extraneous peaks in hplc
DESCRIPTION
The mystery of extra peaks in HPLC chromatogramsTRANSCRIPT
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Unexpected Peaks in Chromatograms Unexpected Peaks in Chromatograms -- Are Are They Related Compounds, System Peaks or They Related Compounds, System Peaks or
Contaminations? Contaminations?
From the Diary of an HPLC DetectiveFrom the Diary of an HPLC Detective
SHULAMIT LEVIN, MEDTECHNICA
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HPLC in HPLC in PharmaceuticsPharmaceutics
Σ
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Stability Indicating MethodsStability Indicating Methods
• Accurately quantifies the active pharmaceutical ingredient without interference from:– Impurities– Degradation products– Excipients– Other potential impurities– Other active ingredients
FDA Guidelines:FDA Guidelines:Where an analytical method reveals the presence of impurities in addition to the degradation products (e.g., impurities arising from the synthesis of the drug substance), the origin of these impurities should be discussed.
Extra Sensitive to Extraneous Peaks
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2.20.00342.714Impurity 5
5.80.01062.549Impurity 4
2.60.00372.504Impurity 3
6454499.9542.263Butamben
16.40.02292.231Impurity 2
4.10.00542.174Impurity 1
Signal-to-Noise
Area %
Retention Time (Minutes)Compound
Accounting for Every Peak in the Related Compounds’ Profile - Even Down to 0.003 %Area
Butamben
>LOQ
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Sample vs. Blank (Diluent)Sample vs. Blank (Diluent)m
AU
0.00
10.00
20.00
30.00
40.00
50.00
mA
U
0.00
10.00
20.00
30.00
40.00
50.00
Minutes
0.00 10.00 20.00 30.00 40.00 50.00
Blank
Sample
Unexpected peaks can appear in blanks and in all following injectionsIn this case they are excluded from the processing of the sample
? ? ? ? ?
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INJECTION OF PURE SOLVENT:INJECTION OF PURE SOLVENT:Why would there be peaks?Why would there be peaks?
Injection “System Peak” ???Carry over ???
Contamination ???
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System PeaksSystem Peaks“Legitmate” System Peaks
Originate from the Mobile Phase Components Going Through Re-Equilibration
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CONDITIONS FOR APPEARANCE OF CONDITIONS FOR APPEARANCE OF REAL SYSTEM PEAKSREAL SYSTEM PEAKS
• Mobile phase is multi-component (n=>2)
• Mobile phase contains adsorbable components
• Mobile phase’s components respond to the detector(high background)
• Sample or sample-diluent is different from the mobile phase, enough to create equilibrium perturbation.
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EXAMPLE:Two additives in the mobile phase
CHROMATOGRAM
Mechanism of System Peaks FormationMechanism of System Peaks Formation
Vacancy
Example: kExample: k’’(1) = 1(1) = 1Step 1:Step 1:
Equilibrium:Equilibrium:Cs=1Cs=1; ; CmCm = 1= 1
Step 2:Step 2:Injection of Injection of Vacancy:Vacancy:
Cs=1Cs=1; Cm=0; Cm=0
Step 3:Step 3:ReRe--equilibration:equilibration:Cs=0.5Cs=0.5; ; Cm=0.5Cm=0.5
Example: kExample: k’’(2) = 2(2) = 2Step 1:Step 1:
Equilibrium:Equilibrium:Cs=2Cs=2; ; CmCm = 1= 1
Step 2:Step 2:Injection of Injection of Vacancy:Vacancy:
Cs=2Cs=2; Cm=0; Cm=0
Step 3:Step 3:ReRe--equilibration:equilibration:
Cs=1.33Cs=1.33; ; Cm=0.67Cm=0.67
k' = tR- t 0
t0
k’ = CCssφCm
t0
k’=1 k’=2
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Injection of free amino acids into mobile phase containing:Injection of free amino acids into mobile phase containing:Ac buffer, Ac buffer, CuAcCuAc, and , and HeptsulfonateHeptsulfonate. .
