1. The document discusses troubleshooting strategies and common problems in HPLC. 2. It outlines a 5-step troubleshooting strategy of identifying the problem, determining the cause, isolating the exact cause, rectifying the problem if possible, and returning the system to use. 3. Common problems discussed include issues with the mobile phase, pump, injector, detector, and peaks/baseline, along with potential causes and solutions for each.

1. PREPARED BY:
RAVINDRA BABU KOPPERA
PA/2019/118
M.S (Pharmaceutical Analysis)
NIPER Hyderabad.

2. COMPONENTS OF HPLC

3. TROUBLESHOOTING

4. TROUBLESHOOTING STRATEGY AND PROCESS
Strategy
Any troubleshooting strategy involves five steps;
 1. Identification of the problem
 2. Awareness of the cause(s) of the problem
 3. Isolation of the exact cause of the problem
 4. Rectifying the problem if able
 5. Returning the unit to routine use or referring the
problem to your maintenance manager.

5. Troubleshooting process
❖ To execute the strategy a systematic approach, which will
work for any problem, is required. The
❖ systematic approach should follow a logical sequence, so
that the exact cause of the problem can be found.
1. Gather the facts – not theories.
2. Check the simplest things first – it’s easier.
3. Compare the performance obtained to the expected
performance.
4. List possible causes.
5. Work through the possible causes in a step-by-step manner
checking the outcome from any changes made.
6.As a last resort – get help from elsewhere, for example your
instrument supplier help desk or your local technical
support department.

6. TROUBLESHOOTING PROBLEMS
1.MOBIILE PHASE PROBLEMS
2.PUMP PROBLEMS
3. INJECTOR/INJECTION PROBLEMS
4.DETECTOR PROBLEMS

7. MOBILE PHASE PROBLEMS
 Low sensitivity and rising baselines, noise, or spikes on
the chromatogram can often be attributed to the mobile
phase. Contaminants in the mobile phase are especially
troublesome in gradient elution. The baseline may rise, and
spurious peaks can appear as the level of the contaminated
component increases.
▪ Water is the main source of contamination --- use only highly
pure de-ionized water.
▪ Use HPLC grade solvents.
▪ Aqueous buffers --- Fresh and filtered
▪ Use ion-pair reagents carefully– Increase in chain length
increase retention time

8. PUMP PROBLEMS
 Some of the more common symptoms are erratic retention
times, noisy baselines, or spikes in the chromatogram.
 Leaks at pump fittings or seals will result in poor
chromatography. Run the HPLC system constantly at low flow
rates (e.g. 0.1 ml/min) to avoid crystallization effects.
 A sure sign of a leak is a buildup of salts at a pump
connection. Buffer salts should be flushed from the
system daily with fresh deionized water.
 To isolate and repair specific problems related to your
apparatus, use the troubleshooting and maintenance sections
of the operation manual.
 Pump seals require periodic replacement. You should perform
regular maintenance rather than waiting for a problem to
occur.

9. INJECTOR/INJECTION PROBLEMS
 Mechanical problems involving the injector (e.g.,
leaks, plugged capillary tubing, worn seals) are easy
to spot and correct. Use a column filter unit to
prevent plugging of the column frit due to physical
degradation of the injector seal
 Whenever possible, dissolve and inject samples in
the mobile phase. Otherwise, be sure the injection
solvent is of lower eluting strength than the mobile
phase
 Be aware that some auto samplers use separate
syringe wash solutions. Make sure that the wash
solution is compatible with and weaker than the
mobile phase.

10. DETECTOR PROBLEMS
Detector problems fall into two categories – electrical
and mechanical/optical.
▪ For electrical problems, we should contact the
instrument manufacturer , Mechanical or optical
problems can usually be traced to the flow cell.
▪ Detector-related problems include leaks, air bubbles,
and cell contamination
▪ Flow rates or back pressures that exceed the
manufacturer’s recommendation will break the cell
window.
▪ Old or defective lamps as well as incorrect detector
rise time, gain, or attenuation will reduce sensitivity
and peak height

11. PROBLEMS AND SOLUTIONS
 1.NO PEAKS/ VERY SMALL PEAKS
POSSIBLE CAUSE SOLUTION
DETECTOR OFF CHECK DETECTOR
BROKEN CONNECTIONS
TO RECORDER
CHECK
CONNNECTIONS
NO SAMPLE/WRONG
SAMPLE
CHECK
SAMPLES,BUBBLES
WRONG SETTINGS ON
RECORDER OR
DETECTOR
CHECK ATTENUATION

12.  2.NO FLOW
POSSIBLE CAUSE SOLUTION
PUMP OFF Check reservoirs. Check position of the inlet
tubing. Check loop for obstruction or air.
Check degassing of mobile phase. Check
compatibility of the mobile phase
components
FLOW INTERRUPTED Check fittings. Check pump for leaks and
precipitates. Check pumps seals
LEAK Disconnect column and prime pump. Flush
system with 100% methanol or isopropanol.
Contact servicing if necessary.

