HPLC Troubleshooting: Problem Possible Cause Solution

HPLC Troubleshooting
Abhishek Raj
Deputy Manager (R&D)
Quest Pharmaceuticals Pvt. Ltd, Chhata Pipara, Bara, Birgunj, Nepal.
abhishekraj4@gmail.com
Start up - Preliminary checks

Problem
Possible cause
Detector off
No peaks or
very small
peaks
Solution
Check detector
Broken connections
Check connections
to recorder
No sample/Wrong
sample
Check sample. Be sure it is not deteriorated.

Check for bubbles in the vials
Wrong settings on
Check attenuation. Check gain
recorder or detector
Pump off
Start Pump
Flow interrupted
Check reservoirs. Check position of the inlet

tubing. Check loop for obstruction or air. Check
degasing of mobile phase. Check compatibility of
the mobile phase components.
Leak
Check fittings. Check pump for leaks and

precipitates. Check pump seals.
Air trapped in the

system
Disconnect column and prime pump. Flush

system with 100% methanol or isopropanol.
Contact servicing if necessary.
No Flow
Column and Fittings Leaks

Problem
Column end
Possible cause
Loose fitting
Solution
Tighten or replace fitting
leaks
White powder at
loose fitting
Cut tubing and replace ferrule; disassemble

fitting, rinse and reassemble.
Leak at detector Detector-seal failure Replace detector seal or gaskets.

Leak at injection Worn or scratched
valve
valve rotor
Replace valve rotor
Leak at pump
Replace pump seal; check piston for scratches

and, if necessary, replace
Pump seal failure
Change in Retention time

Problem
Possible cause
Solution
Buffer retention times
Use buffer with concentration greater

than 20 mM.
Contamination buildup
Flush column occasionally with

strong solvent
Equilibration time
Pass at least 10 column volumes
insufficient for gradient run
through the column for gradient
or changes in isocratic mobile
regeneration or after solvent changes
phase
First few injections - active
Changing
sites
Retention Times
Condition column by injecting

concentrated sample
Inconsistent on-line mobilephase mixing
Ensure gradient system is delivering a

constant composition; compare with
manually prepared mobile phase;
partially premix mobile phase
Selective evaporation of
mobile-phase component
Cover solvent reservoirs; use lessvigorous helium purging; prepare

fresh mobile phase
Thermostat or insulate column;

Varying column temperature ensure laboratory temperature is
constant.
Decreasing
Retention Times Active sites on column
packing
Use mobil-phase modifier, competing

base (basic compounds), or increase
buffer strength; use higher coverage
column packing.
Column overloaded with

sample
Decrease sample amount or use

larger-diameter column.
Increasing flow rate
Check and reset pump flow rate.
Loss of bonded stationary
Use mobile-phase pH between pH 2
phase or base silica
and pH 8
Thermostat or insulate column;

Varying column temperature ensure laboratory temperature is
constant
Decreasing flow rate
Increasing
Retention Times Changing mobile-phase
composition
Loss of bonded stationary
phase
Slow column
equilibration
time
Check and reset pump flow rate;

check for pump cavitation; check for
leaking pump seals and other leaks in
system
Cover solvent reservoirs; ensure that
gradient system is delivering correct
composition.
Use mobile-phase pH between pH 2
and pH 8
Reversed phase ion pairing long chain ion pairing

Use ion-pairing reagent with shorter
reagents require longer
alkyl chain length
equilibration time
Baseline
Problem
Possible cause
Air bubbles in mobile phase
Void Time Positive-negative noise

difference in refractive
index of injection solvent
and mobile phase
Drifting
baseline
Solution
Degas or use back pressure restricor on
detector
Normal with many samples; use mobile
phase as sample solvent
Use non-UV absorbing mobile phase

Negative direction (gradient
solvents; use HPLC grade mobile phase
elution) - absorbance of
solvents; add UV absorbing compound to
mobile-phase A
mobile phase B.
Use higher UV absorbance detector
Positive direction (gradient wavelength; use non-UV absorbing mobile
elution) - absorbance of
phase solvents; use HPLC grade mobile
mobile phase B
phase solvents; add UV absorbing compound
to modile phase A.
Positive direction contamination buildup and
elution
Flush column with strong solvent; clean up

sample; use HPLC grade solvents
Wavy or undulating -
Monitor and control changes in room
Baseline
noise
temperature changes in
room
temperature; insulate column or use column

oven; cover refractive index detector and
keep it out of air currents.
Continous - detector lamp

problem or dirty cell
Replace UV lamp( each should last 2000 h;

clean and flush flow cell.
Gradient or isocratic
proportioning - lack of
solvent mixing
Use proper mixing device; check

proportioning precision by spiking one
solvent with UV absorbing compound and
mointor UV absorbance detector outputl.
Gradient or isocratic
proportioning malfunctioning
proportioning valvesl
Clean or replace proportioning precision

