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Bulletin 826E
HPLC Troubleshooting Guide
How to identify, isolate, and correct the most common HPLC problems

Although HPLC method development has been im-

proved by advances in column technology and instru-
mentation, problems still arise. In this guide we offer
you a systematic means of isolating, identifying, and
correcting many typical problems.
The important segments of an HPLC system are the
same, whether you use a modular system or a more
sophisticated unit. Problems affecting overall system
performance can arise in each component. Some com-
mon problems are discussed here. Solutions to these
problems are presented in easy-to-use tables.

E000648

CONTENTS:
Isolating HPLC Problems ................................................... 2
How to Prevent Mobile Phase Problems ............................ 2
Isolating Pump Problems .................................................. 3
Injector and Injection Solvents .......................................... 3
Column Protection ........................................................... 3
Getting the Most from Your Analytical Column ................ 3
Solving Detector Problems ................................................ 4
Column Heater, Recorder ................................................. 4
Keeping Accurate Records ................................................ 4
Problem Index .................................................................. 4
HPLC Problems, Causes and Remedies ........................ 5-13
Restoring Your Column's Performance ...................... 14-15
Preventing and Solving Common Fitting Problems .......... 16
A Selection of Column Protection Products ............... 17-20

sigma-aldrich.com
Isolating HPLC Problems To prevent bubbles in the system, degas the mobile phase. Gener-
In an HPLC system, problems can arise from many sources. First ally an in-line degasser is a first choice, but sparging with helium can
define the problem, then isolate the source. be an alternative if the mobile phase does not contain any volatile
components.
Use Table 1 (page 5) to determine which component(s) may be
causing the trouble. A process of elimination will usually enable you Use ion pair reagents carefully. The optimum chain length and
to pinpoint the specific cause and correct the problem. concentration of the reagent must be determined for each analysis.
Concentrations can be as low as 0.2 mM, or as high as 150 mM, or
more. In general, increasing the concentration or chain length
How to Prevent Mobile Phase Problems increases retention times. High concentrations (>50%) of acetoni-
Low sensitivity and rising baselines, noise, or spikes on the chro- trile or some other organic solvents can precipitate ion pair re-
matogram can often be attributed to the mobile phase. Contami- agents. Also, some salts of ion pair reagents are insoluble in water
nants in the mobile phase are especially troublesome in gradient and will precipitate. Avoid this by using sodium-containing buffers
elution. The baseline may rise, and spurious peaks can appear as the in the presence of long chain sulfonic acids (e.g., sodium dodecyl
level of the contaminated component increases. sulfate), instead of potassium-containing buffers.
Water is the most common source of contamination in reversed Volatile basic and acidic modifiers, such as triethylamine (TEA) and
phase analyses. You should use only high purity distilled or deion- trifluoracetic acid (TFA), are useful when you wish to recover a
ized water when formulating mobile phases. However, several compound for further analysis. These modifiers also let you avoid
common deionizers introduce organic contaminants into the water. problems associated with ion pair reagents. They can be added to
To remove these contaminants, pass the deionized water through the buffer at concentrations of 0.1 to 1.0% TEA or 0.01 to 0.15%
activated charcoal or a preparative C18 column. TFA. Increasing the concentration may improve peak shape for
Use only HPLC grade solvents, salts, ion pair reagents, and base and certain compounds, but can alter retention times.
acid modifiers. Cleaning lower quality solvents is time consuming, Recycling the mobile phase used for isocratic separations has
and trace levels of contaminants often remain. These trace contami- become more popular in recent years as a means of reducing the
nants can cause problems when you use a high sensitivity ultraviolet cost of solvents, their disposal, and mobile phase preparation time.
or fluorescence detector. An apparatus such as the Supelco SRS-3000 or SRS-1000 Solvent
Because many aqueous buffers promote the growth of bacteria or Recovery System uses a microprocessor controlled switching valve
algae, you should prepare these solutions fresh, and filter them to direct the solvent stream to waste when a peak is detected. When
(0.2 μm or 0.45 μm filter) before use. Filtering also will remove the baseline falls under the selected threshold, uncontaminated
particles that could produce a noisy baseline, or plug the column. solvent is directed back to the solvent reservoir.
Prevent microorganism growth by adding about 100 ppm of
sodium azide to aqueous buffers. Alternatively, these buffers may
also be mixed with 20% or more of an organic solvent such as
ethanol or acetonitrile.

Figure A. Components of an HPLC System

Injector
Solvent
Column
Return
Debubbler Oven
Back-Pressure Line
Pre-injector Regulator
Filter

Precolumn Detector
Helium
Filter
Supply Pump

Waste
Solvent Recovery
System

Column and
Integrated Guard Data System
Solvent Reservoir Column
and Inlet Filters
Pulse Damper

