Advanced HPLC Method Development Using Quality by Design
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1 Advanced HPLC Method Development Using Quality by Design A comprehensive course in the systematic development of liquid chromatography separations using QbD principles COURSE HANDOUT: [SECTION 2] Separation Basics Fundamental Measurements More About Resolution Controlling Selectivity
2 Advanced HPLC Method Development Using QbD Section 1. Getting Started Section 2. Separation Basics Fundamental Measurements More About Resolution Controlling Selectivity Section 3. HPLC Columns Section 4. Reversed-Phase HPLC of Neutral Analytes Section 5. Reversed-Phase HPLC of Ionizable Analytes Section 6. Equivalent & Orthogonal Columns Section 7. Gradient Separations Section 8. Quality by Design (QbD) Section 9. HPLC and UHPLC Section 10. Quality Issues Section 11. Normal Phase & HILIC Separations Section 12. Chiral Separations 2-1 Hypothetical LC Separation of a Three-Component Mixture SOLVENT FLOW WET DRY C B A 2-2 Retention and Differential Migration in LC M C m A m Equilibrium distribution of compounds C and A between stationary and mobile phases C s S A s 2-3
3 Processes Within the Column Leading to Band Spreading 2-4 The Resulting Chromatogram t R t 0 C A B w b minutes 2-5 The Capacity Factor, k k t R k = t R - t 0 t 0 t 0 minutes k =
4 from the chromatogram Estimating t 0 minutes minutes inject a non-retained solute (e.g., uracil) calculation: t 0 = V m / F V m 0.5 L d c 2 / 1000 (all units in mm), or V m 0.01 L (mm) (for 4.6 mm i.d. columns) 2-7 Relative Retention, α 2-8 Column Plate Number, N t R N = 16 (t R / w b ) 2 N = 5.54 (t R / w 0.5 ) 2 N = (t R / σ) 2 w σ w b 2-9
5 How Big Should N Be? Manufacturers: 5 µm: N ,000/m 3 µm: N ,000/m 150,000/m 2 µm: N ,000/m More realistic estimate: N 300 L / dp where L = column length, mm dp = particle diameter, µm 2-10 Separation Quality great! good ummm no way! 2-11 R S = Resolution, R s t R2 - t R1 0.5 (w b1 + w b2 ) R s = ( ) / (0.12) =
6 Tailing and Resolution R s = 1.5 R s = 1.5 R s = 1.2 R s = 1.5 R s = 1.0 R s = 1.0 R s = 1.2 R s = Advanced HPLC Method Development Using QbD Section 1. Getting Started Section 2. Separation Basics Fundamental Measurements More About Resolution Controlling Selectivity Section 3. HPLC Columns Section 4. Reversed-Phase HPLC of Neutral Analytes Section 5. Reversed-Phase HPLC of Ionizable Analytes Section 6. Equivalent & Orthogonal Columns Section 7. Gradient Separations Section 8. Quality by Design (QbD) Section 9. HPLC and UHPLC Section 10. Quality Issues Section 11. Normal Phase & HILIC Separations Section 12. Chiral Separations 2-14 R vs. k s. k,, N, α 2-15
7 Fundamental Resolution Equation Physics (kinetics) Chemistry (thermodynamics) R S ¼ N ½ (α-1) [k /([ /(k +1)] efficiency selectivity retention 2-16 Effect of k on k Resolution and Peak Height 100% 50% 0% 2-17 Effect of N on Resolution and Peak Height 100% 2-18
8 Measuring Peak Symmetry 2-19 Asymmetry Factor vs. USP Tailing Tailing Factor (5%) Asymmetry Factor (10%) Tailing Factor Excellent 0.9 < TF < 1.2 Undesirable (TF = 2) Awful (TF = 4) 2-21
9 Advanced HPLC Method Development Using QbD Section 1. Getting Started Section 2. Separation Basics Fundamental Measurements More About Resolution Controlling Selectivity Section 3. HPLC Columns Section 4. Reversed-Phase HPLC of Neutral Analytes Section 5. Reversed-Phase HPLC of Ionizable Analytes Section 6. Equivalent & Orthogonal Columns Section 7. Gradient Separations Section 8. Quality by Design (QbD) Section 9. HPLC and UHPLC Section 10. Quality Issues Section 11. Normal Phase & HILIC Separations Section 12. Chiral Separations 2-22 Controlling Selectivity (Reversed-Phase) solvent strength temperature solvent type column type ph additives 2-23 The Importance of Selectivity To obtain a separation which variable first? which likely to be successful? least amount of work? For a QbD method how does each variable affect α? what are robust conditions? what isn t important? 2-24
10 Comparing Selectivity r 2 = 0.98 r 2 = 0.25 log k, variable #1 log k, variable #1 log k, variable #2 log k, variable # log k (orthogonal) Quantifying Orthogonality Orthogonality r 2 = 0.76, SE = 0.18 δlog α avg 1.4 x SE = 0.25 (need 0.1) δ log α log k (original) 2-26 Comparing Orthogonal Leverage parameter change δlog α * avg %B 10% 0.08 t G 3x 0.07 C 20 C 0.07 MeOH (ACN) ACN (MeOH) 0.20 column F s > ph [buffer] 5 units >>0.7** 2x * need 0.1 **ionics only
11 Some Available Variables isocratic %-organic (%B) gradient slope/time organic type (ACN/MeOH) ph additives (ion pair, etc.) buffer type / concentration temperature column type column size particle size flow rate R S ¼ N ½ (α-1) [k /(k /(k +1)] 2-28 Ranking the Variables change in α universal convenient low-uv/lc-ms robustness equilibration Variable % B temperature - (+) solvent type column type 0 (+) ion pairing ph Advanced HPLC Method Development Using QbD Section 1. Getting Started Section 2. Separation Basics Fundamental Measurements More About Resolution Controlling Selectivity Section 3. HPLC Columns Section 4. Reversed-Phase HPLC of Neutral Analytes Section 5. Reversed-Phase HPLC of Ionizable Analytes Section 6. Equivalent & Orthogonal Columns Section 7. Gradient Separations Section 8. Quality by Design (QbD) Section 9. HPLC and UHPLC Section 10. Quality Issues Section 11. Normal Phase & HILIC Separations Section 12. Chiral Separations 2-30
12 For more information and to discuss your training requirements, contact: FOR NORTH AMERICA & EUROPE Dean Graimes T: M: FOR ASIA PACIFIC Jeroen Reiniers T: Frederick House, Princes Court Nantwich, CW5 6PQ, UK 20 Maxwell Road, Maxwell House Singapore
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