Extraneous Peaks in HPLC

Isranalytica2006
Unexpected Peaks in Chromatograms - Are They Related Compounds, System Peaks or Contaminations? From the Diary of an HPLC Detective
levins@medtechnica.co.il
SHULAMIT LEVIN, MEDTECHNICA
Dr. Shulamit Levin 2005, Medtechnica
Isranalytica2006
HPLC in Pharmaceutics
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Stability Indicating Methods

Extra Sensitive to Extraneous Peaks
Accurately quantifies the active pharmaceutical ingredient without interference from:
Impurities Degradation products Excipients Other potential impurities Other active ingredients
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.
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Accounting for Every Peak in the Related Compounds Profile - Even Down to 0.003 %Area
Retention Time (Minutes)
Compound Impurity 1 Impurity 2 Butamben Impurity 3
Area %
0.0054 0.0229 99.954 0.0037 0.0106 0.0034
Signal-toNoise 4.1 16.4 64544 2.6 5.8 2.2
2.174 2.231 2.263 2.504 2.549 2.714
Butamben
Impurity 4 Impurity 5
>LOQ
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Sample vs. Blank (Diluent)

Unexpected peaks can appear in blanks and in all following injections In this case they are excluded from the processing of the sample
50.00 40.00 30.00
Blank
? ? ? ? ?
mAU
20.00 10.00 0.00
50.00 40.00 30.00
Sample
mAU
20.00 10.00 0.00 0.00 10.00 20.00 30.00 40.00 50.00
Minutes
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INJECTION OF PURE SOLVENT: Why would there be peaks?
Injection
System Peak ??? Carry over ??? Contamination ???
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System Peaks
Legitmate System Peaks Originate from the Mobile Phase Components Going Through Re-Equilibration
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CONDITIONS FOR APPEARANCE OF REAL SYSTEM PEAKS

Mobile phase is multi-component (n=>2) Mobile phase contains adsorbable components Mobile phases 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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Mechanism of System Peaks Formation Mechanism of System Peaks Formation

Vacancy EXAMPLE: Two additives in the mobile phase Example: k(1) = 1 Example: k(2) = 2
Step 1:
k' =
tR - t 0 t0 Cs Cm
Step 1:
Equilibrium: Cs=1; Cm = 1
Step 2:
Equilibrium: Cs=2; Cm = 1
Step 2:
k =
Injection of Vacancy: Cs=1; Cm=0

Step 3:
Injection of Vacancy: Cs=2; Cm=0

Step 3:
Re-equilibration: Cs=0.5; Cm=0.5

CHROMATOGRAM
Re-equilibration: Cs=1.33; Cm=0.67
t0
k=1 k=2
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Injection of free amino acids into mobile phase containing: Ac buffer, CuAc, and Heptsulfonate.
Diluent: Water
Levin and Grushka
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Legitimate System Peaks Result from Mobile phase components due to their absence in the injected sample
Levin and Abu-Lafi
Injection of Blank = Water
Conclusion: Use mobile phase composition as the diluent
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An Example for Real System Peaks in Gradients with Trifluoroacetic Acid (TFA) in the Mobile Phase
Peptide Peak
Sample=Peptide Dissolved in Water
Blank = Mobile phase
TFA
Blank = Mobile phase
Zoomed
ACN
Blank =Water
System peaks appear because diluent does not contain TFA
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Real System Peaks in Multi-component Mobile Phase H2O, MeCN, MeOH, THF
Chromatogram and Peaks' UV-VIS Spectra
Cpd 2 - 2.364 nm
240.00 260.00 280.00 300.00 320.00 340.00 220.00 220.00
2.817 nm
240.00 260.00 280.00 300.00 320.00 340.00
220.00
2.981 nm
240.00 260.00 280.00 300.00 320.00 340.00
Cpd 2 - 2.364
0.00 -0.10
2.817 2.981
THF
AU
-0.20 -0.30 -0.40 -0.50 1.00 1.20 1.40
Wavelength: 215nm
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
1.60 1.80 2.00 2.20
2.40 2.60 2.80 3.00
Minutes
Diluent does not contain all mobile phase components
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Contaminations Peaks - Not System Peaks!

