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Agilent Technologies — Technical Presentation (September 2016)
52 slides from Agilent Technologies's technical presentation, Sep 29, 2016
Slide 1: Taking the Trouble Out of Troubleshooting
Taking the Trouble Out of Troubleshooting Is It the Column, Method, or Instrument Rita Steed September 29, 2016 Page 1
Slide 2: What Do We Troubleshoot
What Do We Troubleshoot Typical LC troubleshooting approach asks: – What’s wrong with my column? – What’s wrong with my instrument? But our separations are controlled by more than just the column or instrument. The better question to ask is - Why doesn’t my separation work as expected? And the answe
Slide 3: Why Doesn't My Separation Work as Expected?
Why Doesn’t My Separation Work as Expected? Finding the Answer Discuss some of the most commonly observed column related issues in HPLC • System pressure • Peak shape • Retention/Selectivity Explore reasons for problems Preventive measures Page 3
Slide 4: 1. Pressure Issues
1. Pressure Issues • Plugged Inlet • Large Frit Column Pressure Potential • Column Increase Contamination Observations Problems • Plugged Packing Note: Low pressure is typically a connection or LC issue; unless the column has been improperly used and disassembled or lost all its packing. Page 4
Slide 5: Determine the Cause and Correct
Determine the Cause and Correct Possible Causes • Column inlet frit contaminated/plugged • Frit in purge valve contaminated • Column contaminated • Blockage in a capillary, particularly needle seat capillary • Rotor in injection valve plugged • Guard or in-line filter Check pressure with/without col
Slide 6: Preventing Column Back Pressure Problems
Preventing Column Back Pressure Problems • Filter mobile phase – Filter non-HPLC grade solvents – Filter buffer solutions • Install in-line filter between auto-sampler & column (removes pump seal debris, ALS rotor debris, & sample particulates) • Filter all samples and standards – Syringe; in-line •
Slide 7: Captiva Filtration and its Benefits
Captiva Filtration and it’s Benefits Filtration is basic sample preparation method for all kinds of samples Physically removes particulates from the sample Prevents blocking of capillaries, Pub. No. 5991-1309EN frits, and column inlet (especially for UHPLC) Results in less downtime of the Unfiltered
Slide 8: Why perform Sample Preparation?
Why perform Sample Preparation? To acquire desired sensitivity/selectivity To reduce contamination/carryover issues Use of sensitive and expensive instruments: Protect your investment!!! Pesticides in Avocado without SP Pesticides in Avocado with SP
Slide 9: Contaminant Peaks: Bond Elut SPE and its Benefits
Contaminant Peaks Bond Elut SPE and its Benefits In this example, results after SPE show a decrease in the number of small peaks/removal of interferences and an increase in the intensity of the peak heights HPLC-DAD chromatograms of hydrastine and berberine from goldenseal roots extract before and a
Slide 10: Prevent Pressure Problems by Preventing Microbial Growth
Prevent Pressure Problems by Preventing Microbial Growth Potential problems – Increased system pressure or pressure fluctuations – Increased column pressure, premature column failure – Can mimic application problems – Gradient inaccuracies – Ghost peaks Prevent and/or Reduce Microbial Growth – U
Slide 11: Things to Remember: Troubleshooting Pressure
Troubleshooting Pressure Things to Remember I. Continuously increasing pressure even with no injections - Pump Seals - Mobile Phase Particulates - Mobile Phase Solubility - Mobile Phase Unstable (polymerization) - Column Void Formation (use condition dependent) - Microbial Growth II. Increasing pres
Slide 12: 2. Peak Related Issues
2. Peak Related Issues • Split peaks • Peak tailing • Broad peaks • Poor efficiency (low N) • Inconsistent response • Ghost Peaks Many peak shape issues are also combinations – i.e. broad and tailing or tailing with a change in retention
Slide 13: Split Peaks
Split Peaks Can be caused by: • Column contamination • Partially plugged frit • Column void • Injection solvent effects Page 13
Slide 14: Split Peaks: Column Contamination
