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Transcript of Diagnóstico y corrección de anomalías HPLC /UPLC · Diagnóstico y corrección de anomalías...
Diagnóstico y corrección Diagnóstico y corrección
de anomalías de anomalías
HPLC /UPLCHPLC /UPLC
©2007 Waters Corporation
HPLC /UPLCHPLC /UPLC
CerdanyolaCerdanyola, , EneroEnero 20142014
Course contentCourse content
� Section 1— Potential sources of chromatographic problems
— Performance monitoring
— Band spreading
� Section 2 — Peak shape problems
Section 3
©2007 Waters Corporation 2
� Section 3—Retention time problems
� Section 4—Miscellaneous problems
� Section 5—Column protection & baseline troubleshooting
Problem!
Troubleshooting StrategyTroubleshooting Strategy
Try to simplify:
Inspect the chromatography
Try to categorize
Troubleshoot the easiest to fix items first
©2007 Waters Corporation 3
CHEMISTRY
� COLUMN
� GUARD COLUMN� SOLVENTS� ADDITIVES� SAMPLE & VIALS
� PUMP� INJECTOR� DETECTOR� DATA COLLECTION� CONNECTIONS� TUBING�VIALS
MECHANICAL
•Retention time•Area
•Linearity
•Resolution
Troubleshooting overviewTroubleshooting overview
©2007 Waters Corporation 4
•Solvent- Pump Autoinjector Colum Detector•reservoir
•Tailing •Plate count•Noise/drift
� QCRM’s (suitability standards) are a tool for customers to benchmark their
column and system performance. This provides:
1. Confidence before running any critical assay.
� Preparative chromatography standard- used to show system is suitable for use before extracting valuable samples.
PerfomancePerfomance monitoringmonitoringQCRM’s(Suitability Standards)QCRM’s(Suitability Standards)What are they?What are they?
©2007 Waters Corporation 5
suitable for use before extracting valuable samples. � Neutrals standard used at the end of each assay to test column
efficiency (plate count).
2. A powerful trouble shooting tool that helps customers quickly eliminate and identify causes of failure.
� Eliminate own samples and mobile phase as the cause of error.� Identify column or system issues based on neutrals standard
performance and system information (ACQUITY console)
•
� Nuevo estándar de referencia para sistemas LCMS o MS – Referencia 186006968
� Mezcla de 9 componentes que permiten verificar de forma exhaustiva sistemas LCMS o MS en un amplio rango de métodos y condiciones.
� Mezcla desarrollada por Waters en el centro de I&D y producción de Manchester, y utilizada para la verificación de la instrumentación del Laboratorio de demo.
� El estándar de referencia QCRM para LCMS se suministra en un vial sellado para inyección directa. Contiene 500ul de mezcla 1:10 ACN:Agua calidad LC/MS.
QCRM QCRM –– Quality Control Reference MaterialsQuality Control Reference Materials
©2007 Waters Corporation 6
Contiene 500ul de mezcla 1:10 ACN:Agua calidad LC/MS.
1) Los compuestos da una respuesta variada en ESi (+-) y APCi+2) Cubre un amplio rango de m/z 3) Concentración optimizada para obtener una respuesta adecuada en modo ESI+4) Permite obtener una separación cromatográfica en el rango de condiciones habitualmente utilizadas para verificar la instrumentación.
� Establecer condiciones de referencia:
� Evaluar indicadores clave sobre el estado del sistema mediante comparación con los valores generados cuando el sistema está en óptimas condiciones.
� El resultado obtenido con los materiales de referencia QCRM son específicos del sistema QCRM en el que se ha ejecutado el test. Todas las medidas tienen algún nivel de variabilidad. La observación de tendencias en los resultados son útiles para definir la variabilidad en un sistema, o entre sistemas en uno o varios laboratorios. El test QCRM es una valiosa herramienta para diagnosticar anomalías.
�
QCRM QCRM –– Quality Control Reference MaterialsQuality Control Reference Materials
©2007 Waters Corporation 7
� Los criterios y especificaciones establecidos permiten determinar si el resultado del test QCRM indica un correcto funcionamiento del sistema.
� Criterios típicos del QCRM para sistemas MS son: 1)Exactitud de masa 2)Sensibilidad 3)Respuesta 4)Rango del tiempo de retención oreproducibilidad5)Area de los picos6)Resolución de los picos
Componentes en el QCRM para LCMSComponentes en el QCRM para LCMS
©2007 Waters Corporation 8
� Benchmarking Principle:
� Evaluate or check key performance criteria by comparison with data generated on a new column when the system is known to be in good working order. Sufficient data trending is required to gain an adequate benchmark initial point.
�
System Benchmarking QCRM’sSystem Benchmarking QCRM’s
©2007 Waters Corporation 9
� Criteria and specifications should allow customers to determine if the QCRM results indicate that their system and column is functioning as expected.
� Typical criteria might include:
oRetention time range or reproducibilityo Peak area range or reproducibility o Peak tailing range o Peak resolution o Plate Counto Sensitivity o Response
AU
0.010
0.020
0.030
Experiment procedure and sample Experiment procedure and sample chromatogramchromatogram
•Acetone
•Naphthalene
Acenaphthene
©2007 Waters Corporation 10
0.000
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
•Acenaphthene
•Sample set run three times a day.
psi
6000.00
6500.00
7000.00
psi
6000.00
6500.00
7000.00
psi
6500.00
7000.00
Problem Bad Check Valve: Problem Bad Check Valve: Chromatogram and DataChromatogram and Data
AU
0.00
0.02
0.04
0.06
AU
0.000
0.010
0.020
AU
0.02
0.04
0.06
•Good Check Valve
•Good Check Valve
•Bad Check Valve
•~6600 psi
•~6600 psi
•~6800 psi
©2007 Waters Corporation 11
6000.00
Minutes
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
0.00
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
Acetone Retention Area TailingPlate
Count
Average 0.323 52245 1.18 3144
%RSD 0.69
Naphthalene Retention Area TailingPlate
Count
Average 1.633 155515 1.13 11009
%RSD 0.44
Acenaphthene Retention Area TailingPlate
Count
Average 2.893 101332 1.08 10436
%RSD 0.44
Acetone Retention Area TailingPlate
Count
Average 0.323 50804 1.18 3152
%RSD 0.68
Naphthalene Retention Area TailingPlate
Count
Average 1.631 153239 1.12 10753
%RSD 0.47
Acenaphthene Retention Area TailingPlate
Count
Average 2.893 99538 1.08 10220
%RSD 0.44
•System Benchmarking data• N=45
•Benchmarking data + 9 injections• (after fix)N=54
•~6600 psi
AU
0.000
0.005
0.010
AU
0.000
0.010
AU
0.000
0.005
0.010
Problem Gap in Column Outlet Problem Gap in Column Outlet Fitting: Chromatogram and DataFitting: Chromatogram and Data
AU
0.00
0.02
0.04
0.06
AU
0.00
0.02
0.04
AU
0.00
0.02
0.04
0.06
•Gap present
•No Gap
•No Gap•USP Tailing: 1.12
•USP Tailing: 1.12
•USP Tailing: 1.20
©2007 Waters Corporation 12
Minutes
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.000.00
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
Acetone Retention Area TailingPlate
Count
Average 0.323 52245 1.18 3144
%RSD 0.69
Naphthalene Retention Area TailingPlate
Count
Average 1.633 155515 1.13 11009
%RSD 0.44
Acenaphthene Retention Area TailingPlate
Count
Average 2.893 101332 1.08 10436
%RSD 0.44
Acetone Retention Area TailingPlate
Count
Average 0.323 50599 1.18 3159
%RSD 0.67
Naphthalene Retention Area TailingPlate
Count
Average 1.631 154565 1.12 10751
%RSD 0.45
Acenaphthene Retention Area TailingPlate
Count
Average 2.893 100707 1.08 10247
%RSD 0.44
•System Benchmarking data• N=45
•Benchmarking data + 9 injections• (after fix)N=54
Product Type Mode
(UV, MS)
Part Number
Neutrals Suitability Standard Neutral compounds for most analytical
HPLC/UPLC assays
UV 186006360
Reversed-Phase Suitability
Standard
Neutral, Acid, Base for most
analytical HPLC/UPLC assays
UV 186006363
Preparative Chromatography
Mix
Neutral, Acid, Base for most
purification HPLC assays
UV and MS 186006703
AutoPurification Dye Mix Visible markers for fraction collection
verification
UV and MS 716000765
MassPREP OST Standard Oligo Standard for Oligo applications UV and MS 186004135
System Suitability Part NumbersSystem Suitability Part Numbers
©2007 Waters Corporation 13
MassPREP OST Standard Oligo Standard for Oligo applications UV and MS 186004135
MassPREP Protein Standard Mix Protein Standard for Protein applications UV and MS 186004900
Glycan Performance Test
Standard
Glycan Standard for Glycan applications UV and MS 186006349
BEH200 SEC Protein Standard
Mix
Protein Standard for Size Exclusion UV and MS 186006518
BEH125 SEC Protein Standard
Mix
Protein Standard for Size Exclusion UV and MS 186006519
Cytochrome C Digestion
Standard
Cytochrome C Digestion Standard for
peptide applications
UV and MS 186006371
MassPREPEnolase Standard EnolaseDigestion Standard for peptide
applications
UV and MS 186002325
MassPREP Peptide Mix
Standard
Four Protein Digestion Standard for
peptide applications
UV and MS 186002337
Detector Cell
� How do you get sharp peaks
with excellent resolution?—Well Shaped Bands -- Well Separated
— (Good Mechanical And Chemical Performance)
Isocratic
Band spreadingBand spreading
©2007 Waters Corporation 14
Detector Cell
Waters c 1998
ELUTING FROM THE COLUMNYellowYellow analyte: narrowest band width – sharpest peak
Red analyte: moderate band width – moderate peak width
Blue analyte: Slowest – BROADEST/MOST DILUTED BAND --Broadest Peak (longer on the column – takes more mobile phase to sweep it out of the column -- broader the peak)
Isocratic
Where Band Spreading OccursWhere Band Spreading OccursSimple Isocratic HPLC SystemSimple Isocratic HPLC System
HPLC Column(Stationary Phase)
Data
Mobile Phase
©2007 Waters Corporation 15
Pump
Waste
Data
Detector
InjectorAutoSampler
Sample
Where Band spreading occurs{Flow path of sample from injector to the detector}
Where Band Spreading OccursWhere Band Spreading Occursin gradient systemsin gradient systems
HPLC Column(Stationary Phase)
Data
Mobile Phase B
Pump B
©2007 Waters Corporation 16
Pump AWaste
Detector
InjectorAutoSampler
Sample
Where Band spreading occurs{Flow path of sample from injector to the detector}
•Mixer
Pump B
Mobile Phase A
Instrument Band SpreadInstrument Band SpreadMeasure the “Peak” WidthMeasure the “Peak” Width(No Column In System)(No Column In System)
Note: As the peak width gets wider, indicates band spreading is greater, plate count and resolution goes down Since there is no
column, V is close to 0
“Peak” comes out instantaneously
©2007 Waters Corporation 17
4.4%
close to 0
Measure Width (W) and Convert to Volume in µl
Auto Zero Problem
Base line off-set
Performance of Different Column Diameters Performance of Different Column Diameters with Normal Instrument Band Spreading (80µL)with Normal Instrument Band Spreading (80µL)
6000
7000
8000
9000
10000
Plate Count
4.62.0 mm ID
3.9mm ID
4.6mm ID
Peak Width due toInstrument Band Spreading exceeding Column Band Spreading
3.0mm ID
©2007 Waters Corporation 18
0
1000
2000
3000
4000
5000
6000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Retention Factor
(k)
Plate Count
(N)
4.6
3.9
3.0
2.0
1.01.0 mm ID
2.0 mm ID
Note: You will not get optimum plate count performance on standard instruments as you reduce the column ID, especially below 2.0mm ID
All Columns “would” provide 10,000 plates --Impact greatest for early eluters
System OptimizationSystem Optimization
� Optimize for Narrow Bore:
Instrument Band Spread Reduced from 80µL to <
25µL
—Detector flow cell design/reduced volume
©2007 Waters Corporation 19
—Reduce injector sample loop volume
—Use 0.005” ID tubing/short lengths
—Perfect (pre-cut) connections (with variable depth inlet)
—Time Constants <0.2
—Reduce the number of tubing connections to a minimum
Most common causes of Most common causes of extra band spreadingextra band spreading
� Incorrect tubings
—Diameters
— Lenghts
� Incorrect fittings
©2007 Waters Corporation 20
Effect of Connecting TubingEffect of Connecting Tubingon System Bandspreadingon System Bandspreading
0.009" I.D.
