CAUSE |
REMEDY |
Detector lamp off. |
Turn lamp on. |
Loose/broken wire between detector
and integrator or recorder. |
Check electrical connections and
cables. |
No mobile phase flow. |
See “No Flow”. |
No sample/deteriorated sample/
wrong sample. |
Be sure automatic sampler vials
have sufficient liquid and no air bubbles in the sample. Evaluate system performance
with fresh standard to confirm sample as source of problem. |
Settings too high on detector or
recorder. |
Check attenuation or gain
settings. Check lamp status. Auto-zero if necessary. |
CAUSE |
REMEDY |
Pump off. |
Start pump. |
Flow interrupted/obstructed. |
Check mobile phase level in reservoir(s).
Check flow throughout system. Examine sample loop for obstruction or air
lock. Make sure mobile phase components are miscible and mobile phase is properly
degassed. |
Leak. |
Check system for loose fittings. Check
pump for leaks, salt buildup, unusual noises. Change pump seals if necessary. |
Air trapped in pump head.
(Revealed by pressure fluctuations.) |
Disconnect tubing at guard column (if
present) or analytical column inlet. Check for flow. Purge pump at high flow
rate (e.g., 5-10 mL/min.), prime system if necessary. (Prime each pump head
separately.) If system has check valve, loosen valve to allow air to escape.
If problem persists, flush system with 100% methanol or isopropanol. If
problem still persists, contact system manufacturer. |
CAUSE |
REMEDY |
Leak. |
Check system for loose fittings. Check
pump for leaks, salt buildup, unusual noises. Change pump seals if necessary. |
Mobile phase flow interrupted/ obstructed. |
Check mobile phase level in reservoir(s).
Check flow throughout system. Examine sample loop for obstruction or air lock. Make sure
mobile phase components are miscible and mobile phase is properly degassed. |
Air trapped in pump head. (Revealed
by pressure fluctuations.) |
Disconnect tubing at guard column (if
present) or analytical column inlet. Check for flow. Purge pump at high flow
rate (e.g., 10 mL/min.), prime system if necessary. (Prime each pump head
separately.) If system has check valve, loosen valve to allow air to escape. |
Leak at column inlet end fitting. |
Reconnect column and pump solvent at
double the flow rate. If pressure is still low, check for leaks at inlet
fitting or column end fitting. |
Air trapped elsewhere in system. |
Disconnect guard and analytical column
and purge system. Reconnect column(s). If problem persists, flush system with
100% methanol or isopropanol. |
Worn pump seal causing leaks
around pump head. |
Replace seal. If problem persists,
replace piston and seal. |
Faulty check valve. |
Rebuild or replace valve. |
Faulty pump seals. |
Replace seals. |
CAUSE |
REMEDY |
Problem in pump, injector, in-line
filter, or tubing. |
Remove guard column and analytical
column from system. Replace with unions and 0.010'' I.D. or larger tubing to
reconnect injector to detector. Run pump at 2-5 mL/min. If pressure is
minimal, see No Flow. If not, isolate cause by systematically eliminating
system components, starting with detector, then in-line filter, and working
back to pump. Replace filter in pump if present. |
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, while disconnected from the
detector. If problem persists, column may be clogged with strongly retained contaminants.
Use appropriate restoration procedure. If problem still persists, change
inlet frit or replace column. |
CAUSE |
REMEDY |
Leak. |
Check system for
loose fittings. Check pump for leaks, salt buildup, unusual noises. Change
pump seals if necessary. |
Change in mobile
phase composition. (Small changes can lead to large changes in retention
times.) |
Check make-up of
mobile phase. If mobile phase is machine mixed using proportioning values,
hand mix and supply from one reservoir. |
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 (Especially evident in ion exchange systems). |
Use reliable column
oven. (Note: higher column temperatures increase column efficiency. For optimum
results, heat eluant before introducing it onto column.) |
Column overloading.
(Retention times usually decrease as mass of solute injected on column
exceeds column capacity.) |
Inject smaller
volume (e.g., 10 μL vs. 100 μL) or inject the same volume after 1:10 or 1:100
dilutions of sample. |
Sample solvent
incompatible with mobile phase. |
Adjust solvent.
Whenever possible, inject samples in mobile phase. |
Column problem. (Not
a common cause of erratic retention. As a column ages, retention times gradually
decrease.) |
Substitute new
column of same type to confirm column as cause. Discard old column if
restoration procedures fail |
CAUSE |
REMEDY |
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. If problem persists, column may be clogged
with strongly retained contaminants. Use appropriate restoration procedure.