794-0746

2
Isolating Pump Problems Figure B. Supelguard Columns Prolong the Lifespan
The pump must deliver a constant flow of solvent to the column over of Your Analytical Columns
a wide range of conditions. Modern HPLC pumps incorpor-ate column: SUPELCOSIL LC-PCN, 25 cm x 4.6 mm I.D., 5 μm particles (with
Supelguard LC-PCN guard column) (58378)
single or dual piston, syringe, or diaphragm pump designs. mobile phase: 25:60:15, 0.01 M potassium phosphate (pH to 7 w/85%
phosphoric acid):acetronitrile:methanol
Pumping system problems are usually easy to spot and correct. flow rate: 2 mL/min.
Some of the more common symptoms are erratic retention times, temp.: 30 °C
det.: UV, 215 nm
noisy baselines, or spikes in the chromatogram. Leaks at pump injection: 100 μL reconstituted SPE eluant (20 ng/mL each analyte and
fittings or seals will result in poor chromatography. A sure sign of a int. std. in serum)
leak is a buildup of salts at a pump connection. Buffer salts should
be flushed from the system daily with fresh deionized water. To
2
isolate and repair specific problems related to your apparatus, use
Same analytical
the troubleshooting and maintenance sections of the operation Analytical column plus column after
manual. Pump seals require periodic replacement. You should Supelguard column replacing
perform regular maintenance rather than waiting for a problem to after 100 serum Supelguard column
1. Trimipramine (int. std.)
occur. extract injections 2. Doxepin 3
3. Amitriptyline 1
2 4. Imipramine
5. Desmethyldoxepin
Injector and Injection Solvents 7 6. Nortriptyline
3 4
The injector rapidly introduces the sample into the system with 7. Desipramine
8. Protriptyline (int. std.) 8
minimal disruption of the solvent flow. HPLC systems currently use
1 4 56
variable loop, fixed loop, and syringe-type injectors. These are 5 6 8 7
activated manually, pneumatically, or electrically.
7
Mechanical problems involving the injector (e.g., leaks, plugged
capillary tubing, worn seals) are easy to spot and correct. Use a pre-
column filter to prevent plugging of the column frit due to physical
degradation of the injector seal. Other problems, such as
irreproducible injections, are more difficult to solve.
0 2 4 6 8 0 2 4 6 8
Variable peak heights, split peaks, and broad peaks can be caused Min Min
794-0809, 794-0810
by incompletely filled sample loops, incompatibility of the injection
solvent with the mobile phase, or poor sample solubility. Whenever
possible, dissolve and inject samples in mobile phase. Otherwise, be Getting the Most from Your Analytical Column
sure the injection solvent is of lower eluting strength than the
Regardless of whether the column contains a bonded reversed or
mobile phase (Table 3). Be aware that some autosamplers use
normal phase, ion exchange, affinity, hydrophobic interaction, size
separate syringe washing solutions. Make sure that the wash
exclusion, or resin/silica based packing, the most common problem
solution is compatible with and weaker than the mobile phase. This
associated with analytical columns is deterioration. Symptoms of
is especially important when switching between reversed and
deterioration are poor peak shape, split peaks, shoulders, loss of
normal phase analyses.
resolution, decreased retention times, and high back pressure.
These symptoms indicate contaminants have accumulated on the
Column Protection frit or column inlet, or there are voids, channels, or a depression in
Although not an integral part of most equipment, mobile phase the packing bed.
inlet filters, pre-injector and pre-column filters, and guard columns Deterioration is more evident in higher efficiency columns. For
greatly reduce problems associated with complex separations. We example, a 3 micron packing retained by 0.5 micron frits is more
recommend that all samples be filtered through 0.45 μm or 0.2 μm susceptible to plugging than a 5 or 10 micron packing retained by
syringe filters. We strongly recommend the use of guard columns. 2 micron or larger frits. Proper column protection and sample
Filters and guard columns prevent particles and strongly retained preparation are essential to getting the most from each column.
compounds from accumulating on the analytical column. The Overloading a column can cause poor peak shapes and other
useful life of these disposable products depends on mobile phase problems. Column capacity depends on many factors, but typical
composition, sample purity, pH, etc. As these devices become values for total amounts of analytes on a column are:
contaminated or plugged with particles, pressure increases and
peaks broaden or split. As an example, Figure B presents a clear case Analytical column (25 cm x 4.6 mm) <500 μg
for the use of guard columns. For more about column protection, Semi-preparative column (25 cm x 10 mm) <100 mg
see the product pages of this guide and request Bulletin 781. Preparative column (25 cm x 21.mm) <500 mg

3
Solving Detector Problems Problem Index
Detector problems fall into two categories — electrical and me- Problem Problem No.
chanical/optical. For electrical problems, you should contact the Baseline
instrument manufacturer. Mechanical or optical problems usually drift 12
can be traced to the flow cell. Detector-related problems include noise, irregular 14
leaks, air bubbles, and cell contamination. These usually produce noise, regular 13
spikes or baseline noise on the chromatograms or low sensitivity. Column back pressure
Some cells — especially those used in refractive index detectors — higher than usual 4
are sensitive to pressure. Flow rates or back pressures that exceed lower than usual 3
the manufacturer’s recommendation will break the cell window. Ghost peaks 19
Old or defective lamps as well as incorrect detector rise time, gain,
or attenuation will reduce sensitivity and peak height. Faulty or Peak shapes, incorrect
reversed cable connections can also be the source of problems. broad 15
fronting 10
rounded 11
Column Heater, Recorder split 7
tailing 8, 9
These components seldom cause problems with the system. They
will be discussed in the troubleshooting table (Table 1). Peaks
height change 16
missing 2
Keeping Accurate Records negative 18
Most problems don’t occur overnight, but develop gradually. no peaks 1
unresolved 6
Accurate record keeping, then, is vital to detecting and solving
many problems. Retention times, variable 5
Evaluate every column you receive, when you receive it and at Selectivity change 17
regular intervals thereafter. By keeping a written history of column
efficiency, mobile phases used, lamp current, pump performance,
etc., you can monitor your system’s performance.
Records also help prevent mistakes, such as introducing water into
a silica column, or precipitating buffer in the system by adding too Trademarks
FPLC — Amersham Pharmacia Biotech
much organic solvent. Many analysts modify their HPLC systems in
Iso-Disc, Pelliguard, Sigma-Aldrich, Supelco,
some way. Reliable records are the best way to ensure that a SUPELCOSIL, Supelguard, Trizma — Sigma-Aldrich Co.
modification does not introduce problems. For problems relating to LO-Pulse — Scientific Systems, Inc.
pumps, detectors, automatic samplers, and data systems, consult Rheodyne — Rheodyne, Inc.
your instrument manual’s troubleshooting guide. Swagelok — Crawford Fitting Co.
Teflon — E.I. du Pont de Nemours & Co., Inc.

4
Table 1. HPLC Problems, Probable Causes, and Remedies
Problem Probable Cause Remedy/Comments

Problem No. 1: No Peaks/Very Small Peaks

Normal

1. Detector lamp off. 1. Turn lamp on.

2. Loose/broken wire between detector 2. Check electrical connections
and integrator or recorder. and cables.
3. No mobile phase flow. 3. See “No Flow” (Problem No. 2).
4. No sample/deteriorated 4. Be sure automatic sampler vials have
sample/ wrong sample. sufficient liquid and no air bubbles in
the sample. Evaluate system
performance with fresh standard to
confirm sample as source of problem.
5. Settings too high on detector 5. Check attenuation or gain settings.
794-0747 or recorder. Check lamp status. Auto-zero if
necessary.
Problem

794-0748

Problem

794-0749

Problem No. 2: No Flow

Normal

1. Pump off. 1. Start pump.

2. Flow interrupted/obstructed. 2. 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.
3. Leak. 3. Check system for loose fittings.
Check pump for leaks, salt buildup,
unusual noises. Change pump seals
794-0747 if necessary.
4. Air trapped in pump head. (Revealed 4. Disconnect tubing at guard column
by pressure fluctuations.) (if present) or analytical column inlet.
Check for flow. Purge pump at high
flow rate (e.g., 5-10 mL/min.), prime
Problem
system if necessary. (Prime each
pump head separately.) If system has
check valve, loosen valve to allow air
to escape. If problem persists, flush
794-0748 system with 100% methanol or
isopropanol. If problem still persists,
contact system manufacturer.

5
 Problem Probable Cause Remedy/Comments

Problem No. 3: No Pressure/Pressure Lower Than Usual

Normal 1. Leak. 1. Check system for loose fittings.