W = 68 µµµµl
©2007 Waters Corporation 21
0
0
0
64X
0.020" I.D.
W = 127 µµµµl
32X
0040" I.D.
W = 400 µµµµl
1 minute
16X
Effect of Wrong Detector cell on Effect of Wrong Detector cell on System BandspreadingSystem Bandspreading
Bad RI Cell
W = 236 µµµµl
Prep Cell
©2007 Waters Corporation 22
Good RI Cell
W = 87 µµµµl
Analytical Cell
Effect of Peak Band Spreading on ResolutionEffect of Peak Band Spreading on Resolution
2 x Gain in SensitivityMetabolite in tail of
©2007 Waters Corporation 23
• Customer’s plumbing• Lack of sensitivity
• Improved plumbing• Replaced 10 connections between the injector, switching valve and MS• Removed 4 feet of extra 0.005 inch id tubing
Metabolite in tail of peak now resolved
7.00 7.50 8.00 7.00 7.50 8.00
Course contentCourse content
� Section 1— Potential sources of chromatographic problems
— Overall troubleshooting strategy
— Performance monitoring
� Section 2 — Peak shape problems
Section 3
©2007 Waters Corporation 24
� Section 3—Retention time problems
� Section 4—Miscellaneous problems
� Section 5—Column protection & baseline troubleshooting
ContentContent� Causes of peak shape problems
—Contaminated in-line filter/guard column *
— Column destroyed
— Secondary interactions
— Incorrect sample solvent
— Temperature
— Column overload
©2007 Waters Corporation 25
—Column overload
o Mass overload
o Volume overload
— Other extra-column effects
o Sampling rate*
o Detector time constant/filter value*
Peak Shape ProblemsPeak Shape Problems
� Mechanical problem
— Tubing & fittings
— Column voided
� Chemical problem
— Silanol activity
— Column overload
All peaks affected?
Yes No
©2007 Waters Corporation 26
—Column voided
— Contaminated in-line
filter
— Injector problem
� Chemical problem
— Sample solvent problem
— Column overload
— Co-elution
— Elution from previous
injection
Peak Shape ProblemsPeak Shape Problems
•
All Peaks Affected
fronting peaksdouble peaks
©2007 Waters Corporation 27
� All Peaks Affected
—COLUMN Voided
— COLUMN DESTROYED
o (pH <2 washes off functional group)
o pH >8 dissolves silica base
Normal columnNormal column
Packing Material Flush
©2007 Waters Corporation 28
“Well packed column”
Good ResultsGood Results
©2007 Waters Corporation 29
“Well packed column”
Column collapseColumn collapse
Packing Material Settled
©2007 Waters Corporation 30
“Voided column”
Column Collapse (Voiding)Column Collapse (Voiding)(shock/high pH/‘old’ (used) packing)(shock/high pH/‘old’ (used) packing)
All Peaks Distorted
©2007 Waters Corporation 31
Voids - high back pressure, distorted and/or double peaks
pH Limitations of Silica Based pH Limitations of Silica Based Packing Materials Packing Materials
Hydrolysis of Bonded Ligand
1
Dissolution of Silica particle
©2007 Waters Corporation 32
0 2 4 6 8 10 12 14pH
Hybrid / Polymeric -- Wider pH Range
Peak Shape ProblemsPeak Shape Problems� Contaminated in-line filter/guard column
� Column destroyed
� Secondary interactions
� Incorrect sample solvent
� Temperature
� Column overload
©2007 Waters Corporation 33
� Column overload
—Mass overload
— Volume overload
� Other extra-column effects
— Sampling rate
— Detector time constant/filter value
Poor Peak Shape for Basic Compounds due to Poor Peak Shape for Basic Compounds due to Secondary InteractionsSecondary Interactions
� Integration errors� Reduced resolution � Reduced sensitivity
Acids and Neutrals have Good peak shape --Basic Analytes “Tail”
©2007 Waters Corporation 34
Minutes
0 5 10 15 20 25
Tailing of Bases Tailing of Bases ---- Chemical Problem Chemical Problem
(Column Brand/pH(Column Brand/pH))
Conventional C18
Neutral
Different Silanol Activities forDifferent Column Brands
©2007 Waters Corporation 35
Time (min)50
Conventional C18
Current Generation C18
Neutral
Base
Base
0
Hydrophobic Interaction with Bonded Phase
Ion exchange Interaction with Charged Sites
O-Si
O-SiO-
O-SiO-SiO-
O-Si
O-Si
O-Si
OH
O-Si
O-Si
OH
O-Si(CH
3)2HN+(CH
3)2
HN+
Mobile Phase pH < 3
MixedMixed--mode retentionmode retention
©2007 Waters Corporation 36
O-Si
O-O-Si
O-Si
O-SiO-O--Si
O-Si
O-SiOH
O-Si
O-Si
O-Si
OH
O-Si
O-Si
3 22Phase pH < 3
Si - OH
-Mobile Phase pH > 3
Si – O
BaseBase
Column comparisonColumn comparison
Amitriptyline
Acenaphthene
Propranol Waters µBondapak™ C18 1973, USP TF = 10
©2007 Waters Corporation 37
Minutes0 15 30 45 60 75 90
Minutes5 10 150
Waters Xbridge RP18 2008, USP TF = 1.1
Peak Shape ProblemsPeak Shape Problems
� Contaminated in-line filter/guard column
� Column destroyed
� Secondary interactions
� Incorrect sample solvent
� Temperature
� Column overload
©2007 Waters Corporation 38
� Column overload
—Mass overload
— Volume overload
� Other extra-column effects
— Sampling rate
— Detector time constant/filter value
Effect of sample solventEffect of sample solvent
Sample in methanol
Minocycline
Tetracycline Demeclocycline
©2007 Waters Corporation 39
Sample in mobile phase(0.1% TFA, 4% ACN, 5% MeOH)Minocycline
Tetracycline Demeclocycline
Minutes
Minutes10
10
20
20
30
30
Peak Shape ProblemsPeak Shape Problems
� Contaminated in-line filter/guard column
� Column destroyed
� Secondary interactions
� Incorrect sample solvent
� Temperature
� Column overload
©2007 Waters Corporation 40
� Column overload
—Mass overload
— Volume overload
� Other extra-column effects
— Sampling rate
— Detector time constant/filter value
Column temperature equilibrationColumn temperature equilibration
70°C
70°C
70°C
©2007 Waters Corporation 41
22°C
70°C
70°C45°C 65°C 70°C
Peak Shape ProblemsPeak Shape Problems� Contaminated in-line filter/guard column
� Column destroyed
� Secondary interactions
� Incorrect sample solvent
� Temperature
� Column overload
©2007 Waters Corporation 42
� Column overload
—Mass overload
— Volume overload
� Other extra-column effects
— Sampling rate
— Detector time constant/filter value
500 µL
300 µL100 µL
10 µL
Column/Volume OverloadColumn/Volume Overload
©2007 Waters Corporation 43
Effect of injection volume on peak
distortion
Volume OverloadVolume Overload
©2007 Waters Corporation 44
Note : Peak starts at same time
Wider peaks first
Column/Volume OverloadColumn/Volume Overload
6.25 µg injected in 5 µL
% of column volume: 0.625
Absorbance
Column Volume: 0.8 mL (800 µL)(4.6 X 50mm ODS)
©2007 Waters Corporation 45
Wider peaks first observed at low
retention( )
Peak position shifts to higher retention in proportion to the injection volume( )
2 4Minutes 6
6.25 µg injected in 500 µL
% of column volume: 62.5
2 4Minutes 6
Absorbance
Concentration/Mass OverloadConcentration/Mass Overload
6.25 µg injected
Absorbance
Analytical load of ~6 µgyields efficient peak
shape
Encountered when mass injected onto column exceeds a certain limit– Note earlier lift-off point for preparative load
©2007 Waters Corporation 46
2Minutes
25 mg injected
Absorbance
Preparative load of 25 mg generates mass overload
peak shape
Note that the back of the peaks of the analytical and prep loads are at the same retention (-------)
4 6
2 4Minutes
6
Peak Shape ProblemsPeak Shape Problems
� Contaminated in-line filter/guard column
� Column destroyed
� Secondary interactions
� Incorrect sample solvent
� Temperature
� Column overload
©2007 Waters Corporation 47
� Column overload
—Mass overload
— Volume overload
� Other extra-column effects
— Sampling rate
— Detector time constant/filter value
SummarySummary
� Peak shape problems
— All peaks affected
o Mechanical problem
o Exception : Incorrect sample solvent
— Only one or two peaks affected
©2007 Waters Corporation 48
—Only one or two peaks affected
o Chemical problem
Minutes0 5 10 15 20 25
Course contentCourse content
� Section 1— Potential sources of chromatographic problems
— Overall troubleshooting strategy
— Performance monitoring
� Section 2 — Peak shape problems
Section 3
©2007 Waters Corporation 49
� Section 3— Retention time problems
� Section 4—Miscellaneous problems
� Section 5—Column protection & baseline troubleshooting
Content Content
� Retention Time problems
—Reproducibility versus drifting or incorrect retention times
o Temperature
o Organic %
o Chemistry problem
©2007 Waters Corporation 50
o Chemistry problem
o pH
o Ion-pairing
o De-wetting (Hydrophobic Collapse)
Retention Time ProblemsRetention Time Problems
Reproducibility, incorrect &
- Solvent Composition
- Temperature
- pH Control
Drifting Retention
- Equilibration
- Stationary Phase Stability
©2007 Waters Corporation 51
- pH Control
- Ion Pairing- Column Contamination
- De-wetting/Hydrophobic Collapse (Low % Organic)
•More or Less Retention Time•(All Peaks Affected)
Retention time drift/Retention time drift/incorrect retention timesincorrect retention times
� Pump Flow Rate Problem —Correct flow rate ?— Air in pump— Check valve not working correctly
©2007 Waters Corporation 52
—Check valve not working correctly
� Wrong Column Type: —C8 – less retention — C18 – more retention
� Temperature Problem —warmer – less retention, — colder – more retention
� % Organic In Mobile Phase —more – less retention, — less – more retention)
Content Content
� Retention Time problems