13. 3.VARIABLE RETENTION TIMES
POSSIBLE CAUSE SOLUTION
Leak Check system for loose fittings. Check
pump for leaks, salt buildup, unusual
noises. Change pump seals if
necessary.
Change in mobile phase composition. Check make-up of mobile phase
Air trapped in pump. (Retention times
increase and decrease at random
times.)
Purge air from pump head or check
valves. Change pump seals if
necessary. Be sure mobile phase is
degassed.
Column temperature fluctuations Use reliable column oven.
Column overloading Inject smaller volume
Sample solvent incompatible with
mobile phase.
Adjust solvent. Whenever possible,
inject samples in mobile phase.
Column problem Substitute new column of same type
to confirm column as cause.

14. 4.PRESSURE
(i)Pressure lower than Usual
(ii)Pressure higher than usual
Possible cause Solution
Leak Check for loose fittings
Mobile phase flow
interrupted/obstructed.
Check flow throughout the system
,Degassing
Air trapped inside Purge the system
Possible cause Solution
Problem in pump, injector, in-line
filter, or tubing.
Remove guard column and
analytical column from system.
Replace with unions
Obstructed guard column or
analytical column.
Remove guard column (if present)
and check pressure. Replace guard
column if necessary. If analytical
column is obstructed, reverse and
flush the column,

15. (iii)High back pressure
Possible cause Solution
Column blocked with irreversibly
adsorbed sample
reverse-flush column with strong
solvent to dissolve blockage
Column particle size too small (for
example 3 micrometers)
Use larger particle size (for
example 5 micrometer)
Mobile phase viscosity too high Use lower viscosity solvents or
higher temperature
Salt precipitation Ensure mobile phase compatibility
with buffer concentration; decrease
ionic strength and water-organic
solvent rat

16. 5.PEAK PROBLEMS
(i)Loss of resolution
Possible cause Solution
Mobile phase
contaminated/deteriorated
(causing retention times and/or
selectivity to change).
Prepare fresh mobile phase
Obstructed guard or analytical
column.
Remove guard column (if present)
and attempt analysis. Replace
guard column if necessary. If
analytical column is obstructed,
reverse and flush

17. (ii)Split peaks
Possible cause Solution
Contamination on guard
or analytical column
inlet.
Remove guard column
or flush analytical
column
Sample solvent
incompatible with
mobile phase.
Adjust solvent.
Whenever possible,
inject samples in mobile
phase.
Small (uneven) void at
column inlet.
Repack top of column
with pellicular particles
of same bonded phase
functionality. Continue
using the column in
reverse flow direction.

18. (iii)Tailing peaks
Possible cause Solution
Guard or analytical column
contaminated/worn out.
Remove guard column and
attempt analysis. Replace guard
column if necessary. If analytical
column problem, use appropriate
restoration procedure
Mobile phase
contaminated/deteriorated.
Check make-up of mobile
phase.

19. (iv)Fronting peaks
Possible cause Solution
Column overloaded. Inject smaller volume of
diluted samples
Sample solvent
incompatible with mobile
phase.
Adjust solvent. Whenever
possible, inject samples in
mobile phase.

20. (v)Broad peaks
Possible cause Solution
Mobile phase composition
changed
Prepare new mobile phase.
Mobile phase flow rate too
low.
Adjust flow rate.
Leak (especially between
column and detector).
Check system for loose fittings
Recorder response time too
high.
Reduce response time
Column overloaded Inject smaller volumes

21. 6.BASELINE PROBLEMS
Possible cause Solution
Mobile phase contaminated,
deteriorated, or prepared from low
quality materials.
Check make-up of mobile phase.
Air trapped in system. purging
Weak detector lamp Replace lamp
Column temperature fluctuation Control column and mobile phase
temperature
Slow column equilibration,
especially when changing mobile
phase.
Flush column with intermediate
strength solvent.