Split Peaks Column Contamination Column: StableBond SB-C8, 4.6 x 150 mm, 5 mm Mobile Phase: 60% 25 mM Na2HPO4, pH 3.0 : 40% MeOH Flow Rate: 1.0 mL/min Temperature: 35°C Detection: UV 254 nm Sample: Filtered OTC Cold Medication: 1. Pseudoephedrine 2. APAP 3. Unknown 4. Chlorpheniramine Injection 1 In
Slide 15: Injection Solvent Effects - HILIC
Injection Solvent Effects - HILIC H2O, CH3OH, CH3CN LC/MS Separation of Vitamin B Related Compounds Agilent 1290 Infinity LC System x102 Agilent 6410A LC/MS 1 H2O Agilent ZORBAX RRHD HILIC Plus 2.1 x 50 0.5 PW 1/2 = 0.113 PW 1/2 = 0.056 mm, 1.8 µm PW 1/2 = 0.016 PW 1/2 = 0.107 0 Acetonitrile / 100 m
Slide 16: Determining the Cause of Split Peaks
Determining the Cause of Split Peaks 1. Complex sample matrix or many samples analyzed - likely column contamination or partially plugged column frit. 2. Mobile phase pH > 7 – may have a column void due to silica dissolution (unless specialty column used; Poroshell 120 HpH, Zorbax Extend C18, or pol
Slide 17: Peak Shape: Tailing Peaks
Peak Shape: Tailing Peaks First Question: All Peaks or Some Peaks? Causes Symmetry > 1.2 Some Peaks Tail: Secondary - retention effects. • Residual silanol interaction Small peak eluting on tail of larger peak Normal Tailing All Peaks Tail: Extra-column effects i.e. poor connections, too much
Slide 18: Peak Tailing: Column Contamination
Peak Tailing Column Contamination Column: StableBond SB-C8, 4.6 x 250 mm, 5mm Mobile Phase: 20% H2O : 80% MeOH Flow Rate: 1.0 mL/min Temperature: R.T. Detection: UV 254 nm Sample: 1. Uracil 2. Phenol 3. 4-Chloronitrobenzene 4. Toluene QC test forward QC test after cleaning QC test reverse direction
Slide 19: Effect of pH on Peak Shape
Effect of pH on Peak Shape What Happens Near the Sample pKa Column: ZORBAX SB-C8 4.6 x 150 mm, 5 um Mobile Phase: 40% 5 mM KH2PO4: 60% ACN Flow Rate: 1.0 mL/min. Temperature: RT CH3CHCOOH pH 4.4 pH 3.0 CH2CH(CH3)2 Ibuprofen pKa = 4.4 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 Time (min) Time (min
Slide 20: Determining the Cause of Peak Tailing
Determining the Cause of Peak Tailing • Evaluate mobile phase effects - alter mobile phase pH and/or additives to eliminate secondary interactions • Evaluate column choice - try column with high purity silica or different bonding technology • Reduce sample load – injection volume and concentration •
Slide 21: Peak Shape Problems Due to Extra Column Volume
Peak Shape and Related Problems Due to Extra Column Volume from Connections and Fittings • ECV is volume in the LC system outside of the column o There will always be some in the flow path and the LC system is designed to minimize the impact of this • Connections and fittings, if made improperly, re
Slide 22: What Happens If the Connection's Poorly Made?
Peak Tailing/Fronting What Happens If the Connection’s Poorly Made ? Wrong … too short Mixing Chamber X If Dimension X is too short, a dead-volume, or mixing chamber, will occur This can broaden peaks and/or cause them to split or tails It will typically affect all peaks, but especially early elu
Slide 23: Peak Tailing – Extra Column Effects
Peak Tailing – Extra Column Effects Poor Fitting mAU DAD1 A, Sig=254,4 Ref=360,100 (MERCURY20... DATA; MERCURY2010 ECLIPSE PLUS 3X100,1.8\USUYB0145300037.D) 40 Before troubleshooting Problem: 30 Tailing peaks All peaks tail 20 (top chromatogram). 10 0 0 2 4 6 8 10 min DAD1 A, Sig=254,4 Ref=360,100 (
Slide 24: Peak Shape - Broadening
Peak Shape - Broadening Efficiency is greatly reduced when extra-column volume increases mAU 0.12 x 105 mm Red Tubing (1.2 µL) 30 QC Test Conditions: 55% CH3CN 25 45% H2O 20 Isocratic, 0.6 mL/min 15 1 uL injection of QC Mix 10 N ≈ 10500 23 oC 254 nm 5 0 0.2 0.4 0.6 0.8 1 1.2 1.4 min mAU 0.25 x 700 m
Slide 25: Extra Column Volume
Extra Column Volume = sample volume + connecting tube volume + fitting volume + detector cell volume Mobile Extra Column Data Phase Volume Device The instrument schematic above depicts where extra-column volume can occur, thus effecting instrument and column performance. Note: More on this in “Makin
Slide 26: Effect on Efficiency: Flow Cell Choice
Effect on Efficiency Flow Cell Choice 2.1x100 mm Poroshell 120 EC-C18, PN 695775-902 Flow Cells 25000 Flow Cells are an integral part of HPLC instrumentation. • Choose the best one for the column 20000 used • Don’t assume you have the best one for your column • Peak broadening will compromise Effici