Diluent: WaterDiluent: Water
Levin and Grushka
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Legitimate System Peaks Result from Mobile phase components Legitimate System Peaks Result from Mobile phase components due to their absence in the injected sampledue to their absence in the injected sample
Injection of Blank = WaterInjection of Blank = Water
Levin and Abu-Lafi
Conclusion: Use mobile phase composition as the diluent
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An Example for Real System Peaks in Gradients with An Example for Real System Peaks in Gradients with TrifluoroaceticTrifluoroaceticAcid (TFA) in the Mobile PhaseAcid (TFA) in the Mobile Phase
Blank =Water
Blank = Mobile phase
Blank = Mobile phase
Zoomed
Sample=Peptide Dissolved in
Water
Peptide Peak
TFA
ACN
System peaks appear because diluent does not contain TFASystem peaks appear because diluent does not contain TFA
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Chromatogram and Peaks' UV-VIS Spectra
Wavelength: 215nm
nm220.00240.00 260.00 280.00 300.00 320.00 340.00
Cpd 2 - 2.364nm220.00
240.00 260.00 280.00 300.00 320.00 340.00
2.817nm220.00
240.00 260.00 280.00 300.00 320.00 340.00
2.981
AU
-0.50
-0.40
-0.30
-0.20
-0.10
0.00
Minutes
1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00
Cp
d2
-2.
364
2.81
72.
981
-0.50
-0.40
-0.30
-0.20
-0.10
0.00
1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00
Real System Peaks in Multi-component Mobile PhaseH2O, MeCN, MeOH, THF
THF
Diluent does not contain all mobile phase componentsDiluent does not contain all mobile phase components
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AU
Sample Name: Diluent
2.37
32.
731
3.40
4
4.27
4
6.47
7
18.2
74
-0.003
-0.002
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
MinutesMinutes
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
Mobile phase: ACN : Phosphate Buffer pH 9.5 1:1Wavelength: 280 nm
Contaminations Peaks Contaminations Peaks -- NotNot System Peaks!System Peaks!Phosphate Buffer and Acetonitril Do Not Produce Such System Peaks!
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Chromatogram and Peaks' UV-VIS Spectra
Wavelength: 254.0 nm
Use of Diode-Array Detector for Troubleshooting of Contamination Peaks in Multicomponent Mobile Phase
nm250.00300.00
Cpd 2 - 2.188nm250.00
300.00
2.748nm250.00
300.00
2.867nm250.00
300.00
3.180nm250.00
300.00
4.226
AU
0.000
0.005
0.010
0.015
Minutes1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00
Cp
d2
-2.
188
2.74
8 2.86
7
3.18
0
4.22
6AU
0.000
0.005
0.010
0.015
Minutes1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00
Negative UV spectrum
Aromatic (not in mobile phase)
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Carry Over:Carry Over:Chemical Residues in the systemChemical Residues in the system
qq Residues in the injectorResidues in the injector
qq NonNon--specific irreversible adsorption on columnspecific irreversible adsorption on column
qq Accumulation on systemAccumulation on system’’s surfacess surfaces
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Carry Over in the InjectorCarry Over in the InjectorWashed by Blank InjectionWashed by Blank Injection
A1100 VWD AU - A1100 AU 286 nm
11.3
34
AU
-0.0001
0.0000
0.0001
0.0002
0.0003
0.0004
A1100 VWD AU - A1100 AU 286 nm
AU
-0.0001
0.0000
0.0001
0.0002
0.0003
0.0004
Minutes8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50
Blank Injection 2Contamination is cleaned
Blank Injection 1
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Blank InjectionsBlank Injectionsm
AU
0.00
20.00
40.00
60.00
80.00
100.00
Minutes
0.00 10.00 20.00 30.00 40.00 50.00
Series of Blank Injections
Signals are reduced from Injection to Injection indicate carry over in the injector
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CarryCarry--Over in the InjectorOver in the InjectorLCLC--MS/MSMS/MS
InjectionInjection--CleanClean--up with up with EDTAEDTA solved the problemsolved the problem
1.1. Peak elutes at RT of main componentPeak elutes at RT of main component2.2. MRMMRM of the main componentof the main component
Blank Sample before wash
Blank Sample after wash
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Carry Over:Carry Over:Residues in the systemResidues in the system
qq Residues in the injectorResidues in the injector
qq NonNon--specific irreversible adsorption on columnspecific irreversible adsorption on column
