CONTENT

1. INTRODUCTION
 Locating and Correcting the Problem

 Prevention

2. ABNORMAL PRESSURE
 No Pressure Reading, No Flow

 No Pressure Reading, Flow Is Normal

 Steady High Pressure

 Steady Low Pressure

 Pressure Climbing

 Pressure Dropping to Zero

 Pressure Dropping, But Not to Zero

 Pressure Cycling
CONTENT
3. LEAKS
 Leaky Fittings

 Leaks at Pump

 Injector Leaks

 Column Leaks

 Detector Leaks

4. PROBLEMS WITH THE CHROMATOGRAM

 Peak Tailing

 Peak Fronting

 Split Peaks

 Distortion of Larger Peaks

 Distortion of Early Peaks

 Tailing, Early Peaks More Than Later Ones

 Increased Tailing as k′ Increases

 Acidic or Basic Peaks Tail

 Extra Peaks
CONTENT
 Retention Time Drifts
 Abrupt Retention Time Changes

 Baseline Drift

 Baseline Noise (Regular)

 Baseline Noise (Irregular)

 Broad peaks

 Loss of Resolution

 All Peaks Too Small

 All Peaks Too large

5. PROBLEMS WITH THE INJECTOR

 Manual Injector, Hard to Turn

 Manual Injector, Hard to Load

 Auto injector, Will Not Turn

 Auto injector, Other Problems

CONTENT
6. PROBLEMS DETECTED BY SMELL, SIGHT, OR SOUND
 Solvent Smell

 “Hot” Smell

 Abnormal Meter Readings

 Warning Lamps

 Warning Buzzers

 Squeaks and Squeals

7. KEY PROBLEM AREAS AND PREVENTIVE MAINTENANCE

 Reservoir

 Pump

 Injector

 Column

 Detector

 General
1 INTRODUCTION
Locating and Correcting the Problem
A systematic approach is best to identify any problems when
troubleshooting the high-performance liquid chromatography (HPLC)
system. This guide is organized into five major categories of symptoms to
help quickly identify the source of the problem(s).

• Pressure abnormalities

• Leaks

• Problems with the chromatogram

• Injector problems

• Other problems detected by smell, sight, and sound

When the problem has been corrected, record the incident in the HPLC
system record book to help with future problems.
1 INTRODUCTION

Prevention
Many liquid chromatography (LC) problems can be
prevented with routine maintenance. For example, replacing
pump seals at regular intervals should eliminate pump-seal
failure and its associated problems. Section 7 lists the most
common problem areas for each LC module, as well as
preventive maintenance practices to reduce their frequency.
These suggestions can be modified to fit any particular
model of LC, and should be made a regular part of the
laboratory routine.
2 ABNORMAL PRESSURE
A change in the operating pressure is a sign that there may be a
problem. Choose the category below that best fits the symptoms and
follow the suggestions to correct the problem.
A. No Pressure Reading, No Flow
Possible Cause Solution
1. Power off 1. Turn on power
2. Fuse blown 2. Replace fuse
3. Controller setting or failure 3. a. Verify proper setting
b. Repair or replace controller
4. Broken piston 4. Replace piston
5. Air trapped in pump head 5. Degas solvents: bleed air from
pump, prime pump
6. Insufficient mobile phase 6. a. Replenish reservoir
b. Replace inlet frit if blocked
7. Faulty check valve(s) 7. Replace check valve(s)

8. Major leak 8. Tighten or replace fittings

B. No Pressure Reading, Flow Is Normal

Possible Cause Solution

1. Faulty meter 1. Replace meter
2. Faulty pressure transducer 2. Replace transducer
C. Steady High Pressure

Possible Cause Solution

1. Flow rate set too high 1. Adjust setting
2. Blocked column frit 2. a. Back flush column
(if permitted)
b. Replace frit
c. Replace column
3. Improper mobile phase; precipitated 3. a. Use correct mobile phase
buffer b.Wash column
4. Improper column 4. Use proper column
5. Injector blockage 5. Clear blockage or replace injector
6. Column temperature too low 6. Raise temperature
7. Controller malfunction 7. Repair or replace controller
8. Blocked guard column 8. Remove/replace guard column
9. Blocked in-line filter 9. Remove/replace in-line filter
D. Steady Low Pressure