Phosphate Buffer and Acetonitril Do Not Produce Such System Peaks!
0.006
0.005
Mobile phase: ACN : Phosphate Buffer pH 9.5 1:1 Wavelength: 280 nm

3.404
0.004
0.003 AU 0.002
2.373 2.731
0.000
-0.001
4.274
6.477
0.001
-0.002
Sample Name: Diluent
-0.003 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
Minutes
18.274
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Use of Diode-Array Detector for Troubleshooting of Contamination Peaks in Multicomponent Mobile Phase
Chromatogram and Peaks' UV-VIS Spectra
Cpd 2 - 2.188 250.00 nm
300.00
2.748 250.00 nm
300.00
2.867 250.00 nm
300.00
3.180 250.00 nm
300.00
4.226 250.00 nm
300.00
Aromatic (not in mobile phase)
Negative UV spectrum
Cpd 2 - 2.188
0.015
Wavelength: 254.0 nm
2.748 2.867 3.180 4.226
Minutes
0.010
AU
0.005
0.000
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
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Carry Over:
Chemical Residues in the system
q q q Residues in the injector Non-specific irreversible adsorption on column Accumulation on systems surfaces
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Carry Over in the Injector Washed by Blank Injection

A1100 VWD AU - A1100 AU 286 nm 0.0004 0.0003 AU 0.0002 0.0001 0.0000 -0.0001 A1100 VWD AU - A1100 AU 286 nm 0.0004 0.0003 AU 0.0002 0.0001 0.0000 -0.0001 8.00 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
11.334
Blank Injection 1
Blank Injection 2 Contamination is cleaned
Minutes
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Blank Injections
Signals are reduced from Injection to Injection indicate carry over in the injector
100.00
Series of Blank Injections
80.00
60.00 mAU 40.00 20.00 0.00 0.00 10.00 20.00 30.00 40.00 50.00
Minutes
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Carry-Over in the Injector

LC-MS/MS
1. 2. Peak elutes at RT of main component MRM of the main component
Blank Sample after wash
Blank Sample before wash
Injection-Clean-up with EDTA solved the problem
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Carry Over:
Residues in the system
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Accumulation on Column
MAIN1
0.0010 AU 0.0005
Sample
10.00 11.00 12.00 13.00 14.00 15.00
0.0000 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Minutes 8.00 9.00
SampleName: SST; Vial: 1; Injection: 1; Name: MAIN1; Area: 833913

0.0010
AU
0.0005
0.0000 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Minutes 8.00
9.00
MAIN1
Blank #1
10.00 11.00 12.00 13.00 14.00 15.00
SampleName: DILUENT; Vial: 2; Injection: 1; Name: MAIN1; Area: 5063

Not Cleaned
MAIN1
0.0010
AU
0.0005
Cleaned
Blank #2
10.00 11.00 12.00 13.00 14.00 15.00
0.0000 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Minutes 8.00 9.00
SampleName: DILUENT; Vial: 2; Injection: 2; Name: MAIN1; Area: 4784
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Contamination Peaks
TFA Containing Mobile Phase: TFA, H2O, MeCN - Do Not Contain Aromatic Components!
Chromatogram and UV-VIS Spectra of Contamination Peaks 8.510

340.00 320.00 300.00 280.00 260.00 240.00 220.00 200.00 0.002 0.001
8.510
11.604
Aromatic group
41.142
Aromatic group
nm
11.604
0.000
-0.001 -0.002
Diluent at 280 nm
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00
Minutes
41.142
AU
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Peaks of aromatic compounds in non aromatic mobile phase:

Cannot be Legitimate System Peaks
Chromatogram and UV -VIS Spectra of a Blank Run 1.066
350.00
1.553
2.362
3.695
6.237
6.418
24.320 26.940 27.026
nm
300.00 250.00
6.237 6.418
24.320
2.362
0.20
AU
0.00 -0.20 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
Minutes Sample Name : blank ; Wavelength 214 :
Mobile phase: Gradient of 0.1% TFA in water with ACN
26.940 27.026
1.066 1.553
3.695
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Contamination Peaks - Residues