qq Accumulation on systemAccumulation on system’’s surfacess surfaces
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Accumulation on ColumnAccumulation on Column
SampleName: SST; Vial: 1; Injection: 1; Name: MAIN1; Area: 833913
AU
0.0000
0.0005
0.0010
Minutes
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
MA
IN1
SampleName: DILUENT; Vial: 2; Injection: 1; Name: MAIN1; Area: 5063
AU
0.0000
0.0005
0.0010
Minutes
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
MA
IN1
SampleName: DILUENT; Vial: 2; Injection: 2; Name: MAIN1; Area: 4784
AU
0.0000
0.0005
0.0010
Minutes
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
MA
IN1
Sample
Blank #1
Blank #2Cleaned Not Cleaned
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Chromatogram and UV-VIS Spectra of Contamination Peaks
Contamination PeaksContamination Peaks
nm
200.00220.00240.00260.00280.00300.00320.00340.00
8.510 11.604 41.142A
U
-0.002
-0.001
0.000
0.001
0.002
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00
8.51
0
11.6
04
41.1
42
-0.002
-0.001
0.000
0.001
0.002
Minutes
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00
Diluent at 280 nm
Aromatic group Aromatic group
TFA Containing Mobile Phase: TFA, HTFA Containing Mobile Phase: TFA, H22O, O, MeCNMeCN -- Do Not Contain Aromatic Components!Do Not Contain Aromatic Components!
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-Chromatogram and UV -VIS Spectra of a Blank Run
Sample Name: blank ; Wavelength: 214
nm
250.00
300.00
350.00
1.066 1.553 2.362 3.695 6.237 6.418 24.320 26.940 27.0261.
066
1.55
3 2.36
2
3.69
5
6.23
76.
418
24.3
20
26.9
4027
.026
AU
-0.20
0.00
0.20
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
AU
-0.20
0.00
0.20
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
Peaks of aromatic compounds in non aromatic mobile phase:Peaks of aromatic compounds in non aromatic mobile phase:Cannot be Legitimate System PeaksCannot be Legitimate System Peaks
Mobile phase: Gradient of 0.1% TFA in water with ACNMobile phase: Gradient of 0.1% TFA in water with ACN
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Chromatogram and UV-VIS Spectra of Contaminations’ Peaks
Contamination Peaks Contamination Peaks -- ResiduesResidues
nm
200.00220.00
240.00
260.00280.00300.00320.00340.00
1.274 1.514 1.731 8.500 11.633 34.345 36.595 37.612 41.018 42.128
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
Minutes5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00
1.27
41.
514
1.73
1
8.50
0
11.6
33
34.3
45
36.5
9537
.612 41
.018
42.1
28
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00
Blank=Diluent at 220 nm
Gradient with TFAGradient with TFA
Contamination on-column and/or in the mobile phase
Aromatic compounds – not legitimate components of the mobile phase
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Carry Over:Carry Over:Residues in the systemResidues in the system
qq Residues in the injectorResidues in the injector
qq NonNon--specific irreversible adsorption on columnspecific irreversible adsorption on column
qq Accumulation on systemAccumulation on system’’s surfacess surfaces
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Contaminations Contaminations Origin Origin –– Outside the SampleOutside the Sample
qq NonNon--pure solventspure solvents
qq VialsVials’’ leachablesleachables
qq ReservoirReservoir’’s s leachablesleachables
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AU
% C
om
po
siti
on
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.00
20.00
40.00
60.00
80.00
100.00
Minutes
5.00 15.00 25.00 35.00 45.00 55.00
Gradient’s Profile
220 nm
230 nm
254 nm280 nm
Baseline at GradientBaseline at GradientChange of Baseline with Time at Various Wavelengths: Change of Baseline with Time at Various Wavelengths:
Indication for non pure solventIndication for non pure solvent
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NonNon--Pure/Contaminated Pure/Contaminated Solvents Used in GradientsSolvents Used in Gradients
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
PG
-2.919
TB
HQ
-4.525
BH
A
-6.896 BH
T
- 10.633
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
PG
-2.919
TB
HQ
-4.525
BH
A
-6.896 BH
T
- 10.633
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
Blank
Sample
Contaminations can also come from the Parafilm used to seal reservoirs!!!
