Troubleshooting is a process of identifying problems, determining their causes, and finding solutions. For high performance liquid chromatography (HPLC), there are many potential sources of errors including instrumental issues, procedural problems, and human errors. This can result in symptoms like changes in retention time, peak issues, baseline abnormalities, and pressure fluctuations. Troubleshooting HPLC issues involves preliminary checks, examining the column and fittings for leaks, and investigating potential causes of different problem symptoms to resolve them.

1. TROUBLE
SHOOTING
IN HPLC
VISHAKHA PRAVIN
YENARE

2. TROUBLE SHOOTING
Troubleshooting is a form of problem solving most often
applied to repair of failed products or processes. It is a
logical, systematic search for the source of a problem so
that it can be solved, and so the product or process can be
made operational again. Troubleshooting is needed to
develop and maintain complex systems where the
symptoms of a problem can have many possible causes.

3. TROUBLE SHOOTING
Troubleshooting requires identification of the malfunction(s)
or symptoms within a system. Then, experience is
commonly used to generate possible causes of the
symptoms. Determining which cause is most likely is often
a process of elimination - eliminating potential causes of a
problem. Finally, troubleshooting requires confirmation
that the solution restores the product or process to its
working state.

4. TROUBLE SHOOTING
troubleshooting is
 the identification of, or diagnosis of "trouble" in a
[system]
 a failure of System.
 symptoms of malfunction,
 process of determining the causes of these symptoms.

5. INSTRUMENTATION OF
HPLC

6. PROCESS OF
TROUBLE SHOOTING
 PRELIMIARY CHECKS
 COLUMNS AND FITTINGSLEAKS
 CHANGE IN RETENTION
 TIME
 BASELINE
 PRESSURE
 PEAKS
 EQUILIBRATION
 SENSITIVITY

7. TROUBLE SHOOTING
IN HPLC
There are many causes in which problem are occured in HPLC.
By Instrumental.
By Procedure.
By Human Errors.

8. SOURCES OF ERRORS
Because of this causes user have to face various problem such
as
 Back pressure,
 Leakage,
 Change in Retention Time,
 peak shape
 base line problem.

9. PRELIMINARY CHECKS
Problem Cause Solution
No Peaks or Very Small Peaks Detector off.
Broken
Connections to the
Recorder
Check detectors.
Check Connections.
Check Sample.
Be Sure It Is Not
Deteriorated.
Checks For Bubbles
In The Vials.
Check Attenuation,
check Gains.

10. COLUMN AND FITTINGS
LEAKS PROBLEM
Problem Causes Solution
Column End Leaks Loose Fitting. Tighten /Replace
Fitting Cut Tubing
Replace Ferrule
Disassemble Fitting
Rinse and
Reassemble.
Leak at Injection
Valve.
Worn or Scratched
Valve Rotor.
Replace Valve Rotor.

11. CHANGE IN RETENTION
TIME
Problem Causes Solutions
Increase in retention time Decreasing flow rate.
Changing mobile-phase
composition.
Loss of bonded stationary
phase.
Check and reset pump flow
rate
Cover solvent reservoirs
Use mobile-phase pH
between pH 2 and pH 8.
Decreasing Retention Times. Column overloaded with
sample.
Increasing flow rate.
Loss of bonded stationary
phase or base silica. Varying
column temperature.
Decrease sample amount or
use larger-diameter column.
Check and reset pump flow
rate.
Thermostat or insulate
column; ensure laboratory
temperature is constant.

12. BASELINE
Problems Causes Solutions
Void Time Noise Air bubbles in mobile
phase. Positive-negative -
difference in refractive
index of injection solvent
and mobile phase.
Degas or use back pressure
restrictor on detector.
Normal with many
samples; use mobile phase
as sample solvent.
Drifting baseline Negative direction
(gradient elution) -
absorbance of mobile-
phase
Wavy or undulating -
temperature changes in
room.
Use non-UV absorbing
mobile phase solvents
BASELINE Flush column
with strong solvent;
use HPLC grade solvents.
Monitor and control
changes in room
temperature;
cover refractive index
detector
keep it out of air currents.

13. PRESSURE PROBLEM:-
Problems Causes Solutions
Decreasing Pressure Insufficient flow from
pump. Leak in hydralic
lines from pump to
column. Leaking pump
check valve or seals. Pump
cavitation.
Loosen cap on mobile
phase reservoir. Tighten or
replace fittings; tighten
rotor in injection valve.
Replace or clean check
valves; replace pump seals.
Degas solvent; check for
obstruction in line from
solvent reservoir to pump;
replace inlet-line frit.
Fluctuating pressure Bubble in pump. Leaking
pump check valve or seals.
Degas solvent
Replace or clean check
valves
replace pump seals.

14. PRESSURE PROBLEM
Problems Causes Solutions
High Back Pressure. Column blokage Column
particle size too small
Microbial growth. Mp
viscosity too high. Plugged
frit in in-line filter or guard
column.
Improve sample cleanup;
use guard column;
reverse-flush column with
strong solvent to dissolve
blockage.
Increasing Pressure. Blocked flow lines.
Particulate buildup at
head of column. Water-
organic solvent systems -
buffer precipitation.
Filter sample; use 0.5
micrometer in-line filter;
disconnect and back flush
column; replace inlet frit.
decrease ionic strength or
water organic solvent
ratio.

15. PEAKS PROBLEM
Problem Causes Solutions
Broad peaks. Analytes eluted early due
to sample overload.
Injection volume too large.
Mobile-phase solvent
viscosity too high.
Dilute sample 1:10 and
reinject
use detector with no heat
exchanger in system.
Decrease solvent strength
of injection
Ghost peaks. Contamination.
Elution of analytes
retained from previous
injection.
RP-HPLC -contaminated
water.
Flush column to remove
contamination;
use HPLC-grade solvent.
use antioxidant.
Peak Doubling Blocked Frit.
Co elution of interfering
compound from previous
injection. Column
overloaded.
increase column diameter;
decrease sample amount.
Replace column,

16. PEAK PROBLEM

17. PEAKS PROBLEM
Problems Causes Solutions
Peak Fronting Channeling in column.
Column overloaded.
Replace or repack
column. Use higher-
capacity stationary phase;
increase column
diameter; decrease
sample amount
Tailing Peaks Basic solutes - silanol
interaction
 Silica-based column –
•degradation at high pH.
•degradation at high
temperature.
Use competing base such
as triethylamine
use polymeric column
Use high purity silica-
based column with low
trace-metal content

18. EQUILIBRATION PROBLEM
Problems Causes Solutions
Slow Column Equilibration
Times
Equilibration time slow for
long-chain ion pairing
reagents.
Use shorter alkyl chain
reagent.
SENSITIVITYPROBLEM
Pronlems Causes Solutions
Sensitivity. Peaks are outside Linear
range of detector.
flow lines blocked.
Injector sample loop
underfilled. .
Dilute or concentrate to
bring into linear range.
Check flow and make
sure no blockages.

19. SYSTEM SUITABILITY
PARAMETER
– System suitability tests are an integral part of gas and
liquid chromatographic methods. They are used to verify
that the chromatographic system is adequate for the
intended analysis. The tests are based on the concept that
the equipment, electronics, analytical operations and
samples to be analyzed constitute an integral system that
can be evaluated

20. SYSTEM SUITABILITY
PARAMETER