Possible Cause Solution

1. Flow set too low 1. Adjust flow rate
2. Leak in system 2. Locate leak and correct
3. Improper column 3. Use proper column
4. Column temperature too high 4. Lower temperature
5. Controller malfunction 5. Repair or replace controller

E. Pressure Climbing
Same as Section C
F. Pressure Dropping to Zero
Same as Section A and B
G. Pressure Dropping, But Not to Zero
Same as Section D
H. Pressure Cycling

Possible Cause Solution

1. Air in pump 1. a. Degas solvent
b. Bleed air from pump
2. Faulty check valve(s) 2. Replace check valve(s)
3. Pump seal failure 3. Replace pump seal
4. Insufficient degassing 4. a. Degas solvent
b. Change degassing methods
(use
Degases on-line degasser)
5. Leak in system 5. Locate leak and correct
6. Using gradient elution 6. Pressure cycling is normal due
to viscosity changes
3. LEAKS
Leaks are usually stopped by tightening or replacing a fitting. Be aware,
however, that over tightened metal compression fittings can leak and
plastic finger tights can wear out. If a fitting leak does not stop when
the fitting is tightened a little, take the fitting apart and inspect for
damage (e.g., distorted ferrule or particles on the sealing surface);
damaged fittings should be discarded and replaced.

A. Leaky Fittings
Possible Cause Solution
1. Loose fitting 1. Tighten
2. Stripped fitting 2. Replace
3. Over tightened fitting 3. a. Loosen and retighten
b. Replace
4. Dirty fitting 4. a. Disassemble and clean
b. Replace
5. Mismatched parts 5. Use all parts from same brand
B. Leaks at Pump

Possible Cause Solution

1. Loose check valves 1. a. Tighten check valve
(do not over tighten)
b. Replace check valve
2. Loose fittings 2. Tighten fittings
(do not over tighten)
3. Mixer seal failure 3. a. Replace mixer seal
b. Replace mixer
4. Pump seal failure 4. Repair or replace
5. Pressure transducer failure 5. Repair or replace
6. Pulse damper failure 6. Replace pulse damper
7. Proportioning valve failure 7. a. Check diaphragms,
replace if leaky
b. Check for fitting damage,
replace
8. Purge valve 8. a. Tighten valve
b. Replace purge valve
C. Injector Leaks
Possible Cause Solution
1. Rotor seal failure 1. Rebuild or replace injector
2. Blocked loop 2. Replace loop
3. Loose injection-port seal 3. Adjust
4. Improper syringe-needle 4. Use correct syringe
diameter
5. Waste-line siphoning 5. Keep waste line above surface
waste
6. Waste-line blockage 6. Replace waste line

D. Column Leaks
Possible Cause Solution
1. Loose end fitting 1. Tighten end fitting
2. Column packing in ferrule 2. Disassemble, rinse ferrule,
reassemble
3. Improper frit thickness 3. Use proper frit (see Frit
selection guide
chart)
E. Detector Leaks

Possible Cause Solution

1. Cell gasket failure 1. a. Prevent excessive backpressure
b. Replace gasket
2. Cracked cell window(s) 2. Replace window(s)
3. Leaky fittings 3. Tighten or replace
4. Blocked waste line 4. Replace waste line
5. Blocked flow cell 5. Rebuild or replace
4. PROBLEMS WITH THE CHROMATOGRAM
Many problems in an LC system show up as changes in
the chromatogram. Some of these can be solved by
changes in the equipment; however, others require
modification of the assay procedure. Selecting the
proper column type and mobile phase are keys to
“good chromatography.”
A. Peak Tailing

Possible Cause Solution

1. Blocked frit 1. a. Reverse flush column (if allowed)
b. Replace inlet frit
c. Replace column
2. Column void 2. Fill void
3. Interfering peak 3. a. Use longer column
b. Change mobile-phase and/or
column/Selectivity
4. Wrong mobile-phase pH 4. a. Adjust pH
b. For basic compounds, a lower pH
usually provides more symmetric peaks
5. Sample reacting with 5. a. Add ion pair reagent or volatile
active sites basic modifier
b. Change column
B. Peak Fronting