—Reproducibility versus drifting or incorrect retention times
o Temperature
o Organic %
o pH
©2007 Waters Corporation 53
o pH
o Ion-pairing
o De-wetting (Hydrophobic Collapse)
Effect of TemperatureEffect of TemperatureReversed phase chromatographyReversed phase chromatography
� Reduction of Retention with Increasing Temperature
� 1% to 2% Change per 1° Celsius
� Shifts in Selectivity Usually Small
©2007 Waters Corporation 54
� Shifts in Selectivity Usually Small
•600 C
Effect of Temperature (Isocratic Effect of Temperature (Isocratic Separations)Separations)
•1160 psi•N=2250
•Higher Temperature:
•Efficiency •Back-Pressure
©2007 Waters Corporation 55
•300
C
•500 C
•Minutes•1.00•2.00•3.00•4.00•5.00•6.00•7.00•8.00•9.00•10.00
•400 C
•1920 psi•N= 1680
•Higher Temperature:
•Shorter Run Time
•Sharper Peaks
•Better Sensitivity
•Lower Back Pressure
Effect of Temperature Effect of Temperature
(Isocratic Separations(Isocratic Separations))
23.5°C
•Higher
Temperature:
©2007 Waters Corporation 56
26.5°C•Note selectivity change
Temperature:
•Shorter Run Time
•Sharper Peaks
•Better Sensitivity
•Lower Back Pressure
•Minutes•5 •10 •15 •20 •25 •30
•Minutes•5 •10 •15 •20 •25 •30
Temperature & SelectivityTemperature & Selectivity
•AU
•0.00
•0.05
•0.10
•0.15
•0.15
•1
•2
•3
•4•5
•1
•2
•3
•30°C
• 1 Triamterene
•Small, but significant selectivity changes may be obtained from temperature changes.
©2007 Waters Corporation 57
•AU
•0.00
•0.05
•0.10
•AU
•0.00
•0.05
•0.10
•0.15
•Minutes
•0.00 •1.00 •2.00 •3.00 •4.00 •5.00 •6.00 •7.00 •8.00 •9.00 •10.00•11.00•12.00
•2•1
•1
•3
•4
•5
•2•3
•4
•5•40°C
•50°C
• 2 Althiazide
• 3 Bumetanide
• 4 Benzthiazide
• 5 Ethacrynic Acid
•Column: XTerra® MS C18•3.5 µm, 4.6 mm x 50 mm •Mobile phase:•25% MeOH, 65% Water, •10% Ammonium •bicarbonate buffer, pH 9
Content Content
� Retention Time problems
—Reproducibility versus drifting or incorrect retention times
o Temperature
o Organic %
o pH
©2007 Waters Corporation 58
o pH
o Ion-pairing
o De-wetting (Hydrophobic Collapse)
Effect of solvent compositionEffect of solvent composition(% organic) RP chromatography(% organic) RP chromatography
� Exponential Relationship between k and Volume % Organic
� Retention Time Change of 5% to 15% per 1%
Retention Times vs. % Methanol
30.00
35.00
©2007 Waters Corporation 59
of 5% to 15% per 1% Change in Solvent Composition
� Bonded Phase Collapse in High Water Content (discussed later)
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35 45 55 65
% MethanolRT (min.)
Change in Solvent CompositionChange in Solvent Compositionreversed phase chromatographyreversed phase chromatography
•More organic•shorter retention
•Can be caused by organic component evaporating from reservoir
©2007 Waters Corporation 60
•Less organic•Longer retention
•1 •2 •3 •4•Minutes
•1 •2 •3 •4
•Minutes
Questions????Questions????
•How difficult is it to make up a liter of•60/40 MeOH/Water •Mobile Phase????
•Mobile • Phase • Preparation
©2007 Waters Corporation 61
•How would You prepare this •mobile phase????
•Would we all get the same result????
•60/40 (MeOH/Water)
••400 mL400 mL••xyz grxyz gr
••600 mL600 mL••abc grabc gr
•Options A and A’ •Options B/C
•A) 600 mL•A’) 6?? mL
•??? mL•400 mL
Mobile Phase Composition Mobile Phase Composition is Critical for Reproducibilityis Critical for Reproducibility
©2007 Waters Corporation 62
•Options A and A’: Adding organic/aqueous directly to measuring vessel (not recommended)• Need to specify which solvent is added first: e.g. H2O to MeOH or MeOH to H2O
•
••Standardize pH Adjustment – ie; BEFORE organic is added
•• <1000 mL<1000 mL
••abc grabc gr
••1000 mL1000 mL
•Difference due to•partial volumes*
•Note: Partial Volumes on mixing may give • more or less than initial volumes•*
•*
•* 960mL
•Mobile Phase Composition •is Critical for Reproducibility
•60/40 (MeOH/Water)
••400 mL400 mL••xyz grxyz gr
••600 mL600 mL••abc grabc gr
•Options A and A’ •Options B
•600 mL•640 mL
•440 mL•400 mL
©2007 Waters Corporation 63
•Options A and A’: Adding organic/aqueous directly to measuring vessel (not recommended)• Need to specify which solvent is added first: e.g. MeOH to H2O or H2O to MeOH
•Options B: Separate volumetric measurement •
•
•• <1000 mL<1000 mL•• (960mL)(960mL)
••abc grabc gr
••1000 mL1000 mL
•Difference due to•partial volumes*
•Note: Partial Volumes on mixing may give • more or less than initial volumes
•* 960mL
Mobile Phase
Composition•is Critical for Reproducibility
•60/40 (MeOH/Water)
••400 mL400 mL••xyz grxyz gr
••600 mL600 mL••abc grabc gr
•Options A and A’ •Options B/C
•600 mL•6?? mL
•4?? mL•400 mL
©2007 Waters Corporation 64
•Options A and A’: Adding organic/aqueous directly to measuring vessel (not recommended)• Need to specify which solvent is added first: e.g. MeOH to H2O or H2O to MeOH
•Options B/C: Separate volumetric measurement or weighing is most accurate•
•
•• <1000 mL<1000 mL
••xyz grxyz gr••abc grabc gr
••1000 mL1000 mL
•Difference due to•partial volumes*
•Note: Partial Volumes on mixing may give • more or less than initial volumes
•Standardize pH Adjustment – ie; BEFORE organic is added
•(960 mL)
•Mobile Phase Composition is Critical for Reproducibility
•Clearly specify in your method HOW the Mobile Phase is to be prepared
•60/40 (MeOH/Water)
••400 mL400 mL••xyz grxyz gr
••600 mL600 mL••abc grabc gr
•Options A and A’ •Options B/C
•600 mL•6?? mL
•4?? mL•400 mL
©2007 Waters Corporation 65
•Options A and A’: Adding organic/aqueous directly to measuring vessel (not recommended)• Need to specify which solvent is added first: e.g. MeOH to H2O or H2O to MeOH
•Options B/C: Separate volumetric measurement or weighing is most accurate•
•
•• <1000 mL<1000 mL
••xyz grxyz gr••abc grabc gr
••1000 mL1000 mL
•Difference due to•partial volumes*
•Note: Partial Volumes on mixing may give • more or less than initial volumes
•Standardize pH Adjustment – ie; BEFORE or AFTER organic is added
•*•* 960 mL
Content Content
� Retention Time problems
—Reproducibility versus drifting or incorrect retention times
o Temperature
o Organic %
o pH
©2007 Waters Corporation 66
o pH
o Ion-pairing
o De-wetting (Hydrophobic Collapse)
Retention Time ReproducibilityRetention Time Reproducibility-- pHpH
pH
� Neutrals: No Influence� Acids: Reduced Retention with Increasing pH
©2007 Waters Corporation 67
� Acids: Reduced Retention with Increasing pH� Bases: Increased Retention with Increasing pH� Up to 10% Change in Retention per 0.1 pH Unit
(Largest shift within +/- 1 pH unit of pKa)
20
25
30
35
ReversedReversed--Phase Retention Phase Retention BehaviourBehaviour of of Basic Basic CompoundsCompounds Relative to Changes in pHRelative to Changes in pH
Capacity Factor
•Non-ionized Base
•> ± 2 pH units provides stable retention (better reproducibility at flat portions of curve)
•Range where •pH control•is critical
©2007 Waters Corporation 68
0
5
10
15
20
0 1 2 3 4 5 6 7 8 9 10 11 12
•pH
•Capacity Factor
(k)
•Ionized Base
•pKa
•±2
•Base 1+2
•1
•10
•100
•log (k) •Acid
•(Un-ionized)•Neutral
•Base 1
•Acid(Ionized)
•Base 2•Un-ionized
Impact On Mobile Phase pH on Impact On Mobile Phase pH on Retention Factor (k) ReproducibilityRetention Factor (k) Reproducibility
©2007 Waters Corporation 69
1+2
•(Ionized)
•pH: Powerful tool in methods development for Selectivity –Range where pH control will be critical
•pH•0.1•0 •2 •4 •6 •8 •10 •12 •14
(Ionized)
•N
•A, B1•B2
•B1 •N
•A
•B2 •pH 5.5
•pH6.0
pH pH –– ControlControlTest for Robustness (+/Test for Robustness (+/-- 0.2 pH Units)0.2 pH Units)
•AZT Robustness testing•Imp1
•Imp1
•Imp2•Imp3
•Imp3
•Imp4
•pH 2.3
©2007 Waters Corporation 70
•Imp1
•Imp2
•Imp2 •Imp3
•Imp3
•Imp4
•Imp4
•pH 2.5
•pH 2.7
Content Content
� Retention Time problems
—Reproducibility versus drifting or incorrect retention times
o Temperature
o Organic %
o pH
©2007 Waters Corporation 71
o pH
o Ion-pairing
o De-wetting (Hydrophobic Collapse)
Retention Time Reproducibility
Ion Pairing Reagents(E.g. alkyl sulphonates / tetra alkyl ammonium salts)
� Retention– Increases Proportional to the Concentration of the Pairing Agent at Low Concentration
©2007 Waters Corporation 72
Pairing Agent at Low Concentration– Nearly Independent of the Concentration of the Pairing Agent at High (~10mM/L) Concentration
� Long Equilibration Times– Due to Adsorption of the Reagent on the Stationary Phase (can be up to 500 Column Volumes)
Content Content
� Retention Time problems
—Reproducibility versus drifting or incorrect retention times
o Temperature
o Organic %
o Chemistry problem
©2007 Waters Corporation 73
o Chemistry problem
o pH
o Ion-pairing
o De-wetting (Hydrophobic Collapse)
•Mobile phase must
Proper Wetting of Bonded Chromatographic Proper Wetting of Bonded Chromatographic Surface is Required for RetentionSurface is Required for Retention
•The particles are very porous, like the pores of a sponge –99% of chromatographic surface is inside the pores
• Mobile • Phase
©2007 Waters Corporation 74
•Mobile phase mustbe allowed into the pore
•in order for chromatographic •retention of the analyte
•to take place.