Check pump for leaks, salt buildup,
unusual noises. Change pump seals
if necessary.
2. Mobile phase flow interrupted/ 2. Check mobile phase level in
obstructed. 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.
3. Air trapped in pump head. (Revealed 3. Disconnect tubing at guard column
by pressure fluctuations.) (if present) or analytical column inlet.
Check for flow. Purge pump at high
794-0750 flow rate (e.g., 10 mL/min.), prime
system if necessary. (Prime each
pump head separately.) If system
Problem has check valve, loosen valve to
allow air to escape.
4. Leak at column inlet end fitting. 4. 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.
5. Air trapped elsewhere in system. 5. Disconnect guard and analytical
column and purge system. Recon-
nect column(s). If problem persists,
flush system with 100% methanol or
isopropanol.
6. Worn pump seal causing leaks around 6. Replace seal. If problem persists,
pump head. replace piston and seal.
7. Faulty check valve. 7. Rebuild or replace valve.
794-0751
8. Faulty pump seals. 8. Replace seals.

Problem No. 4: Pressure Higher Than Usual

1. Problem in pump, injector, in-line 1. Remove guard column and analytical

Normal filter, or tubing. 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
Cause 2. If not, isolate cause by
systematically eliminating system
components, starting with
detector, then in-line filter, and
working back to pump.
2. Obstructed guard column or analytical Replace filter in pump if present.
794-0750
column. 2. Remove guard column (if present)
and check pressure. Replace guard
column if necessary. If analytical
Problem column is obstructed, reverse and
flush the column, while disconnected
from the detector (page 14). If
problem persists, column may be
clogged with strongly retained
contaminants. Use appropriate
restoration procedure (Table 2,
page 14). If problem still persists,
change inlet frit (page 16) or replace
column.

794-0752

6
Problem Probable Cause Remedy/Comments

Problem No. 5: Variable Retention Times

1. Leak. 1. Check system for loose fittings.

Normal Check pump for leaks, salt buildup,
unusual noises. Change pump seals
if necessary.
2. Change in mobile phase composition. 2. Check make-up of mobile phase. If
(Small changes can lead to large mobile phase is machine mixed using
changes in retention times.) proportioning values, hand mix and
supply from one reservoir.
3. Air trapped in pump. (Retention times 3. Purge air from pump head or check
794-0753
increase and decrease at random valves. Change pump seals if
times.) necessary. Be sure mobile phase is
degassed.
Problem 4. Column temperature fluctuations 4. Use reliable column oven. (Note:
(especially evident in ion exchange higher column temperatures
systems). increase column efficiency. For
optimum results, heat eluant before
introducing it onto column.)
5. Column overloading. (Retention times 5. Inject smaller volume (e.g., 10 μL vs.
usually decrease as mass of solute 100 μL) or inject the same volume
injected on column exceeds column after 1:10 or 1:100 dilutions of
794-0747 capacity.) sample.
6. Sample solvent incompatible with 6. Adjust solvent. Whenever possible,
mobile phase. inject samples in mobile phase.
7. Column problem. (Not a common 7. Substitute new column of same type
cause of erratic retention. As a to confirm column as cause. Discard
column ages, retention times gradually old column if restoration procedures
Problem decrease.) fail (see page 14).

794-0754

Problem No. 6: Loss of Resolution

Normal 1. Mobile phase contaminated/deterio- 1. Prepare fresh mobile phase

rated (causing retention times and/or (page 2).
selectivity to change).
2. Obstructed guard or analytical column. 2. Remove guard column (if present)
and attempt analysis. Replace guard
column if necessary. If analytical
column is obstructed, reverse and
flush (page 14). If problem persists,
column may be clogged with
794-0755 strongly retained contaminants. Use
appropriate restoration procedure
Problem (Table 2, page 14). If problem still
persists, change inlet frit (page 16)
or replace column.

794-0756

7
 Problem Probable Cause Remedy/Comments

Problem No. 7: Split Peaks

Normal 1. Contamination on guard or analytical 1. Remove guard column (if present)

column inlet. and attempt analysis. Replace guard
column if necessary. If analytical
column is obstructed, reverse and
flush (page 14). If problem persists,
column may be clogged with strongly
retained contaminants. Use appropri-
794-0757 ate restoration procedure (Table 2,
page 14). If problem still persists, inlet
Problem
frit is probably (partially) plugged.
Change frit (page 16) or replace
column.
2. Partially blocked frit. 2. Replace frit (see above)
3. Small (uneven) void at column inlet. 3. Repack top of column with pellicular
particles of same bonded phase
functionality. Continue using the
column in reverse flow direction.
4. Sample solvent incompatible with 4. Adjust solvent. Whenever possible,
mobile phase. inject samples in mobile phase.

794-0758

Problem No. 8: Peaks Tail on Initial and Later Injections

Normal
1. Sample reacting with active sites. 1. 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 (page 2).
2. Wrong mobile phase pH. 2. Adjust pH. For basic compounds,
lower pH usually provides more
794-0747 symmetric peaks.
3. Wrong column type. 3. Try another column type (e.g.,
deactivated column for basic
Problem compounds).
4. Small (uneven) void at column inlet. 4. See Problem No. 7.
5. Wrong injection solvent. 5. Peaks can tail when sample is
injected in stronger solvent than
mobile phase. Dissolve sample in
794-0759
mobile phase.

Problem No. 9: Tailing Peaks

1. Guard or analytical column contamin- 1. Remove guard column (if present)

Normal
ated/worn out. and attempt analysis. Replace guard
column if necessary. If analytical
column is source of problem, use
appropriate restoration procedure
(Table 2, page 14). If problem
persists, replace column.
794-0747 2. Mobile phase contaminated/ 2. Check make-up of mobile phase
deteriorated. (page 2).
3. Interfering components in sample. 3. Check column performance with
Problem standards.

794-0759

8
Problem Probable Cause Remedy/Comments

Problem No. 10: Fronting Peaks

Normal 1. Column overloaded. 1. Inject smaller volume (e.g., 10 μL vs.

100 μL). Dilute the sample 1:10 or
1:100 fold in case of mass overload.
2. Sample solvent incompatible with 2. Adjust solvent. Whenever possible,
mobile phase. 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.
794-0760 3. Shoulder or gradual baseline rise 3. Increase efficiency or change
before a main peak may be another selectivity of system to improve
sample component. resolution. Try another column type
Problem if necessary (e.g., switch from
nonpolar C18 to polar cyano phase).

