HPLC Hints, Tips, Guidelines and Important Considerations Presenters: Wilhad Reuter / Tom Breslin
Important HPLC Solvent Characteristics: ALL injected compounds/diluents must be soluble High purity and degassed Noncorrosive Low viscosity UV transparency Solvents should not alter component s activity in sample Commercial bottled solvents should not be re-filtered Add 1% MeOH or 0.5% ACN to water. Not recommended using pure water > 2 days old Do not use halogenated acids; e.g., HCl Typically excludes IPA For UV detection, solvents must not absorb close to wavelength(s) of interest; above UV cutoff Especially for pharmaceuticals and biomolecules Select such that components elute between 2 to 20 column volumes ( k )
Solvent Degassing Mobile phase degassing is important for accurate pump blending and to avoid bubble formation in the detector flowcell. Techniques: BOILING Hmm!, not advised VACUUM Useful for only 2-42 4 hours; Must combine w/ sonication HELIUM (at least High Purity) SPARGE CONTINUOUS SPARGE SPARGE AND PRESSURIZE ON-LINE VACUUM { Uses too much helium Best choice, when available Sparge for 5 min; then let pressurize Good choice when helium unavailable; 80% as effective as helium degassing
Effect of (Not) Degassing on Retention Time Pump mixed mobile phase at 30 O C; Injections every 6 min and, after Helium stopped, every 4 hours 2.0 1.8 Solvent A = 1% HOAc in MeOH Solvent B = 1% HOAc in H 2 O Mobile Phase = 34% A 0.22% CCA Chlorthalidone 1.6 Retention Time (min) 1.4 1.2 3.9% (RSD) Not Degassed Not Pressurized Degassed and Pressurized 1.5% Stop Helium Pressurization 1.0 0.24% 0.8 3.5% 1.3% 0.6 48 min 16 Hours 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Injection Number
Effect of temperature Bottom line: Avoid ambient temperatures 2.4 2.2 Premixed solvent; starting at 30 Degrees 3.4% CCA Chlorthalidone Retention Time (min) 2.0 1.8 1.6 1.4 1.2 0.31% (RSD) o 30 C w/ Oven Pressurized and Degassed Room Temperature o 22-25 C Pressurized and Degassed 2.5% 0.27% o 30 C w/ Oven Pressurized and Degassed 1.0 0.24% 0.30% 0.8 24 Hours 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 Injection Number
Common Mobile Phase Modifiers Buffers To stabilize phs of mobile phase under reverse phase (RPC) or ionexchange (IEC) chromatography (phosphate, acetate, citrate); ~5-20 mm Triethylamine (TEA) To reduce tailing of basic analytes RPC; ~0.1% Phosphoric, acetic acid or formic acids Ion suppressors To suppress ionization of acidic analytes under RPC; ~0.1% Ion-pairing reagents For separation of ionic compounds using RPC column (C18) Anions: Quaternary ammonium salts (e.g., Tetrabutylammonium hydroxide); ~2-20 mm Cations: Alkyl sulfonates (e.g., Hexane sulfonate); ~2-20 mm
Series 200 Analytical Pump: Low Pressure Mixing Solvent toward column Solvents Solvent into metering chamber 100 µl 107 µl Solvent mixing delay volume: ~800-1000uL, depending on pressure Advantages: Great solvent mixing and built-in solvent compressibility factor
Low-Pressure Mixing Pump Blending Performance
Series 200 Micro-Pump: Binary High-Pressure Mixing To Injector Purge Valve Purge Valve Tee Pump A Pump B Solvent mixing delay volume: ~10-400uL with static mixer, depending on flow rate Advantage: Very low solvent mixing delay; good for high speed gradients and LC/MS
Method Transferability Concerns Low-Pressure Mixing: 1.0mL Delay Volume = High-Pressure Mixing: 0.1mL Delay Volume = 2 ml/min
Using Injection Delay Time (for Method Transferability) Low-Pressure Mixing: 1.0mL Delay Volume = High-Pressure Mixing: 0.1mL Delay Volume = Low-Pressure mixing with 0.45 min Injection Delay Time 2 ml/min
HPLC: Preferred Materials for Wettable Parts STAINLESS STEEL Industry Standard SS 316; May rarely effect biological activity or allow adverse chealation to occur TITANIUM (alternative to SS) PEEK (Poly-Ether Ether-Ether-Ketone) TEFLON / VESPEL / TEFZEL POLYAMIDE / POLYIMIDE QUARTZ-SILICA GLASS Good biocompatibility. Somewhat more brittle than SS Good biocompatibility and often used for basic compounds. Becomes brittle w/ >20% THF or MeCl 2 Used for rotor seals and low- pressure tubing. Note: : Use PEEK for basic compounds Do not use with MeCl 2 Commonly used for flow cells RUBY ` Used for balls in pump check valves SAPPHIRE Used for pump pistons
Pressure Problems PRESSURE TOO HIGH Dirty column Blockages e.g., system filter, guard column or column inlet frit, tubing, injector needle or valve, detector flow cell. >> Check by isolating pressure point(s); like a plumber Check flow rate Different solvent mixture? Are solvent lines switched?