What influence does this have on chromatography?
•If the pores are dry, the analyte cannot get into the pores and it will not be retained by the chromatographic surface.
•Analyte
High High LigandLigand Density CDensity C88 Column: Column: “Hydrophobic Collapse“Hydrophobic Collapse
•Initial Column pre-wetted
•Mobile phase: Aqueous 0.1% Acetic Acid
•Amoxicillin
©2007 Waters Corporation 75
•Minutes•0 •2 •4 •6 •8 •10
Column pre-wetted with organic
•After Flow Stoppage(Pore de-wetting 100%)
•Vo: No retentivity for analyte
•1500 psi
•1500 psi
What is DeWhat is De--wetting or “Hydrophobic wetting or “Hydrophobic Collapse?”Collapse?”
•Low % organic or pure aqueous mobile
phase
©2007 Waters Corporation 76
•De-wetted Pore•Wetted Pore
•C18 Silica-Gel Particle Pores
How does flow stoppage cause this problem How does flow stoppage cause this problem with an HPLC column?with an HPLC column?
•Analytes•Analytes
©2007 Waters Corporation 77
•At flow with pressure on the mobile phase.
•Stopped flow with no pressure on the mobile phase, pores de-wet. Restart flow, pores remain de-wetted with analytes never entering pores (low surface area).
DeDe--WettingWetting
“Hydrophobic Collapse”“Hydrophobic Collapse”
•“Hydrophobic Collapse” is a term that has commonly been used to describe the loss of retention in RP Chromatography. It refers to the long C18 Chains folding or collapsing back
©2007 Waters Corporation 78
It refers to the long C18 Chains folding or collapsing back down to the particle surface, thereby not being fully extended to interact, or retain the analyte.
•In reality, it is the de-wetting of the pores which causes the loss of retentivity.
ReRe--wetting a Stationary Phase wetting a Stationary Phase Once DeOnce De--wetted:wetted:
�Use a mobile phase containing > 40 % methanol or other polar organic solvent
�Difficult to use pressure to force aqueous mobile
©2007 Waters Corporation 79
�Difficult to use pressure to force aqueous mobile phase back into poresTypically not practical because column outlet is at atmospheric pressure
•1 atm (14.7 psi)
•2200 psi
•Inlet •Outlet
•P
•Length
ReversedReversed--Phase Column Selection Phase Column Selection Considerations Relating to the Risk of DeConsiderations Relating to the Risk of De--Wetting/Hydrophobic Collapse Wetting/Hydrophobic Collapse
� Pore Size
©2007 Waters Corporation 80
� Ligand Density
� Ligand Type
Chapter 4Chapter 4Miscellaneous problemsMiscellaneous problems
� Methods Development Suggestions
� Extraneous Peaks
� Mobile phase degassing
� Gradients
� Samples and Vials
©2007 Waters Corporation 81
� Samples and Vials
•Common Complaints:
• “My column is not reproducible” • – “a new column gives different results”
Changes in Retention/ResolutionChanges in Retention/Resolution
©2007 Waters Corporation 82
• “My chromatography changes”
• “Quality Control Laboratory can’t reproduce• my results”
• WHY?
•Changes in Retention/Resolution Method Development
•Please remember:
•HPLC Columns contain packing materials which can be chemically altered depending
©2007 Waters Corporation 83
which can be chemically altered depending on how they are used and treated
(mobile phase, pH, ion-pairing reagents, surfactants, age).
•“Column History”
•Changes in Retention/Resolution Method Development
•Once the column is altered in your laboratory, if you use this “modified particle surface” to create a separation, you may not be able to reproduce that separation on a new column –
©2007 Waters Corporation 84
reproduce that separation on a new column –because the new column was not chemically
altered in the same way.
•“Regeneration” may not always bring you back to the original chromatographic surface, especially, if ligand hydrolysis (loss of ligand) or
surface modification by ion-pairing reagents has occurred
•Methods Development Risk – starting with a “used column”•(in a drawer, on the instrument, donated by a colleague)
•Changes in Retention/Resolution Method Development
•Two Potential Causes
•Column Product is •Column Product is OK but
©2007 Waters Corporation 85
•How do you determine, and control the method development outcome?
•A suggested approach which controls the results, without confounding the reasons.
•Column Product is not manufactured reproducibly
•Column Product is OK but •use/conditions are changing
•the performance
•Changes in Retention/Resolution Method Development
• How Columns are Manufactured
1) A “BATCH” of Packing Material (particle) is prepared• First Silica then Bonding LOT # (Ligand/Endcapping)
(Determines retention/selectivity/resolution)
• 2) Approve “Chromatography”
©2007 Waters Corporation 86
• 2) Approve “Chromatography”
• 3) Pack Columns, LOT # / Test for efficiency/back pressure• Note: A BATCH* may be used to make many different
LOT #’s, and many thousands of columns
• 4) Approve and Ship
•*Note: Some column manufacturers buy packings and only pack columns•They may have little if any control over chromatographic performance.
•Suggested Methods Development Approach for Robustness*
1)
2) Obtain 3 - 4 NEW columns2 from the SAME BATCH (Columns A & B)
• 1 or 2 from ALTERNATIVE BATCH(ES) (C & D)
©2007 Waters Corporation 87
3) Select column A of the “2 from the SAME BATCH” of new columns and develop method conditions
•*Especially important for validated methods which• will be used for many years or in different locations
•Suggested Methods Development Approach for Robustness
• 4) Once conditions are set, run column B, (other NEW column from the SAME BATCH)
• 5) If results are the same – GREAT (no differences)
©2007 Waters Corporation 88
• 5) If results are the same – GREAT (no differences) then move to step 6
If results are NOT THE SAME – then you “modified” column A while you were developing the method. This necessitates starting again. Even a new column from the same batch will not duplicate your chromatography.
• Move back to step 2
••Suggested Methods Development Approach for Suggested Methods Development Approach for RobustnessRobustness
• 6) Try column C from an ALTERNATIVE BATCH (and column D from a third different batch if available)
• 7) If the results are the same – GREAT.
©2007 Waters Corporation 89
• 7) If the results are the same – GREAT. Manufacturer has adequate batch to batch reproducibility – Continue validation procedure.
•If the results are DIFFERENT – the manufacturer does NOT have adequate batch to batch reproducibility. You will need to work with that column vendor to get more reproducible columns or choose a new column vendor/manufacturer.
•Suggested Methods Development Approach for Robustness
••This approach provides you with an
early assessment of method robustness during the initial stages of separation
©2007 Waters Corporation 90
during the initial stages of separation development whilst allowing you to
identify reproducibility problems that may be occurring.
Tips from Case StudiesTips from Case Studies
� When replacing a column that has been used for
many injections you should expect that the surface
has been modified to some degree
� The new column will/may give different results initially (especially if the ligand was hydrolyzed off the old column)
©2007 Waters Corporation 91
•During Methods Development, running a “used” column
•can sometimes show degree of robustness
old column)
� The new column will have more ligand, and therefore changed retention characteristics.
•Tips from Case Studies
* Always use “New Columns” for initial method development work
©2007 Waters Corporation 92
method development work
•Avoid having to redo the development work all over again
Tips from Case StudiesTips from Case Studies
� Mobile Phase Preparation can be highly critical to ultimate method performance.
� Write your method procedures clearly so that no mistakes or misinterpretation can occur.