If problem still persists, change inlet frit or replace column. |
CAUSE |
REMEDY |
Contamination on guard or
analytical column inlet. |
Remove guard column (if present) and
attempt analysis. Replace guard column if necessary. If analytical column is
obstructed, reverse and flush. If problem persists, column may be clogged
with strongly retained contaminants. Use appropriate restoration procedure. If problem still persists, inlet frit
is probably (partially) plugged. Change frit or replace column. |
Partially blocked frit. |
Replace frit. |
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. |
Sample solvent incompatible with mobile
phase. |
Adjust solvent. Whenever possible,
inject samples in mobile phase. |
CAUSE |
REMEDY |
Sample reacting with active sites. |
First check column performance
with standard column test mixture. If results for test mix are good, add ion pair
reagent or competing base or acid modifier. |
Wrong mobile phase pH. |
Adjust pH. For basic compounds, lower
pH usually provides more symmetric peaks. |
Wrong column type. |
Try another column type (e.g., deactivated
column for basic compounds). |
Small (uneven) void at column
inlet. |
See Split Peaks |
Wrong injection solvent. |
Peaks can tail when sample is injected
in stronger solvent than mobile phase. Dissolve sample in mobile phase. |
CAUSE |
REMEDY |
Guard or analytical column
contaminated/worn out. |
Remove guard column (if present) and
attempt analysis. Replace guard column if necessary. If analytical column is
source of problem, use appropriate restoration procedure. If problem persists,
replace column. |
Mobile phase contaminated/ deteriorated. |
Check make-up of mobile phase |
Interfering components in sample. |
Check column performance with standards. |
CAUSE |
REMEDY |
Column overloaded. |
Inject smaller volume (e.g., 10 μL
vs. 100 μL). Dilute the sample 1:10 or 1:100 fold in case of mass overload. |
Sample solvent incompatible with mobile
phase. |
Adjust solvent. Whenever possible,
inject samples in mobile phase. Flush polar bonded phase column with 50
column volumes HPLC grade ethyl acetate at 2-3 times the standard flow rate,
then with intermediate polarity solvent prior to analysis. |
Shoulder or gradual baseline rise before
a main peak may be another sample component. |
Increase efficiency or change selectivity
of system to improve resolution. Try another column type if necessary (e.g.,
switch from nonpolar C18 to polar cyano phase). |
CAUSE |
REMEDY |
Detector operating outside linear dynamic
range. |
Reduce sample volume and/or concentration. |
Recorder gain set too low. |
Adjust gain. |
Column overloaded. |
Inject smaller volume (e.g., 10 μL
vs. 100 μL) or 1:10 or 1:100 dilution of sample. |
Sample-column interaction. |
Change buffer strength, pH, or
mobile phase composition. If necessary, raise column temperature or change column
type. (Analysis of solute structure may help predict interaction.) |
Detector and/or recorder time constants
are set too high. |
Reduce settings to lowest values
or values at which no further improvements are seen. |
CAUSE |
REMEDY |
Column temperature fluctuation.
(Even small changes cause cyclic baseline rise and fall. Most often affects refractive
index and conductivity detectors, UV detectors at high sensitivity or in
indirect photometric mode.) |
Control column and mobile phase temperature,
use heat exchanger before detector. |
Nonhomogeneous mobile phase.
(Drift usually to higher absorbance, rather than cyclic pattern from
temperature fluctuation.) |
Use HPLC grade solvents, high purity
salts, and additives. Degas mobile phase before use, sparge with helium during
use. |
Contaminant or air buildup in
detector cell. |
Flush cell with methanol or other strong
solvent. If necessary, clean cell with 1N HNO3 (never with HCl and never use
nitric acid with PEEK tubing or fittings.) |
Plugged outlet line after
detector. (High pressure cracks cell window, producing noisy baseline.) |
Unplug or replace line. Refer to detector
manual to replace window. |
Mobile phase mixing problem or change
in flow rate. |
Correct composition/flow rate. To avoid
problem, routinely monitor composition and flow rate. |
Slow column equilibration,
especially when changing mobile phase. |
Flush column with intermediate strength
solvent, run 10-20 column volumes of new mobile phase through column before
analysis. |
Mobile phase contaminated,
deteriorated, or not prepared from high quality
chemicals. |
Check make-up of mobile phase |
Strongly retained materials in
sample (high k’) can elute as very broad peaks and appear to be a rising baseline.
(Gradient analyses can aggravate problem.) |
Use guard column. If necessary, flush
column with strong solvent between injections or periodically during
analysis. |
Detector (UV) not set at
absorbance maximum but at slope of curve. |
Change wavelength to UV absorbance
maximum. |
CAUSE |
REMEDY |
Air in mobile phase, detector
cell, or pump. |
Degas mobile phase. Flush system
to remove air from detector cell or pump. |
Pump pulsations. |
Incorporate pulse damper into system. |
Incomplete mobile phase mixing. |
Mix mobile phase by hand or use less
viscous solvent. |
Temperature effect (column at high
temperature, detector unheated). |
Reduce differential or add heat exchanger. |
Other electronic equipment on same
line. |
Isolate LC, detector, recorder to determine
if source of problem is external. Correct as necessary. |
Leak. |
Check system for loose fittings. Check
pump for leaks, salt buildup, unusual noises. Change pump seals if necessary. |
CAUSE |
REMEDY |
Leak. |
Check system for loose fittings. Check
pump for leaks, salt buildup, unusual noises. Change pump seals if necessary. |
Mobile phase contaminated,
deteriorated, or prepared from low quality materials. |
Check make-up of mobile phase. |
Detector/recorder electronics. |
Isolate detector and recorder electronically.