794-0761

Problem No. 11: Rounded Peaks

Normal 1. Detector operating outside linear 1. Reduce sample volume and/or

dynamic range. concentration.
2. Recorder gain set too low. 2. Adjust gain.
3. Column overloaded. 3. Inject smaller volume (e.g., 10 μL vs.
100 μL) or 1:10 or 1:100 dilution of
sample.
4. Sample-column interaction. 4. Change buffer strength, pH, or mobile
phase composition. If necessary,
raise column temperature or change
column type. (Analysis of solute
structure may help predict
794-0762 interaction.)
5. Detector and/or recorder time 5. Reduce settings to lowest values or
Problem constants are set too high. values at which no further improve-
ments are seen.

794-0763

9
 Problem Probable Cause Remedy/Comments

Problem No. 12: Baseline Drift

1. Column temperature fluctuation. (Even 1. Control column and mobile phase

Normal small changes cause cyclic baseline temperature, use heat exchanger
rise and fall. Most often affects before detector.
refractive index and conductivity
detectors, UV detectors at high
sensitivity or in indirect photometric
794-0748
mode.)
2. Nonhomogeneous mobile phase. (Drift 2. Use HPLC grade solvents, high
usually to higher absorbance, rather purity salts, and additives. Degas
Problem than cyclic pattern from temperature mobile phase before use, sparge
fluctuation.) with helium during use.
3. Contaminant or air buildup in detector 3. Flush cell with methanol or other
cell. strong solvent. If necessary, clean
cell with 1N HNO3 (never with HCl
and never use nitric acid with
PEEK tubing or fittings.)
794-0764 4. Unplug or replace line. Refer to
4. Plugged outlet line after detector.
(High pressure cracks cell window, detector manual to replace window.
producing noisy baseline.)
5. Mobile phase mixing problem or 5. Correct composition/flow rate. To
change in flow rate. avoid problem, routinely monitor
composition and flow rate.
6. Slow column equilibration, especially 6. Flush column with intermediate
when changing mobile phase. strength solvent, run 10-20 column
volumes of new mobile phase
through column before analysis.
7. Mobile phase contaminated, dete- 7. Check make-up of mobile phase
riorated, or not prepared from high (page 2).
quality chemicals.
8. Strongly retained materials in sample 8. Use guard column. If necessary,
(high k’) can elute as very broad flush column with strong solvent
peaks and appear to be a rising between injections or periodically
baseline. (Gradient analyses can during analysis.
aggravate problem.)
9. Detector (UV) not set at absorbance 9. Change wavelength to UV absor-
maximum but at slope of curve. bance maximum.

Problem No. 13: Baseline Noise (regular)

Normal 1. Air in mobile phase, detector cell, or 1. Degas mobile phase. Flush system to
pump. remove air from detector cell or
pump.
2. Pump pulsations. 2. Incorporate pulse damper into
system.
794-0748 3. Incomplete mobile phase mixing. 3. Mix mobile phase by hand or use
less viscous solvent.
4. Temperature effect (column at high 4. Reduce differential or add heat
temperature, detector unheated). exchanger.
Problem 5. Other electronic equipment on same 5. Isolate LC, detector, recorder to
line. determine if source of problem is
external. Correct as necessary.
6. Leak. 6. Check system for loose fittings.
794-0765 Check pump for leaks, salt buildup,
unusual noises. Change pump seals
if necessary.

10
Problem Probable Cause Remedy/Comments

Problem No. 14: Baseline Noise (irregular)

1. Leak. 1. Check system for loose fittings.

Normal Check pump for leaks, salt buildup,
unusual noises. Change pump seals
if necessary.
2. Mobile phase contaminated, deterio- 2. Check make-up of mobile phase.
794-0748 rated, or prepared from low quality (page 2).
materials.
Problem 3. Detector/recorder electronics. 3. Isolate detector and recorder
electronically. Refer to instruction
manual to correct problem.
4. Air trapped in system. 4. Flush system with strong solvent.
5. Air bubbles in detector. 5. Purge detector. Install back-
pressure regulator after detector.
794-0766 Check the instrument manual,
particularly for RI detectors (exces-
sive backpressure can cause the
flow cell to crack).
6. Detector cell contaminated. (Even 6. Clean cell.
small amounts of contaminants can
cause noise.)
7. Weak detector lamp. 7. Replace lamp.
8. Column leaking silica or packing 8. Replace column and clean system.
material.

Problem No. 15: Broad Peaks

1. Mobile phase composition changed. 1. Prepare new mobile phase.

Normal 2. Mobile phase flow rate too low. 2. Adjust flow rate.
3. Leak (especially between column and 3. Check system for loose fittings.
detector). Check pump for leaks, salt buildup,
and unusual noises. Change pump
seals if necessary.
4. Detector settings incorrect. 4. Adjust settings.
5. Extra-column effects: 5. a. Inject smaller volume (e.g., 10 μL
a. Column overloaded vs. 100 μL) or 1:10 and 1:100
dilutions of sample.
b. Detector response time or cell b. Reduce response time or use
volume too large. smaller cell.
c. Tubing between column and c. Use as short a piece of
794-0767 detector too long or I.D. too large. 0.007-0.010" I.D. tubing as
practical.
d. Recorder response time too high. d. Reduce response time.
Problem 6. Buffer concentration too low. 6. Increase concentration.
7. Replace guard column.
7. Guard column contaminated/worn out.
8. Replace column with new one of
8. Column contaminated/worn out.
same type. If new column does not
provide narrow peaks, flush old
column (Table 2, page 14), then
retest.
794-0768 9. Void at column inlet. 9. Replace column or open inlet end
and fill void (page 16).
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 75 °C unless higher
temperatures are acceptable to
column manufacturer.

11
 Problem Probable Cause Remedy/Comments

Problem No. 16: Change in Peak Height (one or more peaks)

Normal 1. One or more sample components 1. Use fresh sample or standard to

deteriorated or column activity confirm sample as source of problem.
changed. 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 (Table 2,
page 14). If performance does not
improve, discard old column.
2. Leak, especially between injection port 2. Check system for loose fittings.
794-0769 and column inlet. (Retention also would Check pump for leaks, salt buildup,
change.) unusual noises. Change pump seals
if necessary.
Problem 3. Inconsistent sample volume. 3. 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.
4. Detector or recorder setting changed. 4. Check settings.
794-0770 5. Weak detector lamp. 5. Replace lamp.
6. Contamination in detector cell. 6. Clean cell.

Problem No. 17: Change in Selectivity

Normal
1. Increase or decrease solvent ionic 1. Check make-up of mobile phase
strength, pH, or additive concentration (page 2).
(especially affects ionic solutes).
2. Column changed, new column has 2. Confirm identity of column packing.
different selectivity from that of old For reproducible analyses, use
column. same column type. Establish
whether change took place gradu-
ally. If so, bonded phase may have
stripped. Column activity may have
changed, or column may be
contaminated.
3. Sample injected in incorrect solvent or 3. Adjust solvent. Whenever possible,
794-0771
excessive amount (100-200 μL) of inject sample in mobile phase.
Problem strong solvent.
4. Column temperature change. 4. Adjust temperature. If needed, use
column oven to maintain constant
temperature.