Slide 27: Effect of Detector Response Time
Peak Shape Effect of Detector Response Time It may not be a “bad column” Response Time 0.1 sec Agilent 1100 DAD 1st peak = 1.2 sec Agilent 1100 WPS with ADVR At 20 pts/sec = 24 pts/sec 0.2 sec Column: Poroshell 300SB-C18 2.1 x 75 mm, 5 mm 0.5 sec Mobile Phase: A: 95% H2O, 5% ACN with 0.1% TFA B: 5%
Slide 28: Ghost Peaks – Even with No Sample
Peak Shape Ghost Peaks – Even with No Sample 60 20% - 100% MeOH 15 No Sample Injected 30 15 Problem • Dirty mobile phase • Sample carryover 0 • May imply poor recovery 3 7 15 17 • Peak from an early run (isocratic) September 30, 2016 28
Slide 29: Solvent Contamination
Solvent Contamination ___ Solvent Source 1 ___ Solvent Source 2 ___ Solvent Source 3 ___ Solvent Source 4, Lot 1 ___ Solvent Source 4, Lot 2 September 30, 2016 29
Slide 30: Usual Suspects
Troubleshooting Peak Shape Issues “Usual Suspects” Partially blocked inlet frit Sample solvent strength Injection volume Sample load Secondary interactions Hardware failure (rotor, stator) Ghost peaks Metal interactions/chelation No or insufficient mobile phase pre-heating Extra
Slide 31: 3. Retention Shifts/Selectivity Changes
3. Retention Shifts/Selectivity Changes All Peaks Shift to Lower Retention (acids, bases, neutrals) • Loss of bonded phase • Mobile phase unstable (less likely) • Solvent delivery system (flow rate or mixing) All Peaks Shift to Greater Retention • Loss of organic solvent in aqueous/organic mix • Col
Slide 32: Problem – Selectivity Does Not Appear the Same from Column to Column
Problem – Selectivity Does Not Appear the Same from Column to Column Details • 3 Columns with the same bonded phase were used • They were the same dimensions, but with different particle sizes (and therefore different lots of material) • They were tested on the same day, on the same instrument, with
Slide 33: Inconsistent Selectivity between Particle Sizes of Eclipse Plus C18
Inconsistent Selectivity between Particle Sizes of Eclipse Plus C18, 4.6 x 50 mm, m.p.: A: water, B: acetonitrile (60:40 A:B) Flow : 1.5 mL/min Temp: 25 C mAU Detection: UV 280nm,16, ref=360,20 1.635 0.961 5um Flow cell: 6mm, 5uL 0.712 25 5 6 Data rate: 0.2s 2 3.907 3 0.293 20 α 5,6 = 2.70 1 Inj. Vo
Slide 34: Problem with Proportioning Valve
Problem with Proportioning Valve One channel premixed mobile phase shows similar α m.p.: A: water, acetonitrile (60:40 v/v) 5 um, UXE01033, NEP0652003 Flow : 1.5 mL/min 1.612 1.615 mAU 0.944 0.946 Temp: 25 C 3.879 3.873 0.695 0.696 Detection: UV 280nm,16, ref=360,20 46 bar 20 Flow cell: 6mm, 5uL 0.2
Slide 35: Compare Alpha Values from Proportioned and Premixed Mobile Phase
Compare alpha values from proportioned and premixed mobile phase Column Type Alpha of peaks 5,6 Alpha of peaks 5,6 premixed proportioned 5um 2.70 2.71 3.5um 2.75 2.74 1.8um 2.88 2.74 •Selectivity changes from column to column and lot-to-lot are the hardest to resolve •Problems can be more than just
Slide 36: Change in Volatile Buffer Concentration
Retention Time Shifts and Peak Shape Change in Volatile Buffer Concentration 8.586 mAU 30:70 ACN:Water with 0.1% TFA, pH 2 Amitriptyline Tf = 1.18 7.008 8 6 Imipramine 2.913 Tf = 1.16 4 Berberine 2 Tf = 1.11 0 1 2 3 4 5 6 7 8 9 min 30:70 ACN:Water with 0.01% TFA, pH 2.9 Amitriptyline 5.623 mAU 4.614
Slide 37: Mobile Phase Preparation
Mobile Phase Preparation Small changes in mobile phase strength can have a large affect on retention HPLC grade or better Volume % of solvents can depend on Buffer prep procedure preparation • Be consistent Specified volume ACN added to a 1 L o Document process volumetric and made to volume wi
Slide 38: Retention
Retention Separation Conditions That Can Cause Changes* Condition Change Retention Change Flow Rate +/- 1% tR +/- 1% Temperature +/- 1 deg C tR +/- 1 to 2% % Organic +/- 1% tR +/- 5 to 10% pH +/- 0.01% tR +/- 0 to 1% *excerpted from “Troubleshooting HPLC Systems”, J. W. Dolan and L. R. Snyder, p 442
Slide 39: Retention Shifts
Troubleshooting Retention Shifts Mobile Phase Related Problems - Make fresh, compare to aged · pH · conductivity · chromatographic test Column Related Problems - Test new column - Test current column with test mixture or e.g., Toluene - "Wash" column and retest - Consider effect of sample matrix
Slide 40: Why Doesn't My Separation Work as Expected?