Contamination on-column and/or in the mobile phase
Chromatogram and UV-VIS Spectra of Contaminations Peaks
1.274 340.00 320.00 300.00 nm 280.00 260.00 240.00 220.00 200.00
41.018 42.128 34.345 Aromatic compounds not legitimate components of the mobile phase
1.514
1.731
8.500
11.633
34.345
36.595
37.612
41.018
42.128
0.030 0.025 0.020 AU 0.015 0.010 0.005 0.000 5.00 10.00 15.00 20.00 25.00 30.00
1.5141.274 1.731
Blank=Diluent at 220 nm
8.500 11.633
35.00
36.595 37.612
40.00
45.00
50.00
55.00
Minutes
Gradient with TFA
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Carry Over:
Residues in the system
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Contaminations
Origin Outside the Sample
q Non-pure solvents q Vials leachables q Reservoirs leachables
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Baseline at Gradient
Change of Baseline with Time at Various Wavelengths: Indication for non pure solvent
0.030 0.025 0.020 60.00 100.00
220 nm
80.00
AU
0.015 Gradients Profile 0.010 0.005 0.000 40.00
230 nm 254 nm 280 nm

5.00 15.00 25.00 35.00 45.00
20.00
0.00 55.00
Minutes
% Composition
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Non-Pure/Contaminated Solvents Used in Gradients

Contaminations can also come from the Parafilm used to seal reservoirs!!!
10.633 BHT 10.633 BHT 6.896 BHA 2.919PG 4.525 4.525 TBHQ TBHQ
Sample
0.00
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 Minutes
30.00
Blank
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 Minutes 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
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Sample vs Blank
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.
50.00 40.00 30.00
This is the reason why there are Gradient Grade solvents
Blank
mAU
20.00 10.00 0.00 50.00 40.00 30.00
Sample
mAU
20.00 10.00 0.00 0.00 10.00 20.00 30.00 40.00 50.00 Dr. Shulamit Levin 2005, Medtechnica
Minutes
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Peaks Origin: Contamination accumulated on pumps in-line filter

Pressure Jump Bubble-Detection
Unexpected Baseline Perturbation
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Contaminations
Origin Outside the Sample q Non-pure solvents q Vials leachables q Reservoirs leachables
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Contamination from Vials Surfaces

0.0012 0.0010 0.0008 AU 0.0006 0.0004 0.0002 0.0000 -0.0002 1.00 2.00 3.00
Non-Certified
Minutes
4.00
5.00
6.00
7.00
8.00
0.0014 0.0012 0.0010 0.0008
Change of vial brand In the same experiment!
AU
Certified
0.0006 0.0004 0.0002 0.0000
-0.0002 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
Minutes
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Contamination is developed during the experiment in complex Diluents (Samples Solvent) (Sample
0.020 0.015
AU
0.010
0.005
0.000 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 Minutes 4.00 4.50 5.00 5.50 6.00 6.50 7.00
SampleName: Sample; Vial: 1:F,1; Injection: 1; Name: Contamination

0.020
0.015
AU
0.010
0.005
0.000 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 Minutes 4.00 4.50 5.00 5.50 6.00 6.50 7.00
SampleName: Diluent ; Vial: 1:E,2; Injection: 1; Name: Contamination
Contamination
Contamination
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Contaminations
Origin Outside the Sample q Non-pure solvents q Vials leachables q Reservoir or Columns leachables
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Contamination Peaks in Size Exclusion Chromatography (SEC)

Contamination is NOT a monomer!
Chromatogram at 210 nm
0.030 0.025 0.020 0.015
Monomer
Sample
Contamination
AU
0.010 0.005 0.000 -0.005 -0.010 0.00
Blank
Higher Molecular Weight
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00
Minutes
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Other Reasons for Extraneous Peaks
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Columns Packing Collapse
All Peaks are split
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Suggested Flow-Chart for Troubleshooting

In a Regulated Environment (no change in method!)
Injection
System Peak ??? Carry over ??? Contamination ???
Yes?
No
Mobile phase:
Multicomponent? Adsorbed components (ion pair)? Response in detector (background)?
Yes?
Legitimate System Peaks

Dissolve samples in mobile phase Adjust diluent with mobile phase components
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Suggested Flow-Chart for Troubleshooting Contd.