� Sequence of mixing, and method of measurement (volume,
©2007 Waters Corporation 93
� Sequence of mixing, and method of measurement (volume, weight) of components can dramatically change the chromatography. (pH control and % organic are especially important for some methods)
� Differences will often be observed for a mobile phase prepared in a single reservoir vs. “same” mobile phase made by blending lines using a proportioning/mixing valve
ContentContent
� Methods Development Suggestions
� Extraneous Peaks
� Mobile phase degassing
� Gradients
� Samples and Vials
©2007 Waters Corporation 94
� Samples and Vials
Extraneous PeaksExtraneous Peaks
Isocratic LC - Extra Peak –
Sharp - Contaminant
©2007 Waters Corporation 95
•Contaminant(s) from; sample, vial, septum, injector etc… •Isocratic Method: extra peak with the same peak shape and width ,
•the contaminant came from this injection
Extraneous PeaksExtraneous Peaks
Isocratic LC - Broad -Peak from
Previous Injection
©2007 Waters Corporation 96
Preventing contaminationPreventing contamination
� Use clean, particle-free solvents
— Filter through 0.2µm filter
� Use ultrapure water
— Particle-free, chemically clean, 18-megaohm
� Prevent microbial growth
— Prepare, filter, and degas aqueous mobile phase daily
� Minimize the use of additives
©2007 Waters Corporation 97
� Minimize the use of additives
� Store solvents in clean glass reservoirs with covers
— Prevent airborne contaminants from entering the solvent
� Clean laboratory glassware properly
— First, rinse it with organic solvent and then water.
— Next, rinse it with the solvent that will be put into it.
— DO NOT WASH GLASS BOTTLES IN DETERGENT
Preventing contaminationPreventing contamination
� Prepare and Handle Samples Correctly
— Use clean vials, caps, and plates
� Use Clean Fittings and Tubing
— Connections that come into contact with solvents or sample include
stoppers, O-rings, check valves, and solvent inlet filters (sinkers).
— Be aware that tubing made of polymers (such as polyvinylchloride,
©2007 Waters Corporation 98
— Be aware that tubing made of polymers (such as polyvinylchloride,
or PVC) may contain plasticizers or other contaminants.
� Wear particulate-free, powder-free, non-latex gloves
� Use clean columns
Contaminated mobile phaseContaminated mobile phase
•4 different buffer as A solvent
•(b)
©2007 Waters Corporation 99
•HPLC grade water as A solvent
•Blank gradients 5-80% ACN after 10min (a) and 30min (b) equilibration w. A eluent =10mM phosphate buffer pH 7.0
•(a)
•Blank gradients 5-80% ACN after 30min (a) and 30min (b) equilibration w. A eluent =4 different 10mM phosphate buffers pH 7.0
Contamination sourcesContamination sources
©2007 Waters Corporation 100
ContentContent
� Methods Development Suggestions
� Extraneous Peaks
� Gradients
� Samples and Vials
©2007 Waters Corporation 101
Gradient TransferGradient Transfer
� Common Problem—Gradient method works excellently on the R&D HPLC
System
However!
— The results cannot be duplicated in the Production Quality Control Laboratories
©2007 Waters Corporation 102
Control Laboratories
(resolution, retention times)
� Is the Column at fault?
Effect of Gradient VolumeEffect of Gradient Volume
INJECT
D
E
H
V K
SCM
CAT Q
RG
Y
A WFL
NL
NS
•PTH Amino Acid Standards717 Autosampler
©2007 Waters Corporation 103
INJECT
INJECT
SCM
CA
NS
T R
Q
G
A
Y M
P
WF L NL
I
D
E
H
V
K
•2 mL Sample Loop
•200 µL Sample Loop
Problems with Gradient TransferProblems with Gradient Transfer
� Question: Are the instruments the same?—Brand—Model
� Question: If Model and Brand are different, how is the gradient mobile phase being created?
©2007 Waters Corporation 104
created?—High pressure or low pressure mixing/blending
� Even if the model and brand are the same, the EXACT SAME PLUMBING CONFIGURATION must be used for both
� The problems commonly arise from DIFFERENCES IN SYSTEM DELAY/DWELL VOLUMES
Gradient Transfer Gradient Transfer
� Check Gradient Delay/Dwell Volume of the
different instruments/systems
� Must specify the plumbing - even ID and
length of connecting tubing - should delay
©2007 Waters Corporation 105
length of connecting tubing - should delay
volumes be different, retention times etc. will
also be different
Example of Transfer of a Example of Transfer of a Gradient MethodGradient Method
large delay volume
small delay volume
•0.60
•0.80
•1.00 •1 mL/min
©2007 Waters Corporation 106
�Change in retention time�Change in resolution
Minutes
•0.00
•0.20
•0.40
•0.60
•2 •4 •6 •8 •10 •12 •14 •16 •18 •20
•AU
•Vo
ContentContent
� Methods Development Suggestions
� Extraneous Peaks
� Gradients
� Samples and Vials
©2007 Waters Corporation 107
Troubleshooting StrategyTroubleshooting Strategy
Problem!•Try to simplify:
•Inspect the chromatography
•Try to categorize
•Troubleshoot the easiest to fix items first
©2007 Waters Corporation 108
CHEMISTRY MECHANICAL
� SAMPLES & VIALS� Contamination� Sample Stability
� VIALS� Dimensional Problems�Jams, Pick-up� Needle Damage� Poor Injection Reproducibility� Septum Dislodging� Sample Evaporation� LVI Assembly
Troubleshooting Concerns Troubleshooting Concerns Regarding VialsRegarding Vials
� Vials Can Present both Chemical and Mechanical Problems
� Most important: What HPLC system is using the vial. Sometimes the autosampler appears to be at fault, but in reality it’s the vial.
— Differences in the designs of HPLC systems determine the demands placed on the vial
©2007 Waters Corporation 109
demands placed on the vial
o Needle draw port design
o Needle draw depth design
o Needle venting design
o Vial handling design (Fixed Tray, Robotic Handling)
� Vials are the least expensive component of an HPLC
system, but can be the biggest contributor to problems
that you encounter, such as:
•Sample Vials
©2007 Waters Corporation 110
� Evaporative loss
� Spurious peaks/extractables
� Sample/analyte degradation
� Sample draw volume reproducibility
� Septum dislodging/Coring
� Injector damage/Robotics malfunction
� Excessive residual sample volumes
•Chemical Mechanical
Choosing the CorrectChoosing the CorrectVIALVIAL
� Colourless/Clear glass— Type 1, Class A Borosilicate •Note:
•IMPORTANT:•Test the sample analytes for compatibility/usability with a vial.
•Some compounds may degrade, or become bound to certain surfaces.
©2007 Waters Corporation 111
— Type 1, Class A Borosilicateo Most chemically inert glass available
— Type 1, Class B Borosilicateo More alkaline than Class A
� Amber/Clear glass— All amber glassware is “Type 1, Class B”— Used for light labile samples
� Polypropylene— A non-ionic, non-reactive plastic. Used where
glass is inappropriate. Max. temperature : 135°C
•Note:•Decativated/Silanized glass surface is also available
Problem Problem -- Septa DislodgingSepta Dislodging
•Common problem with “non-bonded”
septa. •Can jam the
©2007 Waters Corporation 112
•Can jam the autosampler
� Don’t immediately blame the needle or needle alignment
� Switch to pre-slit septum
- Less force required to pierce
� Choose bonded septum (LectraBond™)
- Septum is bonded to the cap. Eliminates dislodging
- Electro welded: No chemicals or adhesives
Choosing the Correct SEPTAChoosing the Correct SEPTA
� Wrong choice of Septum may result in :
—Evaporative loss of sample
—Lack of reproducibility for repetitive injections
©2007 Waters Corporation 113
injections
—Septum coring
—Needle damage
—Septum dislodging
—Contaminationo Di-octylphthalate (silicone)
o Siloxane (silicone)
Problem Problem –– Sample Draw Volume Sample Draw Volume Reproducibility Reproducibility -- VentingVenting
� Symptom: Peak area increases after first injection from the same vial (first injection –
low, latter injections OK)
— Possible cause: Inadequate venting upon needle piercing the septum/cap for the
FIRST Time. “Vacuum formation” caused by the septum/cap sealing around the
injection needle.
— Vacuum draws some sample back out of the needleo Situation can be aggravated by over filling the vial
©2007 Waters Corporation 114
o Situation can be aggravated by over filling the vial
(Never fill the vial all the way to the top)
— Test: Remove cap and septum* from the vial.Perform multiple injections. Measure peak area to determine if the septum/cap is the cause.
•*Some auto samplers may have to have the vial sensor disabled
•Vial A•Inj. 1•Area = 200,000
•Vial A•Inj.2•Area = 250,000
Problem Problem –– Sample Draw Volume Sample Draw Volume Reproducibility Reproducibility -- Coring of SeptumCoring of Septum
� Symptom: Peak area varies (increases /decreases) from injection to injection from the same vial
— Possible cause:Coring of Septum by Needle. If using a bottom draw port needle, the draw port could be plugged with septum material
o Check needle draw port for septum material, remove / replace.
©2007 Waters Corporation 115
— Solution: Should self sealing PTFE/silicone septum be used:
o Switch to pre-slit PTFE/silicone septum
o Pre-slit septum will eliminate coring and deliver good resealing capability
“Alternatively”
o Switch to PTFE septum
o PTFE will eliminate coring. However, it will not reseal•Silicone Septum•Material Lodges in Bottom Draw
Needle
Problem Problem ---- Spurious PeaksSpurious Peaks
� Contaminants can come from three sources and can be either sample dependent or detection-method dependent
—Vial
—Cap
—Septum
©2007 Waters Corporation 116
—Septumo Most common contributor
• Chemical compatibility
• Absorbs contaminants from atmosphere (silicone)
• Observance of problem can be detection-method dependent
•Packaging and storage can be very important
Problem Problem -- Spurious PeaksSpurious Peaks
� Simple & Quick Testo Remove cap and septum from vial and run sample to see if spurious peaks still appear.*
• If peaks don’t appear, the problem could be coming form the septum
→Switch to a PTFE septum (most chemically inert material)
� Should peaks still appear, troubleshoot the vial
©2007 Waters Corporation 117
� Should peaks still appear, troubleshoot the vialo Vials are manufactured from different grades of glass. Choose a vial manufactured from a different grade / class of glass
o Packaging used for shipping the vials can also contribute to this problem.