794-0772

12
 Problem Probable Cause Remedy/Comments

Problem No. 18: Negative Peak(s)

Normal 1. Recorder leads reversed. 1. Check polarity.

2. Refractive index of solute less than 2. Use mobile phase with lower
that of mobile phase (RI detector). refractive index, or reverse recorder
leads.
3. Sample solvent and mobile phase 3. Adjust or change sample solvent.
differ greatly in composition (vacancy Dilute sample in mobile phase
peaks). whenever possible.
4. Mobile phase more absorptive than 4. a. Change polarity when using
794-0747 sample components to UV wavelength. indirect UV detection, or
b. Change UV wavelength or use
mobile phase that does not
adsorb chosen wavelength.
Problem

794-0773

Problem No. 19: Ghost Peak

Previous 1. Contamination in injector or column. 1. Flush injector between analyses (a

Sample 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.
2. Late eluting peak (usually broad) 2. a. Check sample preparation.
present in sample. b. Include (step) gradient to quickly
elute component.
794-0760

Normal

(solvent injected after sample)

794-0774
Problem

(solvent injected after sample)

794-0775

Further Recommendations
We also suggest referring to the maintenance and troubleshooting selectivity (pages 14-15), suggestions on how to prevent and solve
sections of your instrument manual. Modern HPLC systems often column hardware problems (page 16), and a selection of column
have self-diagnostic capabilities that help isolate the problem area protection products from the Supelco catalog. Please refer to our
within the instrument. For persistent problems relating to the catalog for our complete line of accessories that prolong column life
column or your particular analysis, please contact Supelco’s Technical and, in general, simplify or improve your HPLC or FPLC® analysis.
Service Department. Finally, phone us to request additional literature about our HPLC
The remaining pages in this guide include procedures for restoring and FPLC products, or use our ChromFax service for immediate
column performance following loss in resolution, retention, or access to all our free technical literature.

13
Restoring Your Column’s Performance
The following procedures should rejuvenate a column whose Note: Volumes listed in Table 2 are for 25 cm x 4.6 mm I.D. columns,
performance has deteriorated due to sample contamination. which have a column volume of 4.15 mL. When restoring a 4.6 mm
Disconnect and reverse the column. Connect it to the pump, but not I.D. column shorter or longer than 25 cm, multiply all volumes in
the detector. Follow the appropriate flushing procedure in this Table 2 by the ratio of the column length to 25 (e.g., for a 15 cm
table, using a flow rate that results in column back pressure of 1500- column: 15/25, or 0.6 times the volumes in Table 2). When restoring
4500 psi, but never higher than the maximum recommended a column of internal diameter other than 4.6 mm, multiply all
pressure in the manufacturer’s instruction manual. If you have a volumes in Table 2 by the ratio of the square of the column I.D. to
SUPELCOSIL column, analyze with the test mix and the conditions (4.6)2 (e.g., for a 3.2 mm I.D. column: (3.2)2/(4.6)2 = 10.24/21.16 =
listed on the data sheet. Efficiency, symmetry, and capacity should 0.48 times the values in Table 2).
be within 10-15% of the test sheet values. If not, repack the column
inlet (page 16) or replace the column.

Table 2. Column Restoration Procedures

Silica Column SUPELCOSIL LC-PCN Column
Flush with the following: A. To Remove Protein
1. 50 mL hexane Flush with 10 column volumes of acetonitrile:water, 50:50, containing
2. 50 mL methylene chloride 0.1% trifluoroacetic acid.
3. 50 mL 2-propanol B. To Remove TCA
4. 50 mL methanol Flush with 10 column volumes of distilled water (adjust pH to 2.5 with
5. 25 mL methylene chloride H3PO4), then with 10 column volumes each of:
6. 25 mL mobile phase
Evaluate column performance according to 1. water (to remove salts)
conditions specified by the manufacturer. 2. methanol (to remove water)
Note: See also the Silica Column Regeneration Solution listed on page 15 for 3. methanol/methylene chloride, 50:50 (a general clean-up
rejuvenating a deactivated silica column. solution)
4. methanol
If column performance still is not acceptable, prepare the mobile phase
Silica-Based Reversed Phase Column buffer at 10X the concentration used for the analysis and recycle through
(alkyl● , phenyl, or diphenyl column, the column overnight.* Reequilibrate the column with mobile phase at
SUPELCOSIL LC-PAH column) the normal buffer concentration and reevaluate.
A. Water Soluble Samples *Use caution: with some buffer types and/or concentrations a 10-fold increase in
Flush with the following: concentration can cause precipitation.
1. Flush with warm (60 °C) distilled water
2. 50 mL methanol
3. 50 mL acetonitrile Silica-Based Ion Exchange Columns
4. 25 mL methanol (strong or weak anion or cation exchange)
5. 25 mL mobile phase Most analyses involving ion exchange systems use ionic mobile
Evaluate column performance. phases. Compounds that may affect column performance are
usually insoluble or only slightly soluble in water. The following
B. Samples Not Soluble in Water procedure should be sufficient to remove these compounds.
Flush with the following:
1. 50 mL 2-propanol Flush with the following:
2. 50 mL methylene chloride 1. 50 mL hot (40-60 °) distilled water
3. 50 mL hexane 2. 50 mL methanol
4. 25 mL isopropanol 3. 50 mL acetonitrile
5. 25 mL mobile phase 4. 25 mL methylene chloride
Evaluate column performance. 5. 25 mL methanol
6. 25 mL mobile phase
Evaluate column performance.
Polar-Bonded Phase Column
(amino, cyano, or diol column or Pirkle-type chiral columns). Silica-Based Columns for RPLC of Proteins and Peptides
Follow the protocol for silica-based reversed phase columns.
For a column used in the reversed phase mode
Alternatively, make one or more 100 μL injections of
(e.g., organic solvent/aqueous buffer mobile phase), follow the
trifluoroethanol (determine the number of injections by evaluating
same cleanup procedure as for silica-based reversed phase
column performance after each injection).
columns. For a column used with nonaqueous mobile phases,
Evaluate column performance.
use the following scheme:
Flush with the following:
1. 50 mL chloroform
2. 50 mL methanol
3. 50 mL acetonitrile
4. 25 mL methylene chloride
5. 25 mL methanol
6. 25 mL mobile phase
Evaluate column performance.
●
C8, C18, etc.

