2008 Page 3 of 24 Skoog Holler and Nieman Principles of Instrumental Analysis 5 th ed.

4 HPLC Background Chem 250 F 2008 Page 4 of 24 Skoog Holler and Nieman Principles of Instrumental Analysis 5 th ed.

5 HPLC Background Chem 250 F 2008 Page 5 of 24 Skoog Holler and Nieman Principles of Instrumental Analysis 5 th ed.

6 HPLC Background Chem 250 F 2008 Page 6 of 24

7 HPLC Background Chem 250 F 2008 Page 7 of 24

8 HPLC Background Chem 250 F 2008 Page 8 of 24

9 HPLC Background Chem 250 F 2008 Page 9 of 24 What changes in a separation if alpha changes? How does that look?

10 HPLC Background Chem 250 F 2008 Page 10 of 24

11 HPLC Background Chem 250 F 2008 Page 11 of 24 What changes in a separation if N changes? How does that look?

12 HPLC Background Chem 250 F 2008 Page 12 of 24

13 HPLC Background Chem 250 F 2008 Page 13 of 24 Theoretical Plates and Plate Count: x i := i ( ) S x, μ, σ 1 1 exp ( 2 π σ 2 σ 2 x μ) 2 := = 10 = = = N: = = 1600 N 25 2 μ σ = = Illustration of different plate counts plates signal plates plates plates retention time or column length

14 HPLC Background Chem 250 F 2008 Page 14 of 24 Plate count depends on retention time: Why does the plate count go up for the more retained peaks? Because more retained peaks are expected to get broader by longitudinal diffusion but these do not. In other words: To produce a narrow peak at longer t R requires a higher performance column. N: = = 9216 N μ σ = = Illustration of different plate counts signal retention time or column length

15 HPLC Background Chem 250 F 2008 Page 15 of 24 The van Deemter Equation: H = plate height A = multipath term B = longitudinal diffusion term C = resistance to mass transfer Plate height as a function of linear flow rate: H A+ B + C u u H12 (,, 0.5, u) u Rationalize why is there an optimum flow rate: Very slow flow allows too much time for diffusional broadening. B/u term Excessive flow rate can exceed the rate of partitioning into and out of the stationary phase. C*u term

16 HPLC Background Chem 250 F 2008 Page 16 of 24 H A+ B + C u u A 2λ d p multipath term B 2γ D M longitudinal diffusion C = complex function of particle size, coating, diffusion coefficient, favored in general by large diffusivity and small particle size resistance to mass transfer

17 HPLC Background Chem 250 F 2008 Page 17 of 24 The General Elution Problem and Gradient Elution Retention and efficiency together. A mixture of molecules with strongly differing k values is hard to separate because: If the solvent is weak enough to separate 1 and 2: a. Separation takes too long b. Peaks 5 and 6 broaden into baseline If the solvent is strong enough to elute 5 and 6 in a reasonable time: Peaks 1 and 2 and 3 and 4 co-elute

18 HPLC Background Chem 250 F 2008 Page 18 of 24 isocratic (LC) and isothermal (GC) separations are vulnerable to the general elution problem the solution to the general elution problem: o solution gradient elution o temperature gradient elution (liquid chromatography) (gas chromatography) the gradient methods o begin with weakly eluting conditions and this helps to separate the weakly retained species o finish with strongly eluting conditions to expedite the elution of strongly retained species and to limit diffusional broadening in LC gradient elution is a bit more difficult because o the columns require a fairly significant equilibration time between runs o the detector baseline can drift significantly if the different solvents have different UV absorbance at the detection wavelength if this is a problem, then one can use o a different wavelength or o a different set of solvents

19 HPLC Background Chem 250 F 2008 Page 19 of 24 Example: GC of a complex mixture. Isothermal: 45 C Isothermal: 145 C T-programmed: C

20 HPLC Background Chem 250 F 2008 Page 20 of 24 High Performance Liquid Chromatography Skoog Holler and Nieman Principles of Instrumental Analysis 5 th ed.

21 HPLC Background Chem 250 F 2008 Page 21 of 24 Types of LC, all can be performed as HPLC in high performance instrumentation if necessary. Normal Phase: stationary phase: hydrophilic (SiO 2 particles) mobile phase: hydrophobic mobile phase (hexane, methanol). weak solvent: hexane strong solvent: methanol retained: polar compounds eluted first: non-polar compounds Reverse Phase: stationary phase: hydrobic (SiO 2 particles coated with hexadecane monolayer) mobile phase: hydrophilic mobile phase (water, methanol) weak solvent: water strong solvent: methanol retained: non-polar compounds eluted first: polar compounds Exclusion Gels GPC Gel permeation chromatography: porous polymer gel particles small molecules penetrate and are retained, large molecules elute more quickly. PAGE polyacrlamide gel electrophoresis: Aqueous gel: -CH 2 -CONH- used for electrophoresis, large molecules experience more drag and are retained.

22 HPLC Background Chem 250 F 2008 Page 22 of 24 HPLC modes continued - Ion Exchange: Stationary phase is an ionic polymer e.g. polystyrenesulfonic acid: SO 3 - * * n The solid or gel phase polymer transiently binds cations from solution. Cations that have a higher affinity for the resin are retained longer and vice-versa.

23 HPLC Background Chem 250 F 2008 Page 23 of 24 HPLC System overview: Injection valve

24 HPLC Background Chem 250 F 2008 Page 24 of 24 Example of Reverse-phase HPLC stationary phase: Ideal qualities of HPLC stationary phase: Very fast partitioning between mobile and stationary phase. 1. Ultimate thin film: single molecular layer. 2. Perfect defect free film with no sites for strong adsorption. Mechanically strong to withstand high pressure w/o collapse. Chemically inert. Partition coefficients of solute analytes are all different in single solvent i.e. the SP provides a measure of selectivity without being limited to just a few chosen analytes.

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