Possible Cause Solution

1. Low temperature 1. Increase column temperature

2. Wrong sample solvent 2. Use mobile phase for injection

solvent

3. Sample overload 3. Decrease sample concentration

4. Bad column 1. a. Reverse flush column (if

allowed)
b. Replace inlet frit
c. Replace column
Fill void
C. Split Peaks

Possible Cause Solution

1. Contamination on guard or 1. a. Remove guard column and attempt
analytical column inlet column inlet analysis
b. Replace guard if necessary
c. If analytical column is obstructed, reverse
and flush
d. If problem persists, column may be fouled
with strongly retained contaminants
e. Use appropriate restoration procedure
f. If problem persists, inlet is probably
plugged
g. Change frit or replace column
2. Sample solvent incompatible 2. Change solvent; whenever possible, inject
with mobile phase samples in mobile phase
D. Distortion of Larger Peaks

Possible Cause Solution

1. Sample overload 1. Reduce sample size

E. Distortion of Early Peaks

Possible Cause Solution
1. Wrong injection solvent 1. a. Reduce injection volume
b. Use weaker injection solvent
F. Tailing, Early Peaks More Than Later Ones

Possible Cause Solution

1. Extra-column effects 1. a. Replumb system (shorter,
narrower tubing)
b. Use smaller volume detector
cell
G. Increased Tailing as k′ Increases

Possible Cause Solution

1. Secondary retention effects, 1. a. Add triethylamine (basic samples)
reversed-phase mode b.Add acetate (acidic samples)
c. Add salt or buffer (ionic samples)
d.Try a different column
2. Secondary retention effects, 2. a. Add triethylamine (basic
normal-phase mode compounds)
b.Add acetic acid
3. Secondary retention effects, 3. Add triethylamine (basic samples)
ion-pair
H. Acidic or Basic Peaks Tail

Possible Cause Solution

1. Inadequate buffering 1. a. Use 50–100 mM buffer concentration
b. Use buffer with pKa equal to pH of
mobile phase

I. Extra Peaks
Possible Cause Solution
1. Other components in 1. Normal
sample
2. Late-eluting peak from 2. a. Increase run time or gradient slope
previous injection b. Increase flow rate
3. Vacancy or ghost peaks 3. a. Check purity of mobile phase
b.Use mobile phase as injection solvent
c. Reduce injection volume
J. Retention Time Drifts

Possible Cause Solution

1. Poor temperature control 1. Thermostat column
2. Mobile phase changing 2. Prevent change (évaporation, reaction,
etc.)
3. Poor column equilibration 3. Allow more time for column equilibration
between runs

K. Abrupt Retention Time Changes

Possible Cause Solution

1. Flow rate change 1. Reset flow rate
2. Air bubble in pump 2. Bleed air from pump
3. Improper mobile phase 3. a. Replace with proper mobile phase
b. Set proper mobile phase mixture on
controller
 L. Baseline Drift

Possible Cause Solution

1. Column temperature fluctuation (even 1. a. Control column and mobile phase
small changes cause cyclic baseline rise Temperature
and fall; most often affects refractive b.Use heat exchanger before detector
index and conductivity detectors, or
UV detectors at high sensitivity or in
direct photometric mode)
2. Nonhomogeneous mobile phase (drift 2. a. Use HPLC-grade solvents, high-purity
usually to higher absorbance, rather than salts, and additives
cyclic pattern from temperature b.Degas mobile phase before use
fluctuation) c. Sparge with helium during use
3. Contaminant or air build-up in 3. a. Flush cell with methanol or other
detector cell strong solvent
b. If necessary, clean cell with 1 N
HNO3 (never with HCl)
4. Plugged outlet line after detector 4. a. Unplug or replace line
(high-pressure cracks cell window, b.Refer to detector manual to replace
producing noisy baseline) window
Possible Cause Solution
5. Mobile-phase mixing problem or change 5. a. Correct composition/flow rate
in flow rate b. To avoid, routinely monitor
composition and flow rate
6. Slow column equilibration, especially 6. a. Flush with intermediate strength
when changing mobile phase solvent
b.Run 10–20 column volumes of new
mobile phase before analysis
7. Mobile phase contaminated, 7. a. Check make-up of mobile phase
deteriorated, or prepared from low-quality b.Use highest grade chemicals and
materials HPLC solvents
8. Strongly retained materials in sample 8. a. Use guard column
(high k) can elute as very broad peaks and b. If necessary, flush column with strong
appear to be a problem solvent between injections or periodically
during analysis
9. Mobile phase recycled but detector not 9. a. Reset baseline
adjusted b.Use new mobile phase when dynamic
range of detector is exceeded
Possible Cause Solution
10. Detector (UV) not set at absorbance 10. Change wavelength to UV
maximum but at slope of curve absorbance maximum
M. Baseline Noise (Regular)
Possible Cause Solution
1. Air in mobile phase, detector 1. a. Degas mobile phase
cell, or pump b. Flush system to remove air from detector
2. Leak 2. a. Section 3
b. Check system for loose fittings
c. Check pump for leaks, salt buildup,unusual
noises
d. Change pump seals if necessary