14
Table 3. Properties of Organic Solvents Commonly Used in HPLC
Miscible with Water? UV Refractive Index Solvent Strength, Viscosity
Solvent Polarity Cutoff● at 20 °C ∈o (silica) at 20 °C, cP

Hexane nonpolar no 200 1.3750 0.00 0.33

Isooctane no 200 1.3910 0.01 0.50
Carbon tetrachloride no 263 1.4595 0.14 0.97
Chloroform no 245 1.4460 0.31 0.57
Methylene chloride no 235 1.4240 0.32 0.44
Tetrahydrofuran yes 215 1.4070 0.35 0.55
Diethyl ether no 215 1.3530 0.29 0.23
Acetone yes 330 1.3590 0.43 0.32
Ethyl acetate poorly 260 1.3720 0.45 0.45
Dioxane yes 215 1.4220 0.49 1.54
Acetonitrile yes 190 1.3440 0.50 0.37
2-Propanol yes 210 1.3770 0.63 2.30
Methanol yes 205 1.3290 0.73 0.60
Water yes –– 1.3328 >0.73 1.00

polar
●
Typical values

Nonbonded Silica Columns Exposed to Polar Solvent Figure C. Regeneration Solution Restores
Samples and mobile phases containing very strongly polar solvents, Silica Column Performance
such as water or alcohols, can deactivate uncoated silica HPLC column: SUPELCOSIL LC-Si, 15 cm x 4.6 mm I.D., 3 μm packing (58981)
mobile phase: 50:50, methylene chloride:methanol:water, 99.4:0.5:0.1 (Fig.
columns. This can drastically affect column performance, particu- C1) 2-propanol:water, (Fig. C2)
larly solute retention and selectivity. (Figure C2). Even prolonged 99.4:0.5:0.1, Silica Column Regeneration Solution, 4 mL/min.
for 10 min., then methylene chloride:methanol:water, 2 mL/
column flushing with a nonpolar solvent only partially restores min. for 10 min. (Fig. C3)
column performance, while wasting chemicals. flow rate: 2 mL/min.
temp.: ambient
A silica regeneration solution quickly and inexpensively restores det.: UV, 254 nm
injection: 10 μL (Fig. C3 obtained after equilibration).
silica column performance by removing trapped polar material.
Pump the solution through the affected column for 10 minutes at
a rate of 4 mL/minute, then flush with mobile phase for 10 minutes
1. Benzene
at a rate of 2 mL/minute. Evaluate column performance by using the 2. Benzanilide
test mixture for evaluating silica columns (Cat. No. 58281). Perfor- C1 – Properly Performing Column 3. Acetanilide
mance should be virtually the same as before the polar solvent was 12
introduced (Figure C3).
C2 – Same Column Exposed to
Silica Column Regeneration Solution, 200 mL 33175 Water and Alcohol
1
C3 – Same Column Treated
2 with Regeneration Solution
Column Test Mixes
Performance evaluation mixes for HPLC columns. 12
3
Well defined test mixes enable you to troubleshoot chromato-
graphic problems, optimize system efficiency, and evaluate col-
umns under conditions where their performance is understood. We
ship our test mixes in amber ampuls to prevent photodegradation,
and we include instructions for proper use and interpretation of
3 3
results.
0 2 4 6
Choose from column-specific or application-specific mixes. Refer to Min
our catalog for our extensive selection of test mixes, or call our
Technical Service Department.
0 2
Min

0 2 4 6
Min
794-0787, -0788, -0789

15
 Preventing and Solving Common Hardware Problems
Preventing Leaks
Leaks are a common problem in HPLC analyses. To minimize leaks isolated the column as the problem source, and other restorative
in your system, avoid interchanging hardware and fittings from procedures have failed, a void in the packing or a persistent
different manufacturers. Incompatible fittings can be forced to fit obstruction on the inlet frit may exist.
initially, but the separation may show problems and repeated As a last resort, open the inlet end of the column. Caution: opening
connections may eventually cause the fitting to leak. If interchang- the inlet end, and more so opening the outlet end, can permanently
ing is absolutely necessary, use appropriate adapters and check all damage the packing bed. Before opening columns, consult the
connections for leaks before proceeding. manufacturer’s literature. (Never open either end of a resin-filled
Highly concentrated salts (>0.2 M) and caustic mobile phases can column).
reduce pump seal efficiency. The lifetime of injector rotor seals also
Use the following procedure to open a column.
depends on mobile phase conditions, particularly operation at high
pH. In some cases, prolonged use of ion pair reagents has a 1. Disconnect the column from the system. To prevent the
lubricating effect on pump pistons that may produce small leaks at packing from oozing out of the column, perform
the seal. Some seals do not perform well with certain solvents. subsequent steps as quickly as possible.
Before using a pump under adverse conditions, read the instrument 2. Using a vise and wrench, or two wrenches, carefully
manufacturer’s specifications. To replace seals, refer to the mainte- remove the inlet end fitting (see Figure D). If the frit remains
nance section of the pump manual. in the fitting, dislodge it by tapping the fitting on a hard
surface. If the frit stays on the column, slide it off rather than
Unclogging the Column Frit lift it off. This will help preserve the integrity of the packing
A clogged column frit is another common HPLC problem. To bed.
minimize this problem from the start, use a precolumn filter and Modular columns may require a special tool
guard column. (e.g., Cat. No. 55216), to remove the frit cap.
To clean the inlet, first disconnect and reverse the column. Connect 3. Examine the old frit. Compression of the frit against the
it to the pump (but not to the detector), and pump solvent through stainless steel tubing will leave a ring around the edge on
at twice the standard flow rate. About 5-10 column volumes of the column side of a properly seated frit. No ring can mean
solvent should be sufficient to dislodge small amounts of particulate the ferrule is seated too near the tubing end. The resulting
material on the inlet frit. Evaluate the performance of the cleaned loose connection can leak silica or act as a mixing chamber.
column using a standard test mixture. 4. Examine the packing bed. If it is depressed or fractured,
you need a new column.
Replacing a Frit at the Column Inlet 5. Replace the frit.
Sometimes neither solvent flushing (see above) nor restoration
6. Replace the end fitting. Screw it down fingertight, then
procedures (see Table 2) restore a column’s performance. If you’ve
tighten 1/4 turn with a wrench.

