Webinar on

HPLC Troubleshooting AND COLUMN

REGENERATION – PRACTICAL APPROACH
(IPAT-2021)

by
Likhit Mandal
Analytical Scientist
Aurobindo Pharma Ltd.
 HPLC System Components

• Pump
• Binary Pump
• Quaternary Pump
• Injector/Autosampler
• Column
• Detector
• Ultra Violet(UV)-Vis(visible) /Photo Diode Array(PDA) Detector
• Refractive Index (RI) Detector
• Fluoresence Detector
• Evaporative Light Scattering (ELSD) Detector
• Data System/Integrator
 FACTORS INFLUENCING HPLC ANALYSIS

 Quality of Solvents and Buffer

 Filtration
 Degassing
 System washing
 System priming and equilibration
 Column Maintenance
 Sample preparation
 Detector related troubleshooting
 Maintenance of HPLC after analysis
 Phase Conversion
 Preparation of mobile phase

• Highest quality or HPLC Grade

Quality of • Water should be Deionized or HPLC Grade

Solvent
and Buffer
 Preparation of mobile phase

• All HPLC solvents should be filtered through a 0.45 μM or 0.22µM filter

before use
Filtration
 Preparation of Mobile phase

• Mobile phase degassed to remove all dissolved gasses.

• Dissolved gas can be removed from solution by (Bubbling with
Degassing inert gas / Sonication/ Vacuum filtration)

If the mobile phase is not degassed, air bubbles can form in the high-pressure
system resulting in problems associated with system instability, spurious baseline
peaks, etc.
 MAINTAINANCE of HPLC SYSTEM BEFORE ANALYSIS

• To ensure the cleaning of suction filter before analysis.

• Possible cause:
• Buffer precipitation due to prior analysis
• Air bubble entrapment
Suction • Corrective Action:
filter • Sonicate the Suction filter in Hot water with 0.1% Nitric acid, followed by Water,
Methanol mixture solvent

• Flush the system respective shipping solvents based on prior analysis

• Flush with Hot Water about 60ºC to remove precipitation related to buffers.
System
washing • Wash with Magic Solvent (Water: Acetonitrile :Methanol :IPA , 25% each + 1% Formic
acid or 1% Nitric Acid)
 TROUBLESHOOTING – Column

• It is advisable to perform a quick visual check of the instrument and column. This
Visual will pick up leaks, loose or disconnected tubing, changes in instrument settings.
inspection
• High pressure : The most common causes of high pressure are blocked tubing around the injector and
column inlet.
• Low pressure : The most common causes of no/low pressure are the solvent inlet lines not being
immersed in solvent, no solvent in the reservoir and leaks before column inlet.
Pressure • Fluctuating pressure : The most common cause of fluctuating pressure is poorly primed lines with badly
degassed solvents

• Non-cyclic noise – Fluid path problems

Baseline • Non-cyclic noise – Detector electronics problems
irregularit • Cyclic noise – Detector related problems and others
ies
 TROUBLESHOOTING – Column
Changes in chromatography
 Early eluting peaks broad
 Possible Cause:
 Sample overload or incorrect system plumbing.
 TROUBLESHOOTING – Column
Changes in chromatography
 All peaks broad
 Possible Cause:
 Due to errors in instrumentation or column.
 Corrective Action:
 Investigating the column and guards first
 TROUBLESHOOTING – COLUMN
Changes in chromatography
 All peaks doubling
 Possible Cause:
 Due to blockage prior to the column or guard voiding
 Partially plugged frit
 Corrective Action:
 First wash the column and guards properly
 TROUBLESHOOTING – COLUMN
Changes in chromatography
 Fronting peaks
 Possible Cause:
 Large injection volumes of a sample that is dissolved in solvents that are incompatible with the
mobile phase being used
 Peak fronting is a voided or contaminated guard or column.
 TROUBLESHOOTING – COLUMN
Changes in chromatography
 Tailing, Broadening and Loss of
Efficiency (N, plates) of Peaks
 Possible Cause:
 Due to column degradation or
inlet Contamination.

 Column “secondary interactions”

 Column packing voids

 Column contamination

 Column aging

 Column loading

 Extra-column effects
 Split Peaks -Injection Solvent Effects

Injecting in a solvent stronger than the mobile phase can cause peak shape
problems, such as peak splitting or broadening.
Note: earlier peaks (low k) most affected
 Peak Tailing -Column “Secondary Interactions”

Peak tailing of amine analytes eliminated with mobile phase modifier (TEA (triethylamine ))
at pH 7
Reducing the mobile phase pH reduces interactions with silanols that cause peak tailing. No TEA
modifier required.
 Peak Tailing
Column Contamination
Peak Tailing/Broadening _Sample Load Effects
 TROUBLESHOOTING – DETECTOR
Baseline irregularities

Possible Cause: Air in the system.