3. Incomplete mobile phase 3. Mix mobile phase by hand or use less

mixing viscous solvent
4. Temperature effect (column 4. Reduce differential or add head exchanger
at high temperature, detector
unheated)
5. Other electronic equipment 5. Isolate LC, detector, or recorder to determine
on same line if source of problem is external;
correct as necessary
6. Pump pulsations 6. Incorporate pulse dampener into system
N. Baseline Noise (Irregular)

Possible Cause Solution

1. Leak 1. a. Section 3
b. Check for loose fittings
c. Check pump for leaks, salt build-up, unusual
noises
d. Change seals if necessary
e. Check for detector cell leak

2. Mobile phase contaminated, 2. Check make-up of mobile phase

deteriorated, or prepared from
low-quality materials
3. Mobile phase solvents 3. Select and use only miscible solvents
immiscible
4. Detector/recorder electronics 4. a. Isolate detector and recorder electronically
b. Refer to instruction manual to correct
problem
Possible Cause Solution
5. Air trapped in system 5. Flush system with strong solvent
6. Air bubbles in detector 6. a. Purge detector
b. Install back-pressure device after
detector
7. Detector cell contaminated (even 7. Clean cell by flushing with 1 N HNO3
small amounts of contaminants can (never with HCl)
cause noise)
8. Weak detector lamp 8. Replace lamp
9. Column leaking silica or packing 9. Replace column
material
10.Mobile phase mixer inadequate or 10. Repair or replace the mixer or mix
malfunctioning off-line if isocratic
 O. Broad Peaks
Possible Cause Solution
1. Mobile-phase composition changed 1. Prepare new mobile phase
2. Mobile-phase flow rate too low 2. Adjust flow rate
3. Leaks (especially between column 3. a. Section 3
and detector) b.Check for loose fittings
c. Check pump for leaks, salt build-up,
and unusual noises
d. Change seals if necessary
4. Detector settings incorrect 4. Adjust settings
5. Extra-column effects: 5. a. Inject smaller column (e.g., 10 μl vs. 100
a. Column overloaded μl) or 1:10 and 1:100 dilutions of sample
b. Detector response time or cell b.Reduce response time or use smaller cell
volume too large c. Use as short a piece of 0.007–0.010- inch
c. Tubing between column and ID tubing as practical
detector too long or ID too large d. Reduce response time
d. Recorder response time too high

6. Buffer concentration too low 6. Increase concentration

7. Guard column contaminated/worn 7. Replace guard column
Possible Cause Solution
8. Column contaminated/worn out; low 8. a. Replace column with new one of
plate number same type
b. If new column provides symmetrical
peaks, flush old column with strong
solvent
9. Void at column inlet 9. Open inlet end and fill void or
replace column
10. Peak represents two or more poorly 10. Change column type to improve
resolved compounds separation
11.Column temperature too low 11. Increase temperature; do not exceed
75ºC unless higher temperatures are
acceptable to column manufacturer
12. Detector time constant too large 12. Use smaller time constant
P. Loss of Resolution
Possible Cause Solution
1. Mobile phase 1. Prepare new mobile phase
contaminated/deteriorated
(causing retention time to change)
2. Obstructed guard or analytical 2. a. Remove guard column and attempt
column analysis
b. Replace guard if necessary
c. If analytical column is obstructed, reverse
and flush; if problem persists, column may be
fouled with strongly retained contaminants
d. Use appropriate restoration procedure; if
problem persists, inlet is probably plugged
e. Change frit or replace column
Q. All Peaks Too Small
Possible Cause Solution
1. Detector attenuation too 1. Reduce attenuation
high
2. Detector time constant too 2. Use smaller time constant
large
3. Injection size too small 3. Use larger sample loop
4. Improper recorder 4. Use correct connection
connection