Figure D. Typical HPLC Column Designs

A. Column with Conventional Endfittings

Column

1/16" Nut 1/16" Ferrule

Porous
Frit Packing 794-0785

B. Modular Column with Reusable Endfittings

Column

Endfitting 794-0786

16
 A Selection of Column Protection Products
Supelco™ Mobile Phase Filtration Apparatus Filtration Apparatus 1 (connects to 1000 mL sidearm flask)
(connect to aspiration line) Includes 250 mL glass reservoir, funnel base and stopper, clamp,
stainless steel holder and screen, 10 Teflon gaskets, 50 Nylon 66
filters (47 mm, 0.45 μm pores).
Filtration Apparatus 2 Filtration Apparatus 2 (connects to aspiration line)
Includes 250 mL glass reservoir, 34/45 tapered funnel base, 34/45
tapered 1000 mL flask and glass cap, clamp, stainless steel holder
250 mL Reservoir
and screen, 10 Teflon gaskets, 50 Nylon 66 filters (47 mm, 0.45 μm
pores).
Clamp
Description Cat. No.
Supelco Mobile Phase Filtration Apparatus
Funnel Base Filtration Apparatus 1 58061
Filtration Apparatus 2 58062-U
Replacement Glass Parts
For Filtration Apparatus 1
Reservoir, 250 mL 58063
1000 mL Flask Reservoir, 500 mL 58074
Funnel Base and Stopper 58064
For Filtration Apparatus 2
Tapered Funnel Base 58068
Tapered Flask, 1000 mL for Apparatus 2 58070-U
Cap Tapered Flask, 2000 mL for Apparatus 2 58075
Cap for Flask for Apparatus 2 58071
Replacement Filter Parts for Both Apparatus
Filter Holder and Screen, SS 58065
910-0113
Gaskets, Teflon (pk. of 10) 58066
Protect your instrument and columns by removing particles and Filter, Nylon 66, 47 mm (pk. of 50)
gases from solvents and other mobile phase components. Nylon 66 0.45 μm pores 58067
membrane filters are compatible with all solvents commonly used 0.20 μm pores 58060-U
Clamp, Spring Action 58053
in HPLC.

Supelco Mobile Phase Degassing System

● 4 Channel
● 0-5 mL/min. Flow Range
● Validation output satisfies system compliance
● Smart sensor detects leaks and communicates with
vacuum pump
● LED indicates degassing status
● Unprecedented 4 year warranty
The Supelco Mobile Phase Degassing system with its smart sensor
not only detects and alerts you to leaks, it also communicates with
the vacuum pump. If a change in vacuum is detected due to mobile
phase flow rate changes, the pump can compensate by changing its
speed. The validation output records vacuum level to satisfy system
compliance and validation requirements. The degassing system has E000923
a Teflon® AF membrane, with NO-OX™ fittings and tubing.
Description Cat. No.
Supelco Mobile Phase Degassing System 55018-U

Refer to the current Supelco catalog for

many additional products, and for prices.
17
Filters
A precolumn filter is essential for protecting HPLC columns against particulate matter which can accumulate on the column frit, leading
to split peaks and high back pressure. Sources of particles include mobile phases (especially when buffers are mixed with organic solvents),
pump and injector seals, and samples. Use a 2.0 μm frit to protect columns containing 5 μm or larger particles, or a 0.5 μm frit for columns
with particles smaller than 5 μm.

Supelco Filter Valco Precolumn Frit and

Direct-connect; protects analytical and Screen Filters3
guard columns. Our precolumn filter can be In-line installation. Efficient,
connected directly, hand-tight, into any HPLC low dead volume filters protect
column or guard column listed in our current your columns from particles
catalog, or with any other column that has without reducing column per-
Valco-compatible endfittings. PEEK cap and formance. The replaceable P000550
body, 2 μm stainless steel frit. For a metal-free 1/8" frit has 0.5 μm pores to
P000551
system, order PEEK/Teflon replacement frits protect 3 μm or 5 μm column packings, the replaceable screen has
(Cat. No. 57430-U). 2 μm pores. Choose the frit filter for higher filtration capacity (most
applications) or the screen filter for less dead volume (e.g., with
Description Cat. No.
microbore columns). Use with 1/16" O.D. tubing; 1/16" fittings
Supelco Precolumn Filter Z22732-3
included.
Frits (pk. of 5)
0.5 μm pores Z29087-4 Description Cat. No.
2 μm pore Z22733-1
Valco Precolumn Filters
PEEK/Teflon, 2 μm1 57430-U
Frit Filter 58420-U
1
Biocompatible, metal-free. Screen Filter 58279-U
Frits (pk. of 10)
0.5 μm pores 59037
2.0 μm pores 59129
Screens (pk. of 10) 58284
3
Frits and screens should not be interchanged in these filters.

P000548
Isolation
SSI High Pressure Precolumn Filter Technologies
In-line installation. The 316 stainless steel filter disc (0.5 μm pores) Precolumn Filter
is easily replaced without removing the column end fitting. Maxi- In-line installation. P000549
mum operating pressure: 15,000 psi (1054 kg/cm²). For 1/16" High capacity inlet fil-
tubing. ter minimizes dead volume and band broadening, to prevent loss of
Description Cat. No. column efficiency while protecting your column. Frit porosity: 0.5
SSI High Pressure Precolumn Filter
μm. Complete as shown.
10-32 Threads2 59269 Description Cat. No.
Waters Threads 59271
Filter Elements and Seals (pk. of 10) Isolation Technologies Precolumn Filter
0.5 μm pores 59273 with 3 mm frit (4.6 mm columns) 57675-U
2 μm pores 59272 with 1.5 mm frit (2.1 mm columns) 57676-U
2
Frits (pk. of 5)
Most HPLC fittings, except SSI and some Waters fittings, have 10-32 threads. 3 mm 57677
1.5 mm 57678
SSI High Pressure
Preinjector Filter
Upchurch Precolumn Filter
Place between the pump
In-line installation. Stainless steel
and injector to provide fi-
nal filtration for the mo- body with inert polyetherether-
ketone (PEEK) endfittings and a
bile phase. Easily replaced P000547 0.5 μm or 2 μm PEEK frit in one
316 stainless steel filter P000555
element (0.5 μm pores). Maximum operating pressure: 15,000 psi endfitting.
(105MPa). For 1/16" O.D. tubing, 10-32 threads2. Description Cat. No.

Description Cat. No. Upchurch Precolumn Filter

0.5 μm frit 55079
SSI High Pressure Preinjector Filter 59262-U 2 μm frit 55078
Replacement Filter Elements and Seals (pk. of 2) Frits (pk. of 10)
0.5 μm pores 59264 0.5 μm 55080-U
2 μm pores 59265 2 μm 55081
2
Most HPLC fittings, except SSI and some Waters fittings, have 10-32 threads.
18
Rheodyne Model 7725 and 7725i Injectors
The Rheodyne Model 7725 injector allows you to inject 1 μL-5 mL samples with accuracy and precision. The rugged, easily maintained design
offers many advanced features:
● Patented continuous flow design (see figure) – flow is uninterrupted when you switch from LOAD to INJECT
● Easy seal adjustment using pressure screw on front of injector
● Wide port angle (30 °), for easy access to fittings
Injector includes a 20 μL sample loop and is supplied with a VESPEL rotor seal that can be replaced with a Tefzel rotor seal for operation
at pH 0-14. Factory set at 5000 psi (345 bar), adjustable to 7000 psi (483 bar). Model 7725i has an internal position sensing switch.