Pressure Problems PRESSURE TOO LOW Leaks Pump malfunction e.g., piston seal, connections, rotor seal e.g., vapor lock, sticking check valves or solenoid valve, broken piston Plugged solvent filter Check flow rate Different solvent mixture? Lines switched?
Autosampler: Basic Injection Cycle Based on Rheodyne injector valve Poor peak shape may result if either the connections to injector ports 2 and 3 are reversed or one fills sample loop volume over 50% (in partial-loop mode). Sample from Syringe
Precision Problems Possibly Related to Autosampler Overfilling the sample vial Recommended: No more than 2/3 full Tightly sealed vials not vented Turn venting option on Excessive sampling speed Especially for viscous and low volume samples (<5µL) L): : Reduce speed to slow Bubbles in the autosampler sampling lines or syringe Degas flush solvent Worn rotor seal in the injection valve Results in poor RSD: Replace seal
Detectors for Any Application PerkinElmer Series 200 Detectors UV/Vis PDA UV/VIS considerations: Can detect predominant compounds being analyzed by HPLC Useful for any compounds that absorb between 190 and 700 nm Very good sensitivity (ppb). Typically, better than RI but not as good as Fluor. Exceptional baseline stability Wavelength programmable during run PDA considerations: Approaches UV/VIS sensitivity (ppb) Dual lamps (Deuterium and Tungsten) for full UV/Vis wavelength range Spectral data useful for confirmation and identification of components Capability of peak purity assessment Network-Direct connection for direct transfer of data and spectra to data directories on the network
Detectors for Any Application PerkinElmer Series 200 Detectors FL RI Fluorescence considerations: Very selective (must fluoresce) Exceptional sensitivity (ppt) May have to derivatize if compounds do not fluoresce in native state. Refractive Index considerations: Universal detection Good sensitivity (ppm) Sensitive to temperature changes (RI must have built-in Peltier temp. control; 35 C usually good for most labs) Can not be used for gradient method
Detectors: What to Consider Lamp Energy: Lamp Hours: Wavelength Accuracy: Reference Wavelength (PDA): Flow Cell Condition: RI Reference Purge Valve: Column Connections: Check periodically Lamp lifetime: typically ~1500 hours for UV/Vis, ~1000 for PDA and 500 for FL detectors Check periodically Choose a wavelength transparent to both sample and solvent On PDA, remove flow cell periodically; hold it toward light and check clarity Shine light into UV/Vis flow cell Check position Check that column is connected to Inlet tubing on detector
Detector Pointers Baseline problems related to detectors may be: Mechanical Leaky flow cell, trapped bubble Optical Aging lamp, dirty flow cell Bad mobile phase Not detector Electrical Call vendor Don't wash columns while they are connected to the detector. When possible, the use of a 50psi back pressure device is recommended after the detector (suppresses outgassing)
Suggested Periodic Performance Testing LC PUMPS Flow, composition calibration and pressure Leak test AUTOSAMPLER Precision, carryover, linearity testing DETECTORS Wavelength check Noise and drift testing Linearity testing
Suggested Spare Parts and Replacement Period LC PUMPS High/low pressure piston seals and backup rings kit (~ once a year); Part# N291-0383 Might want to replace high pressure piston every other year AUTOSAMPLER Injector rotor seal (~ once a year) Injector needle (if properly maintained, should hardly ever need replacing) Might want to replace flush and/or sampling syringe periodically (inspect first) DETECTORS Lamp (order new one only after 1000 hours on current UV/Vis or 500 hours on PDA - they have a shelf life)
Chromatographic Diagnostics NO PEAKS: Lamp off/bad, check wavelength Injector problem Check solvent selection / strength No sample in vial CARRYOVER: Materials incompatibility eg., Teflon vs PEEK Insufficient injector flushes or flush speed Column overloaded Some columns exhibit component carryover For that application, switch to another column vendor
