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 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 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 (0.5 – 2.0 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
Email: [email protected]
Acknowledgements: Russell Grant, PhD, LabCorp Donald Ojeda, Essential Testing
Additional References LC-GC Magazine (FREE!) Ron Majors, LC-GC, 2006,24 (11), 1172-1182 “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 1827-34
Journal of Chromatography B Journal of Chromatography A Clinical Chemistry Clinica Chimica Acta Clinical Biochemistry Steroids