* Some autosamplers may require you to disable the optical vial cap sensor
Included in every package Included in every package -- COACOA
©2007 Waters Corporation 118
Chapter 5Chapter 5Column protection, baseline Column protection, baseline troubleshootingtroubleshooting
©2007 Waters Corporation 119
Chapter 5Chapter 5Column protection, baseline Column protection, baseline troubleshootingtroubleshooting
� Column use, storage & maintenance
� Column protection & regeneration
©2007 Waters Corporation 120
� Column protection & regeneration
� Baseline troubleshooting
Column EquilibrationColumn Equilibration
� Initially purge column with 10-20 column
volumes (Vc) mobile phase to be used in
analysis
Vc= 0.5 x D2 x L
(4.6x150mm : Vc= 1.6ml, Equil. = 16-32ml)
©2007 Waters Corporation 121
� Caution : If our are using a buffer make sure it does not
precipitate in the column when initially flushing the column
w. mobile phase
— Most RP column comes in 100% organic usually acetonitrile
— Initially flush with water/organic and the switch to the mobile
phase
Column EquilibrationColumn Equilibration
� Reversed-Phase (C18, C8 etc.) columns
equilibrate faster than Normal Phase columns
— (order of magnitude = 10)
� Normal phase columns (silica or alumina) may
©2007 Waters Corporation 122
� Normal phase columns (silica or alumina) may
take several hours at flow rates of 1.0 ml/min
—Use dried solvents, or water saturated solvents or solvent
with traces of propanol
Column StorageColumn Storage
� Store in Shipping Solvent for Longer Periods of Time— Flush with water/organic and then 100% organic
� Column should be stored in solvent which manufacturer recommends
� For bonded phases, use organic solvent
©2007 Waters Corporation 123
� For bonded phases, use organic solvent (eg. MeOH or ACN) Using non-aqueous solvents minimizes hydrolysis.
� Some bonded phases (CN) become unstable in polar organic mobile phases. Storage in water or buffer is then ok.— Worst mobile phase for CN column is CH3CN
Column StorageColumn Storage
� Columns which may be stored in water or buffered
solvents:
—CN columns
— Ion exchangers
— Aqueous SEC packings
— However:
©2007 Waters Corporation 124
—However:
o Prevent microbial growth by using 0.05% sodium azide
in mobile phase
OR
o Small quantity of organic solvent (acetonitrile 5% or
methanol 10%)
Column StorageColumn Storage
� Columns which should be stored in Mobile Phase:
—Normal Phase
— Organic SEC (GPC)
©2007 Waters Corporation 125
Techniques to Protect the Techniques to Protect the ColumnColumn
� In-line Filter between the Injector and Column
— Protects against particles
� Guard Column Between Injector and Column
— Protects against particles as well as chemical contamination
� Filtering of the Sample
©2007 Waters Corporation 126
— Protects against particles
� Filtering of the mobile phase (0,2µm filter)
— Protect against particles and microbial growth
� Sample Cleanup through Solid Phase Extraction (SPE)
— Protects against chemical interferences
� Limit High Back Pressures/Pulses (solvent viscosity)
Column RegenerationColumn Regeneration
� Always follow column vendor’s guideline for
regeneration
� Regeneration can bring back a column’s performance
if problem relates to compounds, which are retained
under method conditions, causing changes in
©2007 Waters Corporation 127
under method conditions, causing changes in
chromatography. Washing them off with more
aggressive solvents can return performance
� If surface has been chemically altered, ie hydrolysis of
ligands, then performance may not be restored
Column Regeneration/CleaningColumn Regeneration/Cleaning
Reversed phase
columns:
� Methanol*
Normal phase columns:
� 50/50
Methanol/chloroform
Flush with 20 column volumes. Run columns in reverse flow direction, disconnected from detector. Cleaning efficiency is increased at 35-45ºC
©2007 Waters Corporation 128
� THF
� Hexane
� THF
� Methanol
� Mobile phase*
� Ethyl acetate
� Mobile phase
•
•
••* Make sure mobile phase is miscible w. methanol and no salt precipitation occurs
Column LifetimeColumn Lifetime
� HPLC columns have a finite lifetime.
� Most common cause of column failure is due to high pressure, typically caused by particulates.
� Sources of particulate contamination
— Debris from instrument seals
— Particles in the sample
— Particles in the mobile phase
©2007 Waters Corporation 129
� Chemical Degradation
— Contamination from adsorbed materials
o Strongly retained/irreversible bound
— Microbial growth
— Hydrolysis (cleavage) of bonded ligand (ODS,etc.)
— Dissolution of stationary support (silica, etc.)
Column Regeneration/CleaningColumn Regeneration/Cleaning
� Inject 100 µl DMSO while flushing the column with
100% acetonitrile
� Run a 10-100% B gradient where
A= 0.1% TFA in water
•If you suspect contamination with proteins :
©2007 Waters Corporation 130
A= 0.1% TFA in water
B= 0.1% TFA in acetonitrile
� Flush with 7M guanidine HCl or 7M urea
ContentContent
©2007 Waters Corporation 131
Línea de base
DerivaFalta de equilibrado
Fluctuaciones temperatura ambiente
Mirar condiciones de la aplicación para determinar el equilibrado.Si es gradiente, observar el tiempo de equilibrado entre iny e iny.1. Para calcular vol de equilibrado: ColumnEquilibration Time =((SystemVolume x 3) + (ColumnVolume x 5) / Flow rate) –Injection Cycle Time.
Columna a 5 grados por encima de la ambienteObservar ubicación de equipos respectos unidades de calor o frio.
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Fase móvil
Celda que pierde
Columna contaminada
Lámpara
Equipo contaminado
Descartar contaminación de fase móvil, preparar fase frescaCambiar la botella y cambiar frits.Comprobar que los componentes no tienen lectura en la longitud de onda de trabajo.Solventes y aditivos equilibrados en los gradientesSuficiente lavado para obligar a componentes tardíos a eluir de la columna.
Mirar conexiones y apretar, en caso oportuno. Si fuga la celda por dentro sustituirla
Reemplazar por unión de vol muerto, pinchar f.m, si la línea es estable, se necesita columna nueva
Mirar la energía de la lámpara, si es muy baja, se debe reemplazar.Si la lámpara lleva más de 2500 horas, reemplazarSi vemos curvaturas de la línea de base, posiblemente la celda esté sucia. Limpiar la celda
Si quitando la columna y con nuevas fases móviles, solo bombeando fase móvil sigue habiendo deriva limpieza del equipo (ver protocolo Mantenimiento y Limpieza celdas de detectores Acquity)
Linea de BaseLinea de Base
� Ruido cíclico de línea de base
� spikes
Ruido de línea de base ciclico
Fluctuaciones de temperatura
Mezcla de solventes insuficiente
Columna a 5 grados por encima de la ambienteObservar ubicación de equipos respectos unidades de calor o frio.
Si haciendo la mezcla en una sola botella el problema desaparece es un problema del mezclador-ver vía A y B si funcionan correctamente por separado. Si es asi cambiar mezclador-para molec grandes a flujos bajos, utilizar mixer de mayor volumenPuede ser debido a fases móviles con diferentes Abs a la longitud de onda de trabajo
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� Ruido errático
� Línea plana, no picos
Pulsaciones en la bomba
Filtros de botellas sucios
Inexistencia de restrictor de presión a la salida del último detector
Purgar las bombas con fase móvil. Si no se soluciona purgar con metanol o isopropanol. Sonicar las ckeck valves o sustituirlas por unas nuevas si es necesario.
Provocan burbujas en los tubos de solventes de la botella. Quitar los filtros de la botella y cambiar por unos nuevos
Colocar restrictor de presión a la salida del detector (tubo azul). Si detras hay un detector de masas no es necesario
Linea de baseLinea de base
Spikes Burbujas
Falta de restrictor de presión
Ruido eléctrico
Purgar la bomba con metanol y despues pasar 100% de metanol por todo el sistema incluido el detector para eliminar posibles burbujas
Instalar restrictor de presión a la salida del detector (tubo azul). Genera 250 psi. Si detrás hay un detector de masas no es necesario
Adquirir un cromatograma sin pasar fase móvil por el detector. Si el ruido es exactamente igual puede ser ruido eléctrico.
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Saturación del detector
exactamente igual puede ser ruido eléctrico. Vigilar conexiones a circuitos eléctricos próximos.
Comprobar si diluyendo la muestra sucede lo mismo. Comprobar los parámetros de detector programados en el método
Linea de BaseLinea de Base
Línea de Base Plana Comprobar si fluyen los solventes
La lámpara no se enciende
Celda sucia
Longitud de onda incorrecta
Suficiente volumen en botella, caudal correcto
Cambiarla por una nueva o contactar con Waters
Mirar la energia del detector. Si es muy baja puede que la celda esté sucia. Limpiar la celda. (Protocolo de limpieza de celdas)
Comprobar que la longitud de onda
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Longitud de onda incorrecta
Fugas en el equipo
Fase móvil tiene más lectura que analito
No se ha inyectado
Comprobar que la longitud de onda seleccionada es correcta
Chequear que no hay fugas. Comprobar si el flujo a la salida de celda es correcto. Si no lo es debe de haber una fuga
Cambiar las composiciones de fase móvil
Revisar suficiente volumen de muestra en vial, vial en correcta posición, test de pesada.Hacer prime de sample syringe y realizar el “sample srynge leak test”
ACQUITY UPLC™ TroubleshootingACQUITY UPLC™ TroubleshootingTiempos de retenciónTiempos de retención
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Tiempos de retenciónTiempos de retención
Tiempos de retención erráticosTiempos erráticos Equilibrado de sistema y fase móvil
Pulsaciones de bomba, presión inestable
Fugas en algun punto del equipo
Volumen de inyección o concentración de
Mirar condiciones de la aplicación para determinar el equilibrado.Si es gradiente, observar el tiempo de equilibrado entre iny e iny. Si se usa par iónico, asegurarse que en el primer uso de la columna, está perfectamente equilibrada.
Purgar bombas con metanol. Sonicar check valves.