Steps: by ease of use 1. Review and revise diluents composition 2. Replace vials batch and/or brand 3. Replace in-line filters 4. Replace solvents batch and/or brand 5. Try a new/fresh column 6. Wash system, especially injector
Make sure to try a fresh blank each test!
Not System Peaks?

Not washed by blanks?
Peaks still persist? Replace injectors surfaces Peaks still persist?
Try another instrument/Operator/Lab
Isranalytica2006
Many times the extraneous peak disappears on its own and remains an unsolved mystery

Extraneous Peaks in HPLC

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Extraneous Peaks in HPLC

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Isranalytica2006

SHULAMIT LEVIN, MEDTECHNICA

Dr. Shulamit Levin 2005, Medtechnica

Dr. Shulamit Levin 2005, Medtechnica

Stability Indicating Methods

Dr. Shulamit Levin 2005, Medtechnica

Compound Impurity 1 Impurity 2 Butamben Impurity 3

Signal-toNoise 4.1 16.4 64544 2.6 5.8 2.2

2.174 2.231 2.263 2.504 2.549 2.714

Dr. Shulamit Levin 2005, Medtechnica

Sample vs. Blank (Diluent)

20.00 10.00 0.00

50.00 40.00 30.00

20.00 10.00 0.00 0.00 10.00 20.00 30.00 40.00 50.00

Dr. Shulamit Levin 2005, Medtechnica

INJECTION OF PURE SOLVENT: Why would there be peaks?

System Peak ??? Carry over ??? Contamination ???

Dr. Shulamit Levin 2005, Medtechnica

Dr. Shulamit Levin 2005, Medtechnica

CONDITIONS FOR APPEARANCE OF REAL SYSTEM PEAKS

Dr. Shulamit Levin 2005, Medtechnica

Mechanism of System Peaks Formation Mechanism of System Peaks Formation

Injection of Vacancy: Cs=1; Cm=0

Injection of Vacancy: Cs=2; Cm=0

Re-equilibration: Cs=0.5; Cm=0.5

Re-equilibration: Cs=1.33; Cm=0.67

Levin and Grushka

Dr. Shulamit Levin 2005, Medtechnica

Injection of Blank = Water

Conclusion: Use mobile phase composition as the diluent

Dr. Shulamit Levin 2005, Medtechnica

Sample=Peptide Dissolved in Water

Blank = Mobile phase

Blank = Mobile phase

System peaks appear because diluent does not contain TFA

Dr. Shulamit Levin 2005, Medtechnica

-0.20 -0.30 -0.40 -0.50 1.00 1.20 1.40

1.60 1.80 2.00 2.20

2.40 2.60 2.80 3.00

Diluent does not contain all mobile phase components

Dr. Shulamit Levin 2005, Medtechnica

Contaminations Peaks - Not System Peaks!

Mobile phase: ACN : Phosphate Buffer pH 9.5 1:1 Wavelength: 280 nm

Sample Name: Diluent

Aromatic (not in mobile phase)

Dr. Shulamit Levin 2005, Medtechnica

Dr. Shulamit Levin 2005, Medtechnica

Carry Over in the Injector Washed by Blank Injection

Blank Injection 2 Contamination is cleaned

Dr. Shulamit Levin 2005, Medtechnica

Series of Blank Injections

Dr. Shulamit Levin 2005, Medtechnica

Carry-Over in the Injector

Blank Sample after wash

Blank Sample before wash

Injection-Clean-up with EDTA solved the problem

Dr. Shulamit Levin 2005, Medtechnica

Dr. Shulamit Levin 2005, Medtechnica

SampleName: SST; Vial: 1; Injection: 1; Name: MAIN1; Area: 833913

SampleName: DILUENT; Vial: 2; Injection: 1; Name: MAIN1; Area: 5063

SampleName: DILUENT; Vial: 2; Injection: 2; Name: MAIN1; Area: 4784

Dr. Shulamit Levin 2005, Medtechnica

Chromatogram and UV-VIS Spectra of Contamination Peaks 8.510

Dr. Shulamit Levin 2005, Medtechnica