Corrective Action:
• Thoroughly degassed prior to use
• All lines purged with solvent and the pump thoroughly primed.
• To remove air bubbles into the detector flow cell the cell may require cleaning and/or removal
of air bubbles.
Non-
cyclic
noise –
Fluid path
problem
 TROUBLESHOOTING – HPLC
Baseline irregularities

Possible Cause: Insufficient time to equilibrate the detector before an injection

Corrective Action:
• Check that all the cables securely seated in their sockets and that the correct cable is in the
correct socket.
• Check that all settings have been returned to their position prior to the routine maintenance.
Non-cyclic
noise –
Detector
electronic
problem
 TROUBLESHOOTING – HPLC
Baseline irregularities

Possible Cause: Insufficient time to equilibrate the detector before an injection

Corrective Action:
• Check that all the cables securely seated in their sockets and that the correct cable is in the
correct socket.
• Check that all settings have been returned to their position prior to the routine maintenance.

Cyclic noise
– Detector
related
problems
 TROUBLESHOOTING – DETECTOR
Changes in chromatography
 Retention time changes from injection to injection
 Possible Cause:
 Un-equilibrated system.
 Temperature changes during the analysis
 Corrective Action:
 Detector and Fluid system must be stable prior to starting an analysis
 Column is housed in a thermally controlled environment, such as a column oven/jacket

 Continually increasing or decreasing retention times

 Possible Cause:
 peak retention time drift in one direction is poorly prepared or mixed solvents or a system leak
 Corrective Action:
 Solvents were prepared correctly
 Ensure the solvent flow rate is correct and constant
 Retention Issues

Retention time changes (tr)

Retention factor changes (k’)
Selectivity changes (α)
 Changes in Retention (k) Same Column, Over Time

 May be caused by:

1. Column aging
2. Column contamination
3. Insufficient column equilibration
4. Poor column/mobile phase combination
5. Change in mobile phase
6. Change in flow rate
7. Change in column temperature
8. Different Gradient Delay Volumes
9. Other issues
Mobile Phase Change Causes Change in Retention
Column Aging/Equilibration Causes
Retention/Selectivity Changes
Example Change in Retention/Selectivity
Tip: Dwell Volume Differences Between Instruments Can
Cause Changes in Retention and Resolution
 Measure and Correct for Dwell Volume (VD)

 If VD1 > VD2

Compensate for longer VD1 by adding an isocratic hold to VD2,
such that Hold + VD2 = VD1
 If VD1 < VD2
Delay injection, such that VD2 - delay = VD1
Lot-to-Lot Selectivity Change - pH
 Sample preparation
Sample preparation is about more than just the dissolution of s solid in a liquid. Samples may
require other techniques such as filtration, extraction or derivatization as well as accurate weighing
and /or dissolution.

• This can be performed on-line using a pre-

column filter or as the sample is
Filtration introduced to the vial.

• The most common forms of extraction

are liquid-liquid, solid-liquid and solid
Extraction phase extraction.

• It can occur before or after the column. It can also be performed manually or automatically. It is
generally required for detection purposes, for example, the UV detection of analytes without
Derivatiza chromophores
tion
 TROUBLESHOOTING – HPLC
Changes in chromatography
 No Peaks
 Possible Cause:
 The wrong sample being injected
 Detector not being switched on or a blockage between the injector and detector lines.
 Some part of the sample or mobile phase preparation has been performed incorrectly
 TROUBLESHOOTING – HPLC
Changes in chromatography
 Smaller than expected peaks
 Possible Cause:
 Wrong sample being injected
 Incorrect sample volume being injected
 Blockage between the syringe needle and detector.
 Problems with the syringe plunger sticking in the barrel can occur if the sample contains particulates.
 Viscous sample will require a longer draw time. Insufficient draw time will result
 TROUBLESHOOTING – HPLC
Changes in chromatography
 Flat topped peaks
 Possible Cause:
 Either large injection volumes of dilute sample or by small injection volumes of strong
sample dilution.
 TROUBLESHOOTING – HPLC
Changes in chromatography
 Negative peaks
 Possible Cause:
 Due to difference in refractive index between the sample solvent, sample and mobile
phase
 After routine maintenance when the system has not been reconfigured correctly
 TROUBLESHOOTING – HPLC
Changes in chromatography
 Missing peaks
 Possible Cause:
 Single or multiple missing peaks are usually due to the wrong sample being injected of
the sample degrading
 Loss of resolution due to column/solvent inconsistencies.
 Extra peaks
 Possible Cause:
 Due to contamination or degradation of the sample, mobile phase or column.
 Corrective Action:
 To check if the extra peaks are in the sample alone, perform a blank injection of
sample solvent. The peaks should be absent.
 TROUBLESHOOTING – HPLC
Changes in chromatography
 Peaks misidentified
 Possible Cause:
 Occurs most often in degradation samples or those in which related substance levels
are being measured. This is because the software is “calibrated” using a standard
mixture with specific concentrations of each impurity.
 Most “real-life” samples contain impurities at very low levels so the retention time of
their peaks will be slightly different to those generated by the standard mix.
 Identification windows should be set widely enough to take into account this time
variation with respect to concentration
 Column cleaning and regeneration
Ensure that no buffers/samples are present on the column and that the solvent used prior to the clean-up is
miscible with the first wash solvent. After the clean-up, ensure that the test mobile phase is compatible with the
last solvent in the column