Chequear si hay fugas. Medir el flujo a la salida de detector. Si es menor del marcado puede que haya fugas
Reducir el vol de inyección o bien diluirlo con f.m. Si se utiliza un
©2007 Waters Corporation 137
Volumen de inyección o concentración de muestra demasiado alta, disrupción del equilibrio
Fluctuaciones temperatura
Mezcla inadecuada de los solventes
Contaminación de columna
Reducir el vol de inyección o bien diluirlo con f.m. Si se utiliza un solvente más débil que la f.m, se puede inyectar un 10% del volumen de la columna, pero si este solvente es más fuerte, tan sólo un 1%.
Columna a 5 grados por encima de la temperatura ambienteObservar ubicación de equipos respecto a unidades de calor o frio.Confirmar que el estabilizador se usa.
Para confirmar un problema de mezcla:-hacer la premezcla, filtrar, desgasificar-usar vías diferentes-equilibrar la columna-inyectar un std un mínimo de 3 veces y comparar los tr.Si los tr son reproducibles con la mezcla prehecha, indica un problema de mezcla:-verificar miscibilidad-verificar bomba y mixer-ver “ruido de base cíclico”
-Pasar orgánico a la columna (tener en cuenta posibles precipitaciones!!)-limpiar o reemplazar columna-volver a inyectar-si el tr es errático: mirar solventes, contaminación de f.m o de precolumna o filtro inline
Tiempos de retenciónTiempos de retención
Tiempos de retención Incremento progresivo en el tr Equilibrado de sistema
Chequear columna, fase y composición correctas
F.M no desgasificada correctamente
Mirar condiciones de la aplicación para determinar el equilibrado.Si es gradiente, observar el tiempo de equilibrado entre iny e iny. Si se usa par iónico, asegurarse que en el primer uso de la columna, está perfectamente equilibrada.
Reducir el vol de inyección o bien diluirlo con f.m. Si se utiliza un solvente más débil que la f.m, se puede inyectar un 10% del volumen de la columna, pero si este solvente es más fuerte, tan sólo un 1%.
Cebar todas las líneas
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F.M no desgasificada correctamente
F.m. contaminada
Contaminación de columna
Degradación de la columna
Fugas
Filtro de solventes o inlets de las diferentes líneas bloqueados
Cebar todas las líneasCambiar solventes de líneaDesgasificar offline y volver a equilibrar
Ver f.m en el apartado de deriva
-Limpiar columna según su documento de “care and use”.-en caso necesario reemplazar
Realizar test de columna o pinchar un system suitability.Cambiar por nueva en caso necesario
Inspeccionar las conexiones Realizar el “static leak test”
Chequear la presión que se genera en cada una de las líneas y cambiar tubos si es necesarioLimpiar o cambiar los filtros de botella y el filtro inline del equipo.
Tiempos de retenciónTiempos de retención
Tiempos de retención Decremento progresivo en el tr Equilibrado de sistema
Chequear columna, fase y composición correctas
Mirar condiciones de la aplicación para determinar el equilibrado.Si es gradiente, observar el tiempo de equilibrado entre iny e iny. Si se usa par iónico, asegurarse que en el primer uso de la columna, está perfectamente equilibrada.
Cambiar la fase móvil por una nueva. Cambiar columna si es necesario
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Diluyente de muestra es más fuerte que f.m
Contaminación de columna
Degradación parcial de la columna, pérdida de funcionalización
Reducir el vol de inyección o bien diluirlo con f.m. Si se utiliza un solvente más débil que la f.m, se puede inyectar un 10% del volumen de la columna, pero si este solvente es más fuerte, tan sólo un 1%.
-Limpiar columna según su documento de “care and use”.-en caso necesario reemplazar
Realizar test de columna o pinchar un system suitability.Cambiar por nueva en caso necesario
Tiempos de retenciónTiempos de retención
Tiempos de retención Cambio del tr a un nuevo valor fijo
Fase móvil o columna incorrecta
Temperatura programada en el column heater incorrecta
Comprobar posible malfunción de la bomba
Verificar si la columa es la adecuada. Cambiar por una fase movil nueva.
Comprobar si la temperatura programada es correcta y si no lo es cambiarla
Verificar el caudal midiendolo con probeta a la salida del detector
Vigilar aditivos y usar algún conservante.
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Fase móvil contiene estabilizador
“Volumen de retraso del gradiente” incorrecto. Cambio de tubos por otros de diferente volumen (diámetro interno o longitud)
Partial loop con presión asistida con el weak solvent diferente de fase móvil
Vigilar aditivos y usar algún conservante.
Determinar si ha habido algún cambio a nivel de fluídica y en todo caso reajustar el retraso de gradiente correspondiente
Chequear o cambiar weak wash por una adecuada y caracterizarlo
Resultados cualitativos incorrectosResultados cualitativos incorrectos
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Resultados cualitativos incorrectosResultados cualitativos incorrectos
Más picos de los esperados
Fase móvil contaminada
Muestra degradada o impurezas que vienen del proceso de preparación del equipo
Equipo contaminado
Ver punto de f.m en derivaVigilar la calidad de los solventes usados, filtrarlos
Inyectar std y comprobar que todo está correcto. Inyectar blanco y comprobar que no salen picos extras. Si así es, cambiar columna por una nueva.Vigilar temperatura del compartimento de muestras
Chequear el equipo para diagnosticar contaminación (ver documento de contaminación)
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Carryover: compuestos que se arrastran de una inyección anterior. Puede deberse a:
- Inyector no se limpia suficente en cada inyección
- Columna no se limpia bien en cada inyección
- Lavado de aguja no funciona
- Problema en inyector
Pico ancho en medio de cromatograma
Picos desdoblados o con hombros-colas
Weak wash contaminado
Cambiar strong solvent por uno mas adecuado o aumentar el volumen de lavado.
Incrementar fase de limpieza en gradiente o tiempo entre inyección e inyección
Válvula defectuosa, bomba defectuosa, siphoning solvent. Cambiar pieza defectuosa
Reemplazar la pieza defectuosa
Puede venir de f,m que se acumula en columna y eventualmente se eluye, o bien de muestra previa. Modificar el gradiente con una etapa de lavado agresiva antes de volver a condiciones iniciales.
Ver sección de forma de pico
Hacer una inyección a full loop, si la cromatografia es ok, el weak wash está contaminado.
Resultados cualitativos incorrectosResultados cualitativos incorrectos
Menos picos de los esperados
Muestra degradada o impurezas que vienen del proceso de preparación del equipo
Pérdida de resolución
Pérdida de sensibilidad. Dejamos de ver los picos
Inyectar std y comprobar que todo está correcto. Si hay menos picos, utilizar fase nueva y columna nueva.
Según el detector chequear sensibilidad
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Pérdida de sensibilidad. Dejamos de ver los picos más pequeños. El resto se ven más bajos
Aumento del ruido y algunos picos quedan absorbidos por el ruido
Muestra ha precipitado, también observaremos un incremento de presión
No se realiza bien el gradiente. La bomba con fase orgánica da menos caudal. Los último picos no tienen tiempo de salir.
Según el detector chequear sensibilidad (énergia en un detector UV, perdida de sensibilidad en MS)
Determinar segun el detector si el nivel de ruido es aceptable y similar al habitual.
Preparar muestras nuevas. Preparar estandar y chequear si salen todos los picos
Medir el caudal de cada una de las bombas por separado. Si no escorrecto proceder a su revisión.
Forma de pico y consecuenciasForma de pico y consecuencias
Falta de resolución
Picos Anchos Detectores UV: - Sampling rate muy baja.
- Filtro del detector demasiado alto
Detectores MS: - Velocidad de adquisición.
- Demasiado smoothing
Adquirir a una sampling rate suficiente para obtener 15/20 puntos por pico.
Disminuir el filtro con cuidado de no aumentar mucho el ruido de linea de base.
Fijar scan time o dwell time de modo que se adquieran 15/20 puntos por pico.
Reducir smoothing para evitar la distorsión de
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- Demasiado smoothing
Tubos de entrada y salida de columna inadecuados (diametro interno superior al habitual).
Férrulas de loop o tubos colocadas incorrectamente. Tubos mal cortados
No está instalado el estabilizador de temperatura
Vol de inyección o concentración de muestra demasiado elevada
Diluyente de muestra demasiado orgánico respecto a la fase móvil
Needle wash solvent con demasiada proporción de orgánico
Una parte del método o todo él es isocrático
Reducir smoothing para evitar la distorsión de pico
Chequear los tubos instalados y sustituirlos si es necesario. Para acquity deben ser de 0.005” o menor de diámetro interno.
Chequear y cambiar si es necesario las conexiones. No es necesario cortar los tubos. Se suministran cortados
Instalar el estabilizador
Reducir a la mitad el vol de inyeción para chequear si es el problema, diluir la muestra hasta 10 veces
Chequear y sustituir si es necesario
Needle wash solvent debe tener composición similar a la de la fase móvil inicial
Probar a utilizar un métdo de gradiente
Forma de pico y consecuenciasForma de pico y consecuencias
Falta de resolución
Picos con colas Tubos de entrada y salida de columna inadecuados (diametro interno superior al habitual).
Férrulas de loop o tubos colocadas incorrectamente. Tubos mal cortados
Vol de inyección o concentración demasiado elevada
Chequear los tubos instalados y sustituirlos si es necesario. Para acquity deben ser de 0.005” o menor de diámetro interno.
Chequear y cambiar si es necesario las conexiones. No es necesario cortar los tubos. Se suministran cortados
Debemos escalar la capacidad en masa y volumen a la columna utilizada. Reducir volumen de inyección
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Columna degradada que forma volumen muerto
Precolumna degradada
Analito muy hidrofóbico
Caudal demasiado bajo para el tipo de columna
Modo de inyección inadecuado
Weak wash es incompatible con la cromatografía y con el diluyente de muestra
volumen de inyección
Chequear la columna
Sacar la precolumna y si la cromatografia se recupera, reemplazarla
Incrementar la pendiente de gradiente o la proporción de orgánico
Cambiar el flujo por uno mas adecuado a la columna
Probar con otro modo de inyección. Por ej con Full loop puede que inyectemos demasiada cantidad de muestra en la columna
Needle wash solvent debe tener composición similar a la de la fase móvil inicial y ser compatible con diluyente de muestra.
