HPLC Troubleshooting Guide

Using This Guide Table of Contents

This guide was organized to provide both a systematic approach and a component approach to
Gilson HPLC Components 1
assist you with identifying and solving common HPLC problems. The techniques included in this
guide are specific to Gilson HPLC systems, allowing you to better understand and optimize your
Gilson system in the laboratory. Troubleshooting Getting Started 3
General Guidelines 3
Purpose: Checklist 4
Assists you with quick identification of common problems
Provides a list of potential solutions to problems Isolating & Identifying Problems 5
Software 5
Benefits: Hardware 7
Helps you learn more about your Gilson system System 10
Outlines systematic troubleshooting approaches Training 21
References quick techniques and tips in a convenient booklet Computer 22
Laboratory/Facility 22
As you work with your Gilson HPLC system, use this guide as a reference tool. This guide is
intended to be comprehensive with respect to many different Gilson HPLC system configurations;
however, if you are unable to find the specific answer to your question, Gilsons Customer Service Understanding Carryover 23
Department is available for assistance. How Solvent Selection Affects Carryover 23
How Optimizing Rinse & Injection Procedures Reduce Carryover 23
Gilson Customer Service Department How Probe Selection Affects Carryover 25
800-445-7661 How Plumbing Affects Carryover 29
608-836-1551
service@gilson.com
Probe & Transfer Tubing Selection 31
There is an extensive education and training program available from our Gilson Knowledge
Center. Our program offers complimentary Instrument TechTips and web seminars, along with Troubleshooting Carryover 33
additional options for personalized web-based training, local hands-on workshops, and on-site
training. Contact our Gilson Knowledge Center for additional Information. Troubleshooting Hardware Components 35
Pumps 35
Gilson Knowledge Center Valves 36
training@gilson.com Detectors 37
Automatic Liquid Handling/Injectors 39
Fraction Collectors 40

Putting it All Together 43

Appendix
Appendix A: PEEK Tubing Pressure vs. Flow Rate 44
Gilson, Inc. World Headquarters Appendix B: Column Sizes and Capacitites 45
PO Box 620027 | Middleton, WI 53562-0027, USA Appendix C: Tubing: Internal Diameters & Volumes 46
Tel: 800-445-7661 | Fax: 608-831-4451 Appendix D: Pressure Conversion Chart 47
sales@gilson.com | service@gilson.com | training@gilson.com | oem@gilson.com Appendix E: Solvent Polarity Chart 48

Gilson S.A.S.
19, avenue des Entrepreneurs | BP 145, F-95400 VILLIERS LE BEL, FRANCE
Tel: 33-(0)1-34295000 | Fax: 33-(0)1-34295020

IIi 1ii
 Gilson HPLC Components
Pump
GILSON HPLC COMPONENTS

It creates the mobile phase flow to carry the sample from the injection
loop into the system flow path, and it creates the gradient profile used in
the application.
321 PUMP

Liquid Handler/Injector
It performs a number of pre-injection techniques according to the software
method (i.e., filtering, dilution, etc.) prior to transferring sample
to the injection valve and into the path of the column.

Valves
They can be used in many different applications:
to select solvents for rinsing, dilutions, etc.
to switch between columns (from 2 columns to 6 or more)
VALVEMATE II
VALVE ACTUATOR

to switch between 2 columns with regeneration or backflushing

to switch between multiple fraction collectors
many more applications

Detector
Chosen for the analyte or compound of interest, the signal is brought
into the software package and is shown as a chromatogram. Often
the signal is used to perform peak freaction collection. Many different

Example Gilson HPLC Purification System and Flow Path:

types of detectors can be utilized, both analog and digital to display UV/VIS-151

absorbance, scattered light, spectrum, fluorescence, mass, etc.

Fraction Collector
It isolates compounds from the column eluent by collecting from the
detector outlet tubing into fraction tubes or vessels for the purpose of
purification. The fraction collector can be a separate device in the HPLC
system or integrated into the function of the Liquid Handler/Injector via
a 3-way valve.