Why Doesn’t My Separation Work as Expected? Common Problems High pressure Undesirable peak shape Changes in retention/selectivity Problems are not always associated with the column • May be caused by instrument and experimental condition issues Take the trouble out of troubleshooting • Use pro
Slide 41: The End – Thank You!
The End – Thank You! Page 41
Slide 42: Agilent Technical Support
Agilent Technical Support 800-227-9770 (Toll Free US & Canada) • For LC columns • Select option 3, then option 3, option 2 lc-column-support@agilent.com • For GC Columns Select option 3, then option 3, option 1 gc-column-support@agilent.com • For Sample Prep Select option 3, then option 3, option 3
Slide 43: Appendix
Appendix ilent Technologies
Slide 44: Column Cleaning
Column Cleaning Flush with stronger solvents than your mobile phase Make sure detector is taken out of flow path Reversed-Phase Solvent Choices in Order of Increasing Strength Use at least 10 x Vm of each solvent for analytical columns 1. Mobile phase without buffer salts (water/organic) 2. 100% Org
Slide 45: Initial Column and System Equilibration
Using Buffers Successfully Initial Column and System Equilibration In an appropriate vessel, test highest % organic/buffer ratio to verify that buffer will not precipitate. With stirring, add organic to buffer first, not vice versa. Equilibrate column with, in order: • 100% organic modifier (if bran
Slide 46: Shutdown State and Instrument Flushing
Using Buffers Successfully Shutdown State and Instrument Flushing Shutdown State Next day use—using same buffers • Pump mobile phase very slowly (for example, 0.01 – 0.1mL/min). When flushing column or for longer term column storage • Flush with 20/80 organic/water, then 80/20 organic/water or 100%
Slide 47: High Pressure In-line Filter Kit
High Pressure In-line Filter Kit For 2.1mm Frits use inserts with small cone For 4.6mm Frits use inserts with big cone
Slide 48: Assembling the High Pressure Filter Kit
Assembling the High Pressure Filter Kit Put the first insert into the Place the frit on top of this Then place the second insert frit housing. insert on top of the frit
Slide 49: Assembling the High Pressure Filter Kit
Assembling the High Pressure Filter Kit Close the frit housing, Slide the fitting, back and front Ensure that both capillaries are screw finger tight ferrule onto the capillary. Insert still pushed all the way in into the the capillary into the frit housing bore. Then tighten both fittings at bore,
Slide 50: Use 0.12 mm Tubing Instead of 0.17 mm Tubing
Use 0.12 mm Tubing Instead of 0.17 mm Tubing Inside Diameter (mm) Length (mm) Material Color Connections Part Number Volume (ul) 0.12 180 SS Red 1 end pre-swaged G1313-87304 2.0 0.12 280 SS Red 1 end pre-swaged 01090-87610 3.2 0.12 105 SS Red 1 end pre-swaged 01090-87611 1.2 0.12 150 SS Red pre-swag
Slide 51: SPE Modes
SPE Modes Analyte Adsorption (Bind-Elute ) Matrix Adsorption (Interference Analyte(s) retained (KD >> 1) Removal) Matrix unretained (KD ~ 0) Analyte(s) unretained (KD ~ 0) and/or strongly retained (KD >> 1) Matrix retained (KD >> 1) Pre-concentration factor No pre-concentration advantage Cleaner ext
Slide 52: If your sample needs further cleaning…
If your sample needs further cleaning… QuEChERS Solid-supported liquid extraction (SLE) Bond Elut Solid Phase Extraction (SPE) 52
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