Forma de pico y consecuenciasForma de pico y consecuencias
Falta de resolución
Picos con hombros o frontingVolumen de inyección demasiado grande o sample overload, demasiada concentración
Diluyente de muestra demasiado orgánico
Composición de weak wash demasiado orgánico
Columna o precolumna contaminada o
Diluir la muestra o reducir volumen de inyección.
Reducir volumen de inyección ó disminuir porcentaje de orgánico del diluyente
Needle wash solvent debe tener composición similar a la de la fase móvil inicial
Sacar la precolumna y si la cromatografia se
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Columna o precolumna contaminada o degradada o formando un volumen muerto
Férrulas de loop o tubos colocadas incorrectamente. Tubos mal cortados
No está instalado el estabilizador de temperatura
Dos compuestos o isomeros que coeluyen
Sacar la precolumna y si la cromatografia se recupera, reemplazarla. Limpiar o reemplazar la columna si es necesario
Chequear y cambiar si es necesario las conexiones. No es necesario cortar los tubos. Se suministran cortados
Instalar el estabilizador
Modificación del método
Forma de pico y consecuenciasForma de pico y consecuencias
Falta de resolución
Picos inacabadosParámetros del detector incorrectos
Volumen inyectado o concentración de muestra demasiado elevada
La fase móvil tiene demasiada absorbancia a la longitud de onda elegida reduciendo el rango de trabajo del TUV o PDA
Chequear el método de instrumento
Diluir la muestra o reducir volumen de inyección
Escoger una fase móvil más transparente o cambiar la longitud de onda de trabajo
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Picos negativosLa fase móvil absorbe más que el analito a la longitud de onda elegida
Problemas de frente de solvente
Escoger una fase móvil más transparente o cambiar la longitud de onda de trabajo
Hacer más compatible el solvente de inyección y la fase móvil
SensibilidadSensibilidad
Pérdidas de sensibilidadVolumen de inyección
Columna
Volumen de inyección incorrectamente programado
Inyector mal configurado
Fuga en el inyector
Columna degradada, contaminada o con volúmenes muertos
Chequear el volumen de inyección
Chequear volumen de loop y aguja. Caracteizar de nuevo volumen de loop y aguja
Chequear for fugas en las conexiones del inyector
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Conexiones
Muestra
Fase Móvil
Temperatura de columna programada es demasiado baja, el tr se alarga y el pico se ensancha
Deben estar correctamente, volúmenes muertos darán como resultado formas de pico no deseadas. Férrulas de loop o tubos colocadas incorrectamente.
Concentración insuficiente
No está completamente disuelta o precipita a lo largo del tiempo.
Se evapora o degrada
Fase móvil errónea (pH incorrecto, contaminada..)
Chequear temperatura de columna en el método
Chequear y cambiar si es necesario las conexiones.Chequear si hay fugas en alguna conexión del sistema
Aumentar concentración o volumen de inyección
Chequar su solubilidad y si hace falta elevar la temperatura del compartimento de muestras (a temperaturas bajas puede ser insoluble)
Disminuir la temperatura del compartimento de muestras
Preparar fase móvil nueva
SensibilidadSensibilidad
Pérdidas de sensibilidadAguja
Weak wash solvent incorrecto
Aguja calibrada demasiado alta. Needle draw depth programado erróneamente en el método.
Aguja de peek, los compuestos hidrofóbicos se enganchan a ella
Demasiado orgánico, resulta en mala forma de pico, especialmente para los compuestos más hidrofílicos
Realizar de nuevo calibración Z de la aguja.Comprobar needle draw depth
Probar con otro tipo de aguja
Cambiar la composición del weak wash solvent
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Loop offline
Jeringa de muestra
Platos
Temperatura
El loop se aisla demasiado rápido del paso del caudal y parte de la muestra no llega a ser inyectada en la columna
No se han cebado o hay burbujas.
Fugas en la jeringa
Velocidad de aspiración de la jeringa demasiado alta. Puede coger burbujas
Configuración de platos errónea
Temperatura de columna demasiado baja o estabilizador omitido
Evitar usar el loop offline aunque el flujo sea muy bajo y el loop muy grandeSi se usa pasar caudal por el loop más tiempo (3 veces el volumen total del loop) antes de aislarlo
Purgar el inyector
Chequear si fuga. Realizar test de jeringa
Cambiar la velocidad de aspiración en el método
Comprobar configuración de los platos
Modificar la temperatura. Instalar estabilizador
SensibilidadSensibilidad
Pérdidas de sensibilidadData rate
Filter time constant
Energia de detector
Longitud de onda
Números de puntos por pico insuficientes. Mínimo se necesitan 15 o 20.
Si el filtro es demasiado alto, se reduce el ruido de fondo pero los picos serán más bajos y con colas.
Energia de detector baja debido a celda sucia o lámpara con muchas horas
Longitud de onda programada incorrectamente
Adquirir a una sampling rate suficiente para obtener 15/20 puntos por pico.
Disminuir el filtro con cuidado de no aumentar mucho el ruido de linea de base.
Comprobar energia de la celda. Limpiarla si es necesario. Cambio de lámpara si es necesario
Comprobar la longitud de onda del método
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Material de vidrio
Vial
Vigilar los detergentes, algunos de ellos pueden causar ruido de fondo en el detector o supresión iónica en el masas
Usar vidrio borosilicatado
Comprobar que el compuesto no se adhiere al vidrio
Burbuja de aire en el fondo del vial, habitual en los insertos
Vigilar volúmenes residuales de cada tipo de vial
Usar preslit, el no usarlo puede provocar vacío al inyectar
Usar vidrio desactivado
Poner volumen de muestra superior al volumen residual
SobrepresiónSobrepresión
Puntos a chequear por este orden
Desconectar entrada a celda del detector Si presión baja el problema está en la celda, seguir los protocolos de lavado de la celdaSi presión no baja, está antes de celda
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Desconectar tubo salida de columna Si la presión baja, está en el tubo de conexión de salida de columna a detector, comprobar también tornillos. Cambiar tubo.Si no baja el problema está antes
Desconectar conector entrada de columna Si presión baja, el problema está en columna, lavarla, cambiar frits, o renovar columnaSi no baja, es anterior
Desconectar del prefiltro online o precolumna Si presión baja, sustituir prefiltro o precolumna por una nueva
SobrepresiónSobrepresión
Puntos a chequear por este orden
Desconectar salida de inyector Si presión baja el problema está en el tubo de conexión de inyector a columna o en válvulas del column manager. Limpiar o cambiar tuboSi no baja es anterior
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Antes de loop de inyección Si baja está precipitado en el loop, o válvula de inyección. Realizar lavados o cambiar loop por uno nuevoSi no baja es anterior al loop
Desconectar filtro on line Si baja, el problema está en el filtro. Limpiar o poner uno nuevo
Column manager Para saber si está en alguno de los tubos del column manager, probar con diferentes posiciones de columna
� Precauciones básicas con la celda del detector:
— Limpiar con agua después de haber trabajado con tampones
— No dejar nunca la celda en agua durante mucho tiempo para evitar
crecimiento de microorganismos
— Nunca dejar la celda seca. El paso de luz por una celda seca puede
estropearla irreversiblemente
— Pasar fase móvil por la celda antes de encender el detector cuando éste
Limpieza de la celdaLimpieza de la celda
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— Pasar fase móvil por la celda antes de encender el detector cuando éste
lleve un tiempo apagado.
� Si ya tengo la celda sucia u obturada como la puedo limpiar
— Pasando agua si la suciedad se ha originado trabajando con tampones
— Limpiar con soluciones ácidas. Existe un protocolo de limpieza disponible.
Esta limpieza se ha de realizar sin columna.
Limpieza de columnaLimpieza de columna
� Lavado Básico para columna C18, C8, Phenyl
100% agua, 20 volúmenes de columna
100% metanol, 20 volúmenes de columna
100% acetonitrilo, 20 volúmenes de columna
� Para columnas tipo amida, sb o hilic, seguir protocolos indicados en su
“care and use”
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“care and use”
� Siempre leer los documentos “care and use” de las columnas, localizados
en los documentos insertados en el ecord.
� Para fases móviles con pH muy básicos, contactar con el departamento de
columnas, para elaborar protocolos de lavado individualizados
� Para largos períodos inactivos: guardar en 20% agua, 80% orgánico y bien
tapada. Nunca colgada del equipo.
ACQUITY UPLC™ACQUITY UPLC™
� Prevención de problemas de presión
— Filtrar todos los solventes con filtros de 0.2 µm y prepararlos
diariamente, incluidos weak y strong solvent
— Usar sales de alta calidad para preparar los tampones
— Usar solventes orgánicos de grado HPLC gradiente o superior
— Limpiar con agua después de haber trabajado con tampones
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— Limpiar con agua después de haber trabajado con tampones
— No dejar nunca el equipo con agua durante mucho tiempo para evitar
crecimiento de microorganismos
o Usar 10-20% fase orgánica en agua cuando se deje el equipo parado, MeOH
o IPA son correctos
— Asegurarse de utilizar material limpio (botellas, equipos de filtración,
viales)
— Filtrar las muestras con filtros de 0.2 µm
Compatibilidad de solventesCompatibilidad de solventes
� ACQUITY UPLC™ System recommended Solvents� Methanol Methanol/water mixtures � Water Acetonitrile/water mixtures � Acetonitrile (ACN) Isopropanol (IPA)
� Sample diluents (in addition to the solvents listed above)� Dimethyl sulfoxide (DMSO) Dimethylformamide (DMF)
� Additives / Modifiers� 0.2% formic acid 0.1% trifluoracetic acid (TFA) � 0.1% triethyl amine (TEA) 0.1% hexafluorobuteric acid
©2007 Waters Corporation 156
� 0.1% triethyl amine (TEA) 0.1% hexafluorobuteric acid � 10mM phosphate buffer 10mM ammonium bicarbonate � 50mM ammonium hydroxide 50mM ammonium acetate � 0.1% Ethylenediaminetetraacetic acid (EDTA)
� Cleaners� Phosphoric acid (=30%) Sodium hydroxide (=1M)
� ACQUITY UPLC™ System non-recommended Solvents� Methylene-Chloride Chloroform � Ethyl Acetate Toluene � Chlorinated solvents: (Trichlorobenzene) Strong acids >5%