Refer to instruction manual to correct problem. |
Air trapped in system. |
Flush system with strong solvent. |
Air bubbles in detector. |
Purge detector. Install
backpressure regulator after detector. Check the instrument manual, particularly
for RI detectors (excessive backpressure can cause the flow cell to crack). |
Detector cell contaminated. (Even small
amounts of contaminants can cause noise.) |
Clean cell. |
Weak detector lamp. |
Replace lamp. |
Column leaking silica or packing material. |
Replace column and clean system. |
CAUSE |
REMEDY |
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. Check
pump for leaks, salt buildup, and unusual noises. Change pump seals if
necessary. |
Detector settings incorrect. |
Adjust settings. |
Extra-column effects: |
|
Column overloaded |
Inject smaller volume (e.g., 10 μL
vs. 100 μL) or 1:10 and 1:100 dilutions of sample. |
Detector response time or cell volume
too large. |
Reduce response time or use smaller
cell. |
Tubing between column and detector
too long or I.D. too large. |
Use as short a piece of 0.007-0.010"
I.D. tubing as practical. |
Recorder response time too high. |
Reduce response time. |
Buffer concentration too low. |
Increase concentration. |
Guard column contaminated/worn
out. |
Replace guard column. |
Column contaminated/worn out. |
Replace column with new one of same
type. If new column does not provide narrow peaks, flush old column, then retest. |
Void at column inlet. |
Replace column or open inlet end and
fill void. |
Peak represents two or more poorly
resolved compounds. |
Change column type to improve separation. |
Column temperature too low. |
Increase temperature. Do not exceed
75°C unless higher temperatures are acceptable to column manufacturer. |
CAUSE |
REMEDY |
One or more sample components deteriorated
or column activity changed. |
Use fresh sample or standard to confirm
sample as source of problem. If some or all peaks are still smaller than
expected, replace column. If new column improves analysis, try to restore the old column, following appropriate
procedure. If performance does not improve, discard old column. |
Leak, especially between injection
port and column inlet. (Retention also would change.) |
Check system for loose fittings. Check
pump for leaks, salt buildup, unusual noises. Change pump seals if necessary. |
Inconsistent sample volume. |
Be sure samples are consistent.
For fixed volume sample loop, use 2-3 times loop volume to ensure loop is completely
filled. Be sure automatic sampler vials contain sufficient sample and no air
bubbles. Check syringe-type injectors for air. In systems with wash or
flushing step, be sure wash solution does not precipitate sample components. |
Detector or recorder setting
changed. |
Check settings. |
Weak detector lamp. |
Replace lamp. |
Contamination in detector cell. |
Clean cell. |
CAUSE |
REMEDY |
Increase or decrease solvent ionic
strength, pH, or additive concentration (especially affects ionic solutes). |
Check make-up of mobile phase |
Column changed; new column has different
selectivity from that of old column. |
Confirm identity of column
packing. For reproducible analyses, use same column type. Establish whether
change took place gradually. If so, bonded phase may have stripped.
Column activity may have changed, or column may be contaminated. |
Sample injected in incorrect
solvent or excessive amount (100-200 μL) of strong solvent. |
Adjust solvent. Whenever possible,
inject sample in mobile phase. |
Column temperature change. |
Adjust temperature. If needed, use
column oven to maintain constant temperature. |
CAUSE |
REMEDY |
Recorder leads reversed. |
Check polarity. |
Refractive index of solute less
than that of mobile phase (RI detector). |
Use mobile phase with lower refractive
index, or reverse recorder leads. |
Sample solvent and mobile phase differ
greatly in composition (vacancy peaks). |
Adjust or change sample solvent. Dilute
sample in mobile phase whenever possible. |
Mobile phase more absorptive than sample
components to UV wavelength. |
Change polarity when using indirect
UV detection, or |
Change UV wavelength or use mobile
phase that does not adsorb chosen wavelength. |
CAUSE |
REMEDY |
Contamination in injector or
column. |
Flush injector between analyses (a
good routine practice). If necessary, run strong solvent through column to remove
late eluters. Include final wash step in gradient analyses, to remove
strongly retained compounds. |
Late eluting peak (usually broad) present
in sample. |
Check sample preparation. |
Include (step) gradient to quickly
elute component. |
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