A Model 7725 Injector Reduces Wear

Description Cat. No.
and Tear on Your Columns
Model 7725 Injector 57620-U
Model 7725i Injector 57621
1 2 From Pump Replacement Components
MBB Passage VESPEL Rotor Seal 58830-U
MBB Port Tefzel Rotor Seal 57633
6 To Column Stator Face Assembly 57634
3 Needle Port Cleaner 57635
To Valve Angle Bracket
Loop (for all metal Rheodyne valves) 57636
5
4 RheBuild Kit for 7725/7725i/7726 55049
Sample Loops (wide-angle ports; fittings included)
From 2 μL 57622
Loop 5 μL 57623
796-0423 10 μL 57624
A conventional HPLC valve momentarily interrupts flow during sample injection, 20 μL 57625
subjecting your column to repetitive pressure shocks. Rheodyne’s patented MBB (make- 50 μL 57626
before-break) design makes the new connection before breaking the old one, providing 100 μL 57627
uninterrupted flow. 200 μL 57628-U
500 μL 57629-U
1 mL 57630
2 mL 57631
5 mL 57632
Note: Use VESPEL seals to pH 10, Tefzel seals to pH 14.
Supelco is an authorized Rheodyne dealer.

Optimize SSI LO-Pulse Damper

Technologies Pump A pulse damper controls pump pulsations for a more stable baseline.
Replacement Parts The SSI LO-Pulse damper is a patented unit compatible with single
A preventative mainte- piston reciprocating HPLC pumps (Altex 110A, Eldex pumps, LDC
nance program that in- Mini-Pump VS, SSI Models 200 and 300, etc.). At pressures from
cludes routine replacement 500 psi to 6000 psi (35-420 kg/cm²), it improves precision of
of pump parts that are sub- quantitative analyses and detection limits for trace sample compo-
P000583
ject to wear will help you nents. Fittings and instructions included.
avoid costly downtime. Our extensive selection of Optimize Tech-
Description Cat. No.
nologies check valves, seals, and pistons meet or exceed pump
manufacturers' specifications. For the most up-to-date selection of Pulse Damper 58455
Pulse Damper without Cabinet 58442
pump parts, refer to the current Supelco catalog, or call our
Technical Service Department.

19
SupelcoTM Solvent Recovery Systems Installation Is Simple
SRS-3000 SRS-1000 Autoclean Valve
(option with SRS-3000)

Injector
Detector Valve in
SRS-3000/
SRS-1000
Recycle
Column
Pump
995-0148, 897-0035
Flush Mobile Waste
Solvent Phase Reservoir
Recover and reuse clean mobile phase, Reservoir Waste
dispose of only contaminated mobile phase.
● Reduce solvent purchase and disposal costs

● Save money, mobile phase preparation time,

and the environment
Supelco SRS-3000 and SRS-1000 Solvent Recovery Systems can SRS 3000 or SRS-1000
Data System Solvent Recovery System
save money and time in any isocratic analysis. A microprocessor-
1. Connect SRS-3000 or SRS-1000 unit to detector signal output (cable is included).
controlled solvent switching valve monitors detector output and
2. Connect SRS-3000 or SRS-1000 unit to mobile phase and waste reservoirs and
directs the solvent to the waste reservoir only when a peak is detected. detector (Teflon tubing is included).
When the baseline falls below the threshold you select, the uncon- 3. Set the threshold value and begin saving time and money. 795-0506
taminated solvent is directed back to the mobile phase reservoir. In a
typical isocratic analysis, 80-90% of the mobile phase is uncontami-
nated and can be recycled. Settings for threshold, detection range, Recover 80% or More of the Mobile Phase
and delay time enable you to precisely control the switching valve. Used In an Isocratic Analysis
In addition to the basic features mentioned above, the SRS-3000 3
unit offers validation output (included), an Autoclean option (see 4
Solvent Recycled
below), and storage for up to 10 method files. The validation output 1. Uracil
Solvent to Waste 2. Phenol
provides a continuous, auditable data trail of the solvent recycling 2 3. Benzaldehyde
valve position, for GMP, GLP, or ISO-9000 protocols. The valve 4. N,N-Diethyl-m-toluamide
5. Toluene
position is recorded by superimposing tick marks over a separate 6. Ethylbenzene
copy of the chromatographic signal. 1
Autoclean Valve - The SRS-3000 system also is available with a
valve that enables you to select a different solvent to flush the HPLC
5
system. The Autoclean valve is especially useful if you are using a
6
single pump with mobile phases containing buffer or other salts.
The Autoclean valve installs between the mobile phase reservoir and Threshold
the pump. It has two inlet lines, one for the mobile phase and one
for the wash solvent. The valve can be factory installed, or you can 0 1 2 3 4 5 6
order it separately and install it yourself. Min
713-0516
Economy-Priced Unit - The economically priced SRS-1000 includes
the same solvent-saving features as the SRS-3000 unit. A simpler display
and no advanced features (no validation output, Autoclean option, or Description Cat. No.
method storage memory) allow us to keep the price substantially lower. SRS-3000 Solvent Recovery System1 57431
Both Systems are Ready to Use - Both systems include a control SRS-3000 System with Autoclean1 57432
unit with switching valve, a power cord, a 2-lead signal cable Switching valve assembly for SRS-3000 unit 57435
SRS-1000 Solvent Recovery System1
(+/–), Teflon tubing and fittings, and an instruction manual. 110 VAC 506125
In addition to these components, the SRS-3000 system with the 220 VAC 506133
Autoclean valve has the wash valve, additional tubing and fittings, 1
CE approved.
a wash start cable, and a pump remote stop cable.
The SRS-3000 and SRS-1000 units meet all CE requirements. The
SRS-1000 units also meet UL and CSA requirements.

World Headquarters Order/Customer Service (800) 325-3010 • Fax (800) 325-5052 Accelerating Customers’
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High Technology and Service
©2009 Sigma-Aldrich Co. All rights reserved. SIGMA, , SAFC, , SIGMA-ALDRICH, ALDRICH, , FLUKA, , and SUPELCO, are trademarks belonging to Sigma-Aldrich Co.
and its affiliate Sigma-Aldrich Biotechnology, L.P. Sigma brand products are sold through Sigma-Aldrich, Inc. Sigma-Aldrich, Inc. warrants that its products conform to the information
contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply.
Please see reverse side of the invoice or packing slip.

T100826E
AIV