An Introduction to Liquid Chromatography Brian A. Rappold Scientific Director Essential Testing (Collinsville, IL) Financial Disclosures: Grant/Research Support: None Salary/Consultant Fees: Essential Testing, LLC Stocks/Bonds: Laboratory Corporation of America, Quest Diagnostics Honorarium/Expenses: AACC Intellectual Property/Royalty Income: None Liquid Chromatography Stationary Detection Phase Sample The Liquid Chromatograph Solvent Degasser* LC Pumps Autosampler Reservoir Waste Black Box Detector Bypass Valve* Column Guard Column* * OPTIONAL Important Terms Weak Solvent – Loading solvent (Mobile Phase A) Strong Solvent – Eluting solvent (Mobile Phase B) Efficiency – A measure of peak width related to band broadening Particle Size – The mean diameter of particles in an HPLC column Selectivity – A measure of how well the tops of 2 peaks are separated Resolution – A measure of how well the baselines of 2 peaks are separated Retention – A measure of strength of interactions between the analyte, the stationary phase and the mobile phase; how long an analyte is in a column Asymmetry – A measure of the shape of a chromatographic peak. Tailing and fronting are 2 modes of asymmetry Dwell Volume – The volume of the HPLC system prior to and including the column Extra-Column Volume – The volume of the tubing after the column Band Broadening – Diffusion of the analyte longitudinally in a liquid stream Styles of LC Reverse-Phase – “RP”, most common style for LC-MS/MS HILIC – Relatively new, opposite of reverse-phase LC Ion Exchange – “IEX”, Widely used for LC-UV/ECD assays (amino acids, plasma catecholamines) RP - LC Hydrophobic Interactions (Nonpolar-Nonpolar) Nonpolar Groups Polar Groups Plus induced dipole-dipole interactions (i.e. cyano phases) π-π overlap (i.e. phenyl phases) and unbonded silanol H- interactions (all silanol-backbones) Example RP Stationary Phases C18 (ODS)* Cyano* Phenyl* Amide* *Not all columns of the same flavor have the same interactions!!! “Comparison Guide to C18 Reversed Phase HPLC Columns”, MacMod, 4th ed, available online HILIC Hydrophilic Interactions (Polar-Polar) Nonpolar Mobile Phase Polar-Aprotic Solvent (ACN) Polar Analyte Retention Elution Polar Analyte Aqueous Layer -+-+-+--+-+-+- Polar Stationary Phase RP or NP/HILIC Mode? Nature of analyte is major decision point. NP/HILIC ESI sensitivity better for charged analytes – elute in organic rich solvent. Selectivity of NPLC is multi-modal – adsorptive/ pi-pi/ ionic/ H-bonding/ electrostatic/ Partition/ dipole-dipole/ Solubility + RP Mode HILIC Mode Androstenedione Metanephrine Thyroxine Nicotine Pressure 1980’s – “HPLC” 200 bar (~3,000 psi) 1990’s – “HPLC” 400 bar (~6,000 psi) Late 90’s Early 00’s– “HPLC” 600 bar (~9,000 psi) 2004– “UHPLC” 1000 bar (~15,000 psi) ≈ 30-40 psi UHPLC Pressure accumulation is non-proportional at higher pressure regimes 600 Bar System 1000 Bar System Time (min) Time (min) Neat Solution, Pressure Max = 505 Neat Solution, Pressure Max = 783 “Bad” Sample, Pressure Max = 582 “Bad” Sample, Pressure Max = 935 Δ = 77 bar Δ = 152 bar Solvents Weak/Loading Solvent Strong/Eluting Solvent RPLC –Methanol RPLC–H2O RPLC – Acetonitrile (Polar) RPLC – Tetrahydrofuran (Relatively Nonpolar) HILIC – Acetonitrile HILIC – H2O (Polar Aprotic) (Polar) Key Considerations Viscosity (Back Pressure) Safety (High Temperature MS Source) Quality (HPLC Grade or Better) Mass Spec Sensitivity (Gas Phase Interactions/Desolvation) Buffers and Additives Common Buffers Common Additives Ammonium Acetate (+/-) Lithium (+) Ammonium Formate (+/-) Silver (+) Ammonium Bicarbonate (-) Ammonium Fluoride (-) Formic Acid (+/-) Acetic Acid (+/-) Key Considerations Miscibility (Including Salt-based Partitioning) Quality (MS grade or Better) Adduct Formation (Changes in Sensitivity/Fragmentation) Mass Spec Sensitivity (Gas Phase Interactions/pKa) Steps in LC • Step 1: Load – Sample is introduced to LC system – Analytes adsorb to the head of the column • Step 2: Elution – Analytes desorb from column based on “solubility” in mobile phase • Step 3: Wash – Strong solvent used to wash away unwanted residual molecules • Step 4: Re‐equilibrate – The system is returned to initial conditions Separation Styles Gradient Isocratic Changes in Strong Solvent Over Identical Solvent Composition Elution Window Over Elution Window % B % B Time Time 1 2 3 4 1 2 3 4 Step 1: Load Step 1: Load Step 2: Elution Step 2: Elution Step 3: Wash Step 3: Wash Step 4: Re‐equilibrate Step 4: Re‐equilibrate Gradient and Isocratic Gradients Isocratic Selectivity (single analyte) 5 5 Selectivity (Multi-analyte) 5 2 Solvent Consumption 3 3 Ruggedness/Robustness 4 2 Ease of Development 2 4 Selectivity per unit time 5 2 Key Considerations Gradients will be amenable to higher volumes, faster analysis