• Flush with Tetrahydrofuran Methanol Methylene chloride Benzene-free n-

Normal hexane
phase
media
• Flush with HPLC grade water; inject 4 aliquots of 200 μL DMSO during this flush
Reversed Methanol Chloroform Methanol
phase
media

• Flush with HPLC grade water Methanol Chloroform

Anion
exchange
media
 Column cleaning and regeneration
Ensure that no buffers/samples are present on the column and that the solvent used prior to the clean-up is
miscible with the first wash solvent. After the clean-up, ensure that the test mobile phase is compatible with the
last solvent in the column

• Flush with HPLC grade water; inject 4 aliquots of 200 μL DMSO during this flush
Cation Tetrahydrofuran
exchange
media
• Weakly retained proteins : Flush with 30 mL, 0.1M, pH = 3 phosphate buffer
Protein • Strongly retained proteins : Flush for 60 minutes using 100% water to 100% acetonitrile
size gradient
exclusion
media
• Suitable for applications running mobile phases containing trifluoroacetic acid. Flush the
Removal of column with acetonitrile that has been heated to 75°C. the column should also be maintained at
Trifluoro this temperature
acetic acid
 Column cleaning and regeneration
Ensure that no buffers/samples are present on the column and that the solvent used prior to the clean-up is
miscible with the first wash solvent. After the clean-up, ensure that the test mobile phase is compatible with the
last solvent in the column

• Invert the column and flush at 1 mL/min with 50 mL tetrahydrofuran/water (1:1)

containing 0.1% trifluoroacetic acid tetrahydrofuran/water (1:1) containing 0.1%
triethylamine or sodium hydroxide tetrahydrofuran/water (1:1) containing 0.1%
trifluoroacetic acid methanol/water (95:5) to re-equilibrate re-invert the column
Acid/Base
regeneration

• Flush at 1 mL/min with 50 mL acetone 120 mL dibutylether 50 mL acetone

aqueous mobile phase until equilibrated
Strong Organic
regeneration
Acid Wash Can Improve Peak Shape
 Cleaning Procedure of column
 Cleaning Reverse phase columns
 When the mobile phase dose not contain any buffered mobile phase or ion-pairing
reagents use high concentration organic solvent to remove the highly lipophilic
contaminants. Increase the content of organic solvent up to 100 %. Then , flush
the column with 5 column volumes.. When observing excessive back pressure,
reduce and adjust the flow.
 Example column Dimension: 4.6mm, I.D x 250 mm
Method flow rate: 1 mL/min
Method Mobile Phase: Acetonitrile /Water = 65/35
Clean the column with 100 % ACN at 1 mL/min for at least 30 minutes.
 Cleaning Procedure of column

 When Mobile phase contains Ion pairing reagent

 When the mobile phase contain any ion pairing reagent, clean the column with a
water/organic solvent mixture, using the same content as in the buffered mobile phase
containing an ion pairing reagent. For Example, clean the column with 10 % ACN in
water for at least 30 minutes . Then clean it with 50/50 % Water/ACN at least 30
minutes.
 Example column Dimension: 4.6mm, I.D x 250 mm
Method flow rate: 1 mL/min
Method Mobile Phase: 10mM KH2PO4+ (Adjust pH: 2.5 with OPA)/ACN = 80/20
 Clean the column with 10 % ACN in water at 1 mL/min for at least 30 minutes.
 Clean the column with 50/50 % Water/ACN at least 30 minutes.
 Cleaning Procedure of column
 Cleaning HILIC Columns:
 Under HILIC mode, polar analytes are retained with high organic mobile phases.
The following describes the elution strength of solvents used in HILIC mode.
Tetra hydrofuran < Acetonitrile < 2-Propanol < Ethanol < Methanol < Water
--------------------------------------------------------------------------→
(Weak Solvents) (Strong Solvents)
 Cleaning Procedure of column

 Cleaning Reverse phase columns

 Normal separation depends upon polar adsorptive interactions, which the bonded
phase is polar and the mobile phase is non-polar. Polar analytes will be more
strongly retained than non-polar analytes in normal-Phase chromatography. Clean
the column with polar solvents to remove highly polar contaminants.
The following describes the elution strength of solvents used in normal-phase
mode.
Hexane < Chloroform < < Tetra hydrofuran < 2-Propanol < Ethanol
--------------------------------------------------------------------------→
(Weak Solvents) (Strong Solvents)
 Column regeneration and Storage

 Residual ion-pairing reagents, acids or buffer salts in the column will promote and
encourage hydrolysis of the bounded phase resulting in shorter column lifetimes.
Additionally storage in highly aqueous mobile phase for a long period can lead to
splitting peaks or less column efficiency as the sample matrices such as lipids can
accumulate in the column. To prolong column life times, clean the column every
single time after the analysis or whenever possible.
THANK YOU