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TROUBLESHOOTING-GETTING STARTED
Troubleshooting - Getting Started HPLC Troubleshooting Checklist
General Guidelines
Determine what the source of problem(s) is/are by identifying potential areas contributing to the
problem.
Take a moment to observe your system at the time of the problem to record some
initial findings before moving on.
The checklist on the next page will assist you.
When you are ready to try solutions, try one solution at a time not several.
It maybe more time consuming, but the real problem will be uncovered.
Uncovering the true problem will save you time by knowing what it is and developing
a plan on how to prevent it from happening again.
Check each part of the system independently.
Verify general function of each component to identify problem areas.
Start verification with the pumping system and work through the flow path to identify
each component is operating optimally.
Perform a blank injection.
Visually watch this injection to verify proper operation of each hardware component.
Verify the data signal shows normal baseline noise and contains no significant
compound absorbance.
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Potential Problems
1. Is there a software error message on the computer screen?
If yes, capture a screenshot/image of the error message (simultaneously press
the Alt and Print Screen keyboard buttons to capture the image, then paste it
into a document file).
Notes:
2. Does any hardware system component show an error or show a flashing red error
light? If yes, reference the hardware user guide for a listing of error messages
and common error numbers/codes.
If yes, record in the notes below the error number displayed, the system
component, and the serial number
Notes:
3. Are there any visual signs of the problems, such as a system leak, broken test
tubes, or a bent probe?
If yes, record in the notes below what was observed.
Notes:
4. Is there a notable change in the chromatography?
If yes, record the change and when the change occured.
Notes:
5. Record any additional observations or remarks in the notes field.
Notes:
ISOLATING & IDENTIFYING PROBLEMS
Isolating & Identifying Problems
Software
In general, software that is programmed to run your HPLC system can be
a potential source of problems, and with a few easy troubleshooting tips,
you can isolate the potential source to help identify where the problem
exists.
Error Messages & Log File
Gilsons Trilution LC Software has the capability to run many Methods
within a single Application. Each time a Method runs, an entry into the log
file is created, documenting the time and action of each component in the
Gilson HPLC system.
Access the log file from the Project Library screen. Double-click to open the Results folder within
the Application. Then, right click on the run name, and select View Log.
The log file can be viewed or saved as a .txt file. Use the Edit/Find option to search for the word
error. Any errors in the log will be highlighted by time/date of occurrence.
A large part of troubleshooting software errors is understanding when the error occurs in the
course of a running Method and then predicting its reoccurrence in subsequent runs for the same
Method. The log file will indicate the Method and what task in the Method was running when the
error occurred. Many times the error message will point to a simple mistake within the Method;
however, not every error recorded is directly related to software. Some hardware errors are also
recorded in the log file.
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Software Troubleshooting Tips Checklist:
Software Troubleshooting Tips Checklist
1. Note the specific Trilution LC Method that produced the error.
2. Locate when in the Method the error occured and was recorded from the log file.
3. Open the Method in question.
Review the time of each task and compare it to the recommended Method
Skeleton document (found on the Quick Start Tutorial CD-ROM that is
shipped with the software installation CDs).
Tasks should be separated no less than 0.02 minutes apart prior to
data collection.
Tasks should be separated no less than 0.1 minutes apart after data
collection.
Home tasks and all liquid handling tasks (i.e., dilute, rinse, etc.)
should have a coordinating synchronize task to the end of the liquid
handling task.
Injection tasks should follow the recommendations in the Method
Skeleton document.
Verify the bed layout contains the appropriate elements (i.e., riser blocks,
racks, etc.) .
Verify the configuration is using the correct information for each
configured instrument.
4. Make any necessary changes to the Method, and save these changes.
5. Try to reproduce the error by re-running the Method in question after removing
the probe from the liquid handler (be aware that liquid may come from the
transfer tubing onto the bed of the liquid handler with the probe is removed).
6. If the same error is reproduced even with Method changes, the problem was not
identified. Re-open the method to re-evaluate.
7. If a new error occurs as a result of Method changes, you may be closer to
identifying the problem. Evaluate where the error occurs and re-open the method
to re-evaluate.
8. If evaluation of the system and the Method has proven a hardware error occurred,
it is possible there was a software error that caused the hardware to error. Prior to
moving to the hardware section of this guide, first review the next section for
Common Software Errors to Identify.
 Common Software Method Errors to Identify:
Problem Potential Cause
Missing synchronizes to home
and liquid handling tasks.
Tasks scheduled too close
together (not according to
the timelines suggested in the
Probe Bending Method Skeleton document).
Bed Layout uses incorrect
elements.
Task uses a z-depth that is
Z-Target Error on too low for the liquid handler
Liquid Handler configuration to reach (current
probe and clamp).
1. Verify all Gilson components with GSIOC communication properly scan in through Gilsons
GSIOC Utility. There should be a GSIOC Utility icon on your computer desktop. Double-click
to open or go to Start/All Programs/Gilson Applications/Utilities.
Solution
Verify that all home and liquid handling tasks have a
coordinating synchronize to end of task.
Gilson GSIOC Utility
Space tasks at minimum:
0.02 min apart prior to data collection
0.1 min apart after data collection
All GSIOC components will display in the Unit ID box when the scan is complete. Verify this list
Verify that riser blocks and rack risers have been
against your Gilson HPLC GSIOC system components.
added to the bed layout according to the placement
on the liquid handler. Verify riser is/is not present If there is a missing component, verify the proper GSIOC cabling in the system. GSIOC cables
by right-clicking on an element and selecting Hide. should be connected with the long 9-pin female connector attached to the short 9-pin male
Enlarge the screen to display the underlying riser (if
connector on the next GSIOC cable. The short 9-pin female connector (shown as the left-most
present). Right-click again and select Show All.
connector in the image below) should be connected to the Gilson instrument. All GSIOC
components should be daisy-chained to one another. If the system has an interface module, the
For configurations using a 125 mm probe and a clamp
first connection to the computer will be either a 508 Interface Module or 506C Interface Module
height > 175 mm, set the z-depth at 20 mm or higher
via an RS-232 cable. Close Gilsons GSIOC Utility.
from the bottom of the vessel/well and test.