R. All Peaks Too large

Possible Cause Solution
1. Detector attenuation too 1. Use larger attenuation
low
2. Injection size too large 2. Use smaller sample loop
3. Improper recorder 3. Use correct connection
connection
5. PROBLEMS WITH THE INJECTOR
These problems are usually detected while using the injection valve. Leaky
injection valves are discussed in Section 3 (Leaks).
A. Manual Injector, Hard to Turn

Possible Cause Solution

1. Damaged rotor seal 1. Rebuild or replace valve
2. Rotor too tight 2. Adjust rotor tension

B. Manual Injector, Hard to Load

Possible Cause Solution

1. Valve misaligned 1. Adjust alignment
2. Blocked loop 2. Replace loop
3. Dirty syringe 3. Clean or replace syringe
4. Blocked lines 4. Clear or replace lines
C. Auto injector, Will Not Turn

Possible Cause Solution

1. No air pressure (or power) 1. Supply proper pressure
(power)
2. Rotor too tight 2. Adjust
3. Valve misaligned 3. Adjust alignment

D. Auto injector, Other Problems

Possible Cause Solution

1. Blockage 1. Clear or replace blocked portion
2. Jammed mechanism 2. See service manual
3. Faulty controller 3. Repair or replace controller
 6. PROBLEMS DETECTED BY SMELL, SIGHT, OR SOUND
All senses must be used to identify LC problems. Get in the habit of taking a few
minutes each day to expose all senses (except taste!) to the LC to get a “feel” for
how the LC performs normally. This will help to locate problems quickly. For
example, often a leak can be detected by smell before it is seen. The majority of
problems are identified by sight; most of these are included in the proceeding
section.

A. Solvent Smell

Possible Cause Solution

1. Leak 1. Section 3
2. Spill 2. a. Check for overflowing waste container
b. Locate spill and clean u
B. “Ho t” Smell

Possible Cause Solution

1. Overheating module 1. a. Check for proper ventilation, adjust
b.Check temperature setting, adjust
c. Shut module off, see service manual

C. Abnormal Meter Readings

Possible Cause Solution

1. Pressure abnormality 1. Section 2
2. Column oven problem 2. a. Check settings, adjust
b. See service manual
3. Detector lamp failing 3. Replace lamp
D. Warning Lamps

Possible Cause Solution

1. Pressure limit exceeded 1. a. Check for blockage

b. Check limit setting, adjust

E. Warning Buzzers

Possible Cause Solution

1. Solvent leak/spill 1. Locate and correct

F. Squeaks and Squeals

Possible Cause Solution

1. Bearing failure 1.Replace
2. Poor lubrication 2. Lubricate as necessary
7. KEY PROBLEM AREAS AND PREVENTIVE MAINTENANCE
The chart below lists the most common problems that occur with each
LC module. In the right-hand column are listed preventive
maintenance practices that can reduce the failure rate. The numbers in
parentheses are suggested intervals between maintenance. The LC’s
operator and service manuals may have additional suggestions for
preventive maintenance.

A. Reservoir
Problem Preventive Maintenance
1. Blocked inlet frit 1. a. Replace (3–6 months)
b. Filter mobile phase, 0.5
μm filter
2. Gas bubbles 2. Degas mobile phase
B. Pump

Problem Preventive Maintenance

1. Air bubbles 1. Degas mobile phase
2. Pump seal failure 2. Replace (3 months)
3. Check valve failure 3. Filter mobile phase; use inlet-line
frit; keep spare

C. Injector

Problem Preventive Maintenance

1. Rotor seal wear 1. a. Do not over tighten
b. Filter samples
D. Column
Problem Preventive Maintenance
1. Blocked frit 1. a. Filter mobile phase
b. Filter samples
2. Void at head of column 2. a. Avoid mobile phase pH >8
b.Use guard column
c. Use precolumn (saturator
column)

E. Detector
Problem Preventive Maintenance
1. Lamp failure; decreased 1. Replace (6 months) or keep
detector response; increased spare lamp
detector noise
2. Bubbles in cell 2. a. Keep cell clean
b.Use restrictor after cell
c. Degas mobile phase
F. General

Problem Preventive Maintenance

1. Corrosive/abrasive damage 1. Flush buffer from LC and clean
when not in use