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 www.analytical-training-solutions.org
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
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 = 0 1 2 3 4 2-6
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 0.5 4 σ w b 2-9
How Big Should N Be? Manufacturers: 5 µm: N 80-100,000/m 3 µm: N 100-150,000/m 150,000/m 2 µm: N 175-250,000/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 = (3.15-2.95) / (0.12) = 1.7 2-12
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 = 1.0 2-13 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
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
Measuring Peak Symmetry 2-19 Asymmetry Factor vs. USP Tailing Tailing Factor (5%) Asymmetry Factor (10%) 1.0 1.0 1.2 1.3 1.5 1.7 1.7 2.0 2.0 2.6 2.5 3.5 2-20 Tailing Factor Excellent 0.9 < TF < 1.2 Undesirable (TF = 2) Awful (TF = 4) 2-21
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
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 #2 2-25 log k (orthogonal) 1.6 1.2 0.8 0.4 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 0.0-1 -0.5 0.0 0.5 1.0 1.5 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 > 65 0.19 ph [buffer] 5 units >>0.7** 2x 0.02 2-27 * need 0.1 **ionics only
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 0 + + + + + temperature - (+) + + + + + solvent type + + + 0 + 0 column type 0 (+) + 0 + + + ion pairing + - - 0 - - ph ++ - 0 0 - + 2-29 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
For more information and to discuss your training requirements, contact: FOR NORTH AMERICA & EUROPE Dean Graimes dean.graimes@sepscience.com T: +44 203 490 6949 M: +44 7721 097 390 FOR ASIA PACIFIC Jeroen Reiniers jeroen.reiniers@sepscience.com T: +65 6408 9751 Frederick House, Princes Court Nantwich, CW5 6PQ, UK 20 Maxwell Road, 09-17 Maxwell House Singapore 069113 www.analytical-training-solutions.org