valves; partially remix solventsl.
Occasional sharp spikes external electrical

interference
Use voltage stabilizer for LC system; use

independent electrical circuit.
Periodic - pump pulses
Service or replace pulse damper; purge air

from pump; clean or replace check valves.
Random - contamination
buildup
Flush column with strong solvent; clean up

sample; use HPLC grade solvent
Spikes - bubble in detector
Degas mobile phase; use back pressure

restrictor at detector outlet.
Spikes - column
temperature higher than
boiling point of solvent
Use lower column temperature.
Pressure
Problem
Decreasing
Pressure
Possible cause
Solution
Insufficient flow from pump
Loosen cap on mobile phase

reservior
Leak in hydralic lines from pump

to column
Tighten or replace fittings; tighten

rotor in injection valve
Leaking pump check valve or seals
Replace or clean check valves;

replace pump seals.
Pump cavitation
Degas solvent; check for

obstruction in line from solvent
reservoir to pump; replace inlet-line
frit
Fluctuating
pressurre
Bubble in pump
Degas solvent; purge solvent with

helium
Leaking pump check valve or seals
Replace or clean check valves;

replace pump seals
Column blocked wth irreversibly

adorbed sample
Improve sample cleanup; use guard

column; reverse-flush column with
strong solvent to dissolve blockage
Column particle size too small (for Use larger particle size (for
example 3 micrometers)
example 5 micrometer)
High Back
Pressure
Microbial growth on column
Use at least 10% organic modifier

in mobile phase; use fresh buffer
daily; add 0.02% sodium azide to
aqueous mobile phase; store
column in at least 25% organic
solvent without buffer
Mobile phase viscosity too high
Use lower viscosity solvents or

higher temperature
Plugged frit in in-line filter or

guard column
Replace frit or guard column
Plugged inlet frit
Replace endfitting or frit assembly
Use correct solvent with column;

change to proper solvent
compositionl consult
Polymetric columns - solvent
manufacturer's solventchange causes swelling of packing
compatibility chartl use a column
with a higher percentage of crosslinking
Salt precipitation (especially in
reversed-phase chromatography
with high concentration of organic
solvent in mobile phase)
concentration of organic solvent in
mobile phase)
Ensure mobile phase compatibility

with buffer concentration; decrease
ionic strength and water-organic
solvent ratio; premix mobile phase
When injector disconnected from

column - blockage in injector
Clean injector or replace rotor
Blocked flow lines
Systematically disconnect
components from detector end to
column end to find blockage;
replace or clean blocked
component
Particulate buildup at head of
Filter sample; use .5 micrometer in-
Increasing
Pressure
column
line filter; disconnect and backflush

column; replace inlet frit
Water-organic solvent systems buffer precipitation
Ensure mobile phase compatibility

with buffer concentration; decrease
ionic strength or water organic
solvent ratio
Peaks
Problem
Possible cause
Solution
Analytes eluted early due

Dilute sample 1:10 and reinject
to sample overload
Use smallest possible cell volume consistent
Detector-cell volume too
with sensitivity needs; use detector with no heat
large
exchanger in system
Broad
peaks
Ghost
Injection volume too

large
Decrease solvent strength of injection solvent to

focus solute; inject smaller volume
Large extra column

volume
Use low- or zero-dead-volume endfittings and

connectors; use smallest possible diameter of
connecting tubing (<0.10 in. i.d.); connect
tubing with matched fittings
Mobile-phase solvent
viscosity too high
Increase column temperature; change to lower

viscosity solvent
Peak dispersion in
injector valve
Decrease injector sample loop size; introduce

air bubble in front and back of sample in loop
Poor column efficiency
Use smaller-particle-diameter packing, lowerviscosity mobile phase, higher column

temperature, or lower flow rate
Retention time too long
Use gradient elution or stronger isocratic

mobile phase
Sampling rate of data

system too low
Increase sampling frequency.
Slow detector time

constant
Adjust time constant to match peak width
Some peaks broad - late

elution of analytes
retained from previous
injection
Flush column with strong solvent at end of

run; end gradient at higher solvent
concentration
Contamination
Flush column to remove contaminatint; use
HPLC-grade solven
Elution of analytes
retained from previous
injection
Flush column with strong solvent at end of

run; end gradient at higher solvent
concentration
Ion-pair chromatography Prepare sample in mobile phase; reduce