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Sample Sample vsvs BlankBlankm
AU
0.00
10.00
20.00
30.00
40.00
50.00
mA
U
0.00
10.00
20.00
30.00
40.00
50.00
Minutes
0.00 10.00 20.00 30.00 40.00 50.00
Blank
Sample
If Solvents are not entirely pure their impurities are adsorbed on the Stationary Phase at the beginning of the run and then elute from the column as the gradient develops.
This is the reason why there are “Gradient Grade” solvents
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PeakPeak’’s Origin: s Origin: Contamination accumulated on pumpContamination accumulated on pump’’s ins in--line filterline filter
Pressure JumpBubble-Detection
Unexpected Baseline Perturbation
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Contaminations Contaminations Origin Origin –– Outside the SampleOutside the Sample
qq NonNon--pure solventspure solvents
qq VialsVials’’ leachablesleachables
qq ReservoirReservoir’’s s leachablesleachables
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Contamination from VialsContamination from Vials’’ SurfacesSurfaces
AU
-0.0002
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
0.0012
0.0014
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
AU
-0.0002
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
0.0012
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
Non-Certified
CertifiedChange of vial brand
In the same experiment!
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SampleName: Sample; Vial: 1:F,1; Injection: 1; Name: Contamination
AU
0.000
0.005
0.010
0.015
0.020
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Con
tam
inat
ion
SampleName: Diluent ; Vial: 1:E,2; Injection: 1; Name: Contamination
AU
0.000
0.005
0.010
0.015
0.020
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Con
tam
inat
ion
Contamination is developed during the experiment in Contamination is developed during the experiment in complex Diluents complex Diluents (Sample(Sample’’s Solvent)s Solvent)
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Contaminations Contaminations Origin Origin –– Outside the SampleOutside the Sample
qq NonNon--pure solventspure solvents
qq VialsVials’’ leachablesleachables
qq Reservoir or ColumnReservoir or Column’’s s leachablesleachables
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Contamination Peaks in Size Exclusion Contamination Peaks in Size Exclusion Chromatography (SEC)Chromatography (SEC)
Chromatogram at 210 nm
AU
-0.010
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
0.030
Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00
Contamination is NOT a monomer!
Blank
MonomerSample
Contamination
Higher Molecular Weight
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Other Reasons Other Reasons for Extraneous Peaksfor Extraneous Peaks
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ColumnColumn’’s Packing Collapses Packing Collapse
All Peaks are split
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Suggested FlowSuggested Flow--Chart for TroubleshootingChart for TroubleshootingIn a Regulated Environment (no change in method!)In a Regulated Environment (no change in method!)
Mobile phase:Multicomponent?
Adsorbed components (ion pair)?Response in detector (background)?
Yes?Yes?
Legitimate System Peaks•Dissolve samples in mobile phase•Adjust diluent with mobile phase components
Injection “System Peak” ???Carry over ???
Contamination ???
Yes?Yes?
NoNo
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Suggested FlowSuggested Flow--Chart for Troubleshooting Contd.Chart for Troubleshooting Contd.
Steps: by ease of use1. Review and revise diluent’s composition2. Replace vials’ batch and/or brand3. Replace in-line filters4. Replace solvents batch and/or brand5. Try a new/fresh column6. Wash system, especially injectorMake sure to try a fresh blank each test!
Peaks still persist?Peaks still persist?
Not System Peaks?
Replace injector’s surfaces
Try another instrument/Operator/Lab
Peaks still persist?Peaks still persist?
Not washed by Not washed by blanks?blanks?
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Many times the extraneous peak disappears on its own and
remains an unsolved mystery…