An Introduction to Liquid Chromatography

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1 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 Sample Stationary Phase Detection

2 The Liquid Chromatograph Solvent Reservoir Degasser* LC Pumps Autosampler 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)

3 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 Aqueous Layer Elution Polar Analyte Polar Stationary Phase

Selectivity of NPLC is multi-modal adsorptive/ pi-pi/ ionic/ H-bonding/ electrostatic/ Partition/ dipole-dipole/ Solubility +

4 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) psi

5 UHPLC Pressure accumulation is non-proportional at higher pressure regimes 600 Bar System 1000 Bar System Time (min) Neat Solution, Pressure Max = 505 Bad Sample, Pressure Max = 582 Δ = 77 bar Time (min) Neat Solution, Pressure Max = 783 Bad Sample, Pressure Max = 935 Δ = 152 bar Solvents Weak/Loading Solvent RPLC H 2 O (Polar) Strong/Eluting Solvent RPLC Methanol RPLC Acetonitrile RPLC Tetrahydrofuran (Relatively Nonpolar) HILIC Acetonitrile (Polar Aprotic) HILIC H 2 O (Polar) 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 Ammonium Acetate (+/-) Ammonium Formate (+/-) Ammonium Bicarbonate (-) Ammonium Fluoride (-) Formic Acid (+/-) Acetic Acid (+/-) Common Additives Lithium (+) Silver (+) Miscibility (Including Salt-based Partitioning) Quality (MS grade or Better) Adduct Formation (Changes in Sensitivity/Fragmentation) Mass Spec Sensitivity (Gas Phase Interactions/pK a )

6 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 Changes in Strong Solvent Over Elution Window Isocratic Identical Solvent Composition Over Elution Window % B % B Time Time Step 1: Load Step 1: Load Step 2: Elution Step 3: Wash Step 4: Re equilibrate Step 2: Elution Step 3: Wash 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 Gradients will be amenable to higher volumes, faster analysis and higher quality data

7 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 Increase Resolution Increase Resolution Increase Pressure Decreases Pressure Increase Lower Dwell Volume Pressure Increase mm mm µm

6 mm Lower Pressure Higher Flow Rate 50 mm Good Balance Between Resolution and Dwell Volume 30 mm Lower Dwell Volume Lower Resolution 3 µm Higher Resolution/ Efficiency 2.

8 Dimension Compromising Internal Diameter Length Particle Size 2.1 mm 100 mm 5 µm Common i.d. For Higher Resolution Common d p For Most Columns Higher Dwell Most Particles Volume 3 mm Lower Pressure Higher Flow Rate 4.6 mm Lower Pressure Higher Flow Rate 50 mm Good Balance Between Resolution and Dwell Volume 30 mm Lower Dwell Volume Lower Resolution 3 µm Higher Resolution/ Efficiency 2.x µm SPP Sub-2µm Efficiency, Less Pressure Length (mm) Balance of the Dimensions Internal Diameter (mm) Need Fully Porous Particles, Vol (ml) Fused Core Particles, Vol (ml) Dwell Time at 500 ul/min seconds seconds seconds seconds seconds seconds seconds seconds seconds Change Higher Resolving Power, Less Pressure Faster Cycle Time/Higher Flow Rate Increase I.D. (pressure ) Decrease d p (resolution ) Increase I.D. (pressure ) 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

9 Viscosity Higher Flow Rate Lower Pressure Faster Analysis Temperature Solubility Less Retention Different Eluent Content May Inhibit Resolution 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) Time (min) Assay Specific Include Examples in SOP s Mobile Phase Specific Useful Information 0 4 min min. Multiplexing 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 2 = Divert channel to MS/Acquire 3 = Divert channel to waste 4 = System ready for channel 2 Staggered Parallel LC-MS/MS Throughput = samples/hour Detector idle 5% of the time Long Methods Are Not Amenable To Mutliplexing Validate Possible Variations In Multiplexing (Single Channel, 2-Channel, 3-Channel)

10 1 st Column 2-Dimensional LC Multiple Interfering Species Ion Suppression/Matrix Effects Transfer Window Maximizing Selectivity (First Dimension) and Sensitivity (Second Dimension) 2 nd Column Pump #2 Difficult to Troubleshoot Orthogonality/Retention Variations Are Key (Different column in either dimension) Deuterium-Isotope Effect D Deuterium Radius* = femto meters Hydrogen Radius* = femto meters H Internal Standard Gabapentin D10 Analyte Gabapentin 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. Proactive Maintenance Reactive Filters/Frits Valves Valve Seals Auto-sampler Filter Stones Over-Pressure Leaking Tubing Leaking Fittings Column Degradation One Bad Sample When (Tracking and Scheduling) Who (In-House/External Service)

11 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 Pressure Traces and Failure Examples Some Common LC Myths 1) You can t reverse the direction of an HPLC column 2) All C18 columns are the same 3) Guard columns do not affect the separation 4) Higher temperature always leads to better separation 5) Higher carbon load = better Reverse Phase column 6) Always more efficiency with smaller particles 7) Residual silanols responsible for peak tailing 8) Silica columns can only be used from ph 2-7 9) Modern HPLC columns should withstand 1000 injections 10) Columns should be capped to prevent packing damage 11) The injection solvent should be the same as the mobile phase 12) Injection volume is limited by internal diameter 13) Mobile phases must be degassed 14) Water must be present for all HILIC separations 15) 3-5 (RP) or 8-10 (HILIC) column volumes required for reconditioning 16) Less than 50 mm buffer can be introduced to a mass spectrometer

12 Rules of Thumb (And Fingers) Reduce Extra-Column Volume No Involatiles Allowed (sodium, phosphate, etc.) Columns Are Consumables Multiplex As Much As Possible (Where Feasible) Don t Hand Cut Steel Tubing Cut PEEK Tubing Flush Check All Fittings (Don t Just Set and Walk Away) Use In-Line Filters ( µm filters) Use Bypass Valves to Keep Your MS Clean Gradients Are Reproducible Sufficiently Wash Columns Between Samples Check for Ionization Suppression in Real Samples Don t Elute Molecules at 100% Allow for Pressure Overhead (20-30%) Re-Optimize MS Conditions for Each LC Method Applied Chromatography is Largely an Empirical Process Brappold@etlab.org Acknowledgements: Russell Grant, PhD, LabCorp Donald Ojeda, Essential Testing Additional References LC-GC Magazine (FREE!) Ron Majors, LC-GC, 2006,24 (11), Comparison Guide to C18 Reversed Phase HPLC Columns, MacMod, 4th ed, available online Rappold BA, Grant RP, HILIC-MS/MS method development for targeted quantitation of metabolites: Practical considerations from a clinical diagnostic perspective. J Sep Sci, 34 (24), 2011 MSACL/ASMS Short Course Development and Validation of LC-MS/MS assays in clinical diagnostics, Instructors: Russell Grant and Brian Rappold Scott RPW, Principles and Practice of Chromatography, Chrom-Ed Series, Library For Science, 2003 Annesley, TM, Methanol Associated Matrix Effects in Electrospray Ionization Tandem Mass Spectrometry, Clin Chem, 53(10), 2007, pgs Journal of Chromatography B Journal of Chromatography A Clinical Chemistry Clinica Chimica Acta Clinical Biochemistry Steroids

High Performance Liquid Chromatography

High Performance Liquid Chromatography What is HPLC? It is a separation technique that involves: Injection of small volume of liquid sample Into a tube packed with a tiny particles (stationary phase).