and higher quality data Column Selection My Favorite Column is….. Column Selection My Favorite Column is….. “the one with particles packed inside a tube which affords the appropriate selectivity with the best sensitivity for the analyte(s) of interest” Column Dimensions Internal Diameter Length Particle Size Column Volume Resolution Resolution Increase Increase Increase Pressure Pressure Pressure Decreases Increase Increase Lower Dwell Volume 1 – 4.6 mm 30 – 150 mm 1.7 – 5 µm Dimension Compromising Internal Diameter Length Particle Size 2.1 mm 100 mm 5 µm Common i.d. For Higher Resolution Common dp For Most Columns Higher Dwell Most Particles Volume 3 mm 50 mm 3 µm Lower Good Balance Higher Resolution/ Pressure→Higher Between Resolution Efficiency Flow Rate and Dwell Volume 4.6 mm 30 mm 2.x µm SPP Lower Lower Dwell Volume Sub-2µm Efficiency, Pressure→Higher Lower Resolution Less Pressure Flow Rate Balance of the Dimensions Internal Fully Porous Fused Core Length Dwell Time at 500 Diameter Particles, Vol Particles, Vol (mm) uL/min (mm) (mL) (mL) 30 2.1 0.068 0.047 8.1 seconds 30 3 0.138 0.095 16.5 seconds 30 4.6 0.324 0.224 38.9 seconds 50 2.1 0.113 0.078 13.5 seconds 50 3 0.230 0.159 27.6 seconds 50 4.6 0.540 0.374 64.8 seconds 100 2.1 0.225 0.156 27 seconds 100 3 0.459 0.318 55.1 seconds 100 4.6 1.080 0.748 129.6 seconds Need Change Higher Resolving Power, Less Increase I.D. (pressure ↓) Pressure Decrease dp (resolution ↑) Increase I.D. (pressure ↓) Faster Cycle Time/Higher Flow Rate Decrease L (volume ↓) Particles, Pores and Paths Fully Porous Particle Superficially Porous Particle Diffusion Path Diffusion Path Diffusion Path related to peak width Resolution Sensitivity Temperature Viscosity Solubility Higher Flow Rate Less Retention Lower Pressure Different Eluent Content Faster Analysis May Inhibit Resolution Key Considerations Temperature max for most columns is 60°C (some up to 100°C) Column Ovens must be calibrated to NIST-certified thermometers Pressure Traces H2O/Methanol Gradient H2O/Acetonitrile Gradient Pressure (bar) Pressure Time (min) Key Considerations Assay Specific – Include Examples in SOP’s Mobile Phase Specific Multiplexing Useful Information Traditional LC-MS/MS Throughput = 15 samples/hour Detector idle 75% of the time Why acquire all the noise? 1 = Inject/acquisition details to MS 04 min.2 = Divert channel to MS/Acquire 3 = Divert channel to waste 1 2 3 4 4 = System ready for channel 2 Staggered Parallel LC-MS/MS Throughput = 45-60 samples/hour Detector idle 5% of the time 0 4 min. Key Considerations Long Methods Are Not Amenable To Mutliplexing Validate Possible Variations In Multiplexing (Single Channel, 2-Channel, 3-Channel) 2-Dimensional LC 1st Column Multiple Interfering Species Ion Suppression/Matrix Effects Maximizing Selectivity (First Dimension) and Sensitivity (Second Dimension) Transfer nd Window 2 Column Pump #2 Key Considerations Difficult to Troubleshoot Orthogonality/Retention Variations Are Key (Different column in either dimension) Deuterium-Isotope Effect D H Deuterium Radius* = 2.1402 femto meters Hydrogen Radius* = 0.8775 femto meters Analyte Internal Gabapentin Standard Gabapentin D10 Ionization Differences? Matrix Effects? * Note to Physicists: Calculated as the root mean square of electron scattering as a function of nuclear cross-section and not intended to imply deviations in electronic energy levels. Values have been confirmed by quantum electrodynamics. Maintenance Proactive Reactive Filters/Frits Over-Pressure Valves Leaking Tubing Valve Seals Leaking Fittings Auto-sampler Column Degradation Filter Stones “One Bad Sample” Key Considerations When (Tracking and Scheduling) Who (In-House/External Service) Trouble Shooting 101 Rule 1: Engage the Brain Mentally remove unaffiliated components (i.e. over pressure does not require mass spec recalibration) Rule 2: Divide and Conquer Separate each component in sequence and test Rule 3: Use Historical Information System suitability tests, pressure traces, error logs Rule 4: Avoid Random Acts of Replacement Costs time and money – don’t replace unless you’re sure Rule 5: Maintain Your Preventative Maintenance Schedule and Document Rule 6: Share Your Pain Inform others in the lab about the cause and correction LC Goals Consistent Retention Times: Test Multiple Columns (within and between lots) Optimize Your Methods: Empirically Test Wash and Equilibration Times Accelerate Your Methods: Flow Rate Increase During Wash and Equilibration Stress Your Methods: Determine the “What, When and How” of Method Failure Method Risk Management: Determine Interferences and Zones of Suppression Have Method Backups: Test Alternative Columns/Mobile Phases Before You Need Them Document Your Methods: Explicit Descriptions in SOP’s, Including