- upset equilibrium
injection volume
peaks
Negative
peaks
Oxidation of
trifluoroacetic acid in
peptide mapping
Prepare trifluoroacetic acid solutions fresh

daily; use antioxidant
Reversed-phase
chromatography contaminated water
Check suitability of water by running different

amounts through column and measure peak
height of interferences as function of
enrichment time; clean water by running it
through old reversed-phase column; use
HPLC-grade water.
Unknown interferences
in sample
Use sample cleanup or prefractionation before

injection.
Refractive index
detection - refractive
index of solute less than
that of mobile phase
Reverse polarity to make peak positive
UV-absorbance detection
Use mobile phase with lower UV absorbance;
- absorbance of solute
if recycling solvent, stop recycling when
less than that of mobile
recycled solvent affects detection
phase
Peak
Doubling
Blocked Frit
Replace or clean frit; install 0.5-um porosity

in-line filter between pump and injector to
eliminate mobile-phase contaminants or
between injector and column to eliminate
sample contaminants
Coelution of interfering
compound
Use sample cleanup or prefractionation; adjust

selectivity by changing mobile or stationary
phase
Coelution of interfering Flush column with strong solvent at end of

compound from previous ran; end gradient at higher solvent
injection
concentration
Column overloaded
Column void or
channeling
Use higher-capacity stationary phase; increase

column diameter; decrease sample amount
Replace column, or, if possible, open top
endfitting and clean and fill void with glass
beads or same column packing; repack column
Injection solvent too

strong
Use weaker injection solvent or stronger

mobile phase
Use injection volume equal to one-sixth of

Sample volume too large column volume when sample prepared in
mobile phase for injection
Peak
Fronting
Tailing
Peaks
Unswept injector flow

path
Replace injector rotor
Channeling in column
Replace or repack column
Column overloaded
Use higher-capacity stationary phase; increase

column diameter; decrease sample amount
Basic solutes - silanol

interactions
Use competing base such as triethylamine; use

a stronger mobile phase; use base-deactivated
silica-based reversed-phase column; use
polymeric column
Beginning of peak
doubling
See peak doubling
Chelating solutes - trace

metals in base silica
Use high purity silica-based column with low

trace-metal content; add EDTA or chelating
compound to mobile phase; use polymeric
column
Silica-based column degradation at high pH
Use polymeric, sterically protected, or highcoverage reversed-phase column; install silica

gel saturatorcolumn between pump and
injector
Silica-based column degradation at high

temperature
Reduce temperature to less than 50 C
Silica-based column silanol interactions
Decrease mobile-phase pH to suppress silanol

ionization; increase buffer concentration;
derivatize solute to change polar interactions
Unswept dead volume
Minimize number of connections; ensure

injector rotor seal is tight; ensure all
compression fittings arecorrectly seated
Replace column, or, if possible, open top

endfitting and clean and fill in void with glass
Void formation at head of
beads or samecolumn packing; rotate injection
column
valve quickly; use injection valve with
pressure bypass; avoid pressure shock
Spikes
Bubbles in mobile phase Degas mobile phase; use back-pressure

restrictor at detector outlet; ensure that all
fittings are tight

Column stored without
caps
Store column tightly capped; flush reversedphase columns with degassed methanol

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HPLC Troubleshooting

Start up - Preliminary checks

Check sample. Be sure it is not deteriorated.

Check reservoirs. Check position of the inlet

Check fittings. Check pump for leaks and

Air trapped in the

Disconnect column and prime pump. Flush

Column and Fittings Leaks

Cut tubing and replace ferrule; disassemble

Leak at detector Detector-seal failure Replace detector seal or gaskets.

Replace valve rotor

Replace pump seal; check piston for scratches

Pump seal failure

Change in Retention time

Buffer retention times

Use buffer with concentration greater

Flush column occasionally with

Condition column by injecting

Inconsistent on-line mobilephase mixing

Ensure gradient system is delivering a

Cover solvent reservoirs; use lessvigorous helium purging; prepare

Thermostat or insulate column;

Use mobil-phase modifier, competing

Column overloaded with

Decrease sample amount or use

Increasing flow rate

Check and reset pump flow rate.

Loss of bonded stationary

Use mobile-phase pH between pH 2

phase or base silica

Thermostat or insulate column;

Check and reset pump flow rate;

Reversed phase ion pairing long chain ion pairing

Void Time Positive-negative noise

Use non-UV absorbing mobile phase

Flush column with strong solvent; clean up

Monitor and control changes in room

temperature; insulate column or use column

Continous - detector lamp

Replace UV lamp( each should last 2000 h;

Use proper mixing device; check

Clean or replace proportioning precision

Occasional sharp spikes external electrical

Use voltage stabilizer for LC system; use

Periodic - pump pulses

Service or replace pulse damper; purge air

Flush column with strong solvent; clean up

Spikes - bubble in detector

Degas mobile phase; use back pressure

Use lower column temperature.

Insufficient flow from pump

Loosen cap on mobile phase

Leak in hydralic lines from pump

Tighten or replace fittings; tighten

Leaking pump check valve or seals

Replace or clean check valves;

Degas solvent; check for

Degas solvent; purge solvent with