Chromatographic Diagnostics Normal noise Expanded; always a good practice before starting a run
Chromatographic Diagnostics 5 min Air bubbles in solvent Degas solvent with in-line degasser or 5min helium sparge then pressurize Aging/bad detector lamp Replace lamp (typically, order new lamp after 500 hours on current one) Damaged pulse dampener on pump Replace
Chromatographic Diagnostics 5 min Contamination in system (solvent, degasser, pump, AS, column, detector) Remove column(s)/guard column and flush any buffers out of system; Rinse all solvent lines with HPLC water (may require co-solvent flush); Place all solvent lines into common bottle of 10% Ammonium Hydroxide; Flush for 20 min at 3mL/min. then switch to HPLC water for 2 minutes; Replace solvent bottle with 10% Nitric Acid and do another 20 min flush; Replace solvent bottle with HPLC water and do another 10 min flush; May also want to perform 20 min./1ml/min Isopropanol flush (usually not necessary); otherwise, move back to original solvents with clean bottles
Chromatographic Diagnostics Noise from detector, surroundings or poor analog cable grounding Monitor lines and recommend adding line conditioner Aging/bad detector lamp Replace lamp Air bubbles in solvent or dirty flow cell Degas solvent / rinse flow cell with strong co-solvent; The latter may require base/acid rinse (previous slide) May arise from fines from column > flush thoroughly without detector or change vendor Especially observed on RI and electrochemical detectors
Chromatographic Diagnostics A B Void volume; solvent front Unretained peak volume/time Elution volume k = 1 (capacity factor) Refractive index effect (typically from flow cell) Replace flow cell or retire detector
Chromatographic Diagnostics Column voids/channeling Bad column: Discard
Chromatographic Diagnostics Sample overload Inject less sample, dilute sample or change diluent to match mobile phase Forgot to add anti-tailing modifier to mobile phase Add Triethylamine (TEA) or similar Deteriorating column Change column Components may be too basic or acidic If using ion exchange column, switch to C18 and ion pairing mode
Example of Fronting Fronting Peak Caused by sample overload or component miscibility problem Inject less sample, dilute sample or change diluent to match mobile phase Check sample miscibility
Chromatographic Diagnostics All could be caused by sample overload Inject less sample, less concentrated sample or change diluent to match mobile phase Change to another column with higher sample capacity
Chromatographic Diagnostics Proportionally increasing retention times Low flow rate If same peak height, solvent leak before injector Column temperature may be lower; use column oven Possible mobile phase change (modifier, ph, molarity); however, not always proportional Possible column deterioration; however, usually not proportional
Chromatographic Diagnostics Disproportional retention time change Mobile phase change (modifier, ph, molarity); Column deterioration; replace column Change in column temperature; use column oven
Chromatographic Diagnostics Proportional drop in peak height Air in autosampler flush line Check lamp energy: lamp energy may be getting significantly lower. Time to change lamp Check wavelength (not always proportional) Sample absorbing to column or other material; change all teflon contact points in autosampler to PEEK material (rotor seal, etc.) or change to other vendor s column Autosampler: leaky rotor seal or plugged needle Volatile components: use capped vials (not always proportional)
Thank you for attending!
Chromatographic Diagnostics Component interconversion or isomerization Check ph, especially relative to pka(s) of components Check Temperature