Fraction
Collection did Follow recommendations in Method Skeleton
not begin Incorrect order of tasks in document to have the Fraction Collection Settings
No Fractions Method. task or Set Fraction Site/Set Multiple Fraction Sites
508
Collected task at the correct time in the Method. INTERFACE MODULE

Message
RS-232 GSIOC POWER

Hardware
Hardware problems can result from software programming
mistakes, general installation plumbing or cabling
mistakes, improper preventive maintenance, or
equipment malfunction requiring service/repair. Isolating
and identifying the source of the problem is done by
systematically eliminating a series of common system
2. Verify all Gilson components with Ethernet communication properly scan
in through Gilsons Ethernet Utility. There should be an Ethernet Utility
icon on your computer desktop. Double-click to open or go to Start/ Gilson Ethernet Utility
All Programs/Gilson Applications/Utilities.
333 PUMP
Verify that all Ethernet components appear in the list. To verify communication, highlight
UV/VIS-151

334 PUMP
the component and press the Connect button. When communication is active, the icon
checks. When the system checks have been eliminated as next to the component name will change and all column information will appear. Verify this
the source of the problem, often the true source of the problem is evident. for all components.

Communication & Cabling

Gilsons HPLC systems communicate via the Trilution software package, with system components
being uniquely identified in the system. If there is an error message indicating a system
component is not found within Trilution, you will first want to verify that the system components
are properly powered on. Each component has an indicator light for power.

After power has been verified to each component, communication can once again be verified.
Close Trilution software. Note: Verification of the communication mode for each component can be
done using the appropriate Gilson user guide.

78 89
 If there is a missing component, verify the proper Ethernet cabling in the system with
the router. All Ethernet components should be directly connected to the router. The
computer should also be connected to the router.
If the Address column displays information other than an IP address of 192.XXX.X.XXX,
then power off the Ethernet components. Close Gilsons Ethernet Utility. Wait 5 full
minutes, and then power the Ethernet components. Open Gilsons Ethernet Utility,
and reconnect the Ethernet components. If this sequence of events does not allow
communication with all Ethernet components, you may also need to reset the router
via a small button located next to the power connection on the router.
Close Gilsons Ethernet Utility.
Misalignment/Offset
Visual observation of the system indicates if there is a misalignment or offset that needs to be
corrected on the Gilson liquid handler:
Probe is bent
Further inspection of the probe shows the tip is not completely straight or free of burrs
Rack is askew on the platform
Inspection of the injection port shows the probe has hit off-center
Each of Gilsons liquid handler user guides details the process and programs used to properly
align or offset to correct any X, Y positioning issues. If you do not have the tools indicated in the
user guide, you may want to call a Gilson Service Representative to assist with this process.
Component Malfunction
Gilson hardware products are reliable and rugged, but due to the general nature of hardware
over time, there may be a malfunction that occurs within the life of the product. The key to
understanding when there is a malfunction is very important, and every Gilson hardware product
will provide clues on the potential problem. The clues each Gilson hardware product provides is
directly related to knowing answers to the questions on the next page.
The information you capture and record in the checklist is very important to have when
contacting a Gilson Service Representative. Your call will be the next step in order to fully identify
and work on the best solution to the situation.
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10
Yes No Identifying Hardware Component Problems Checklist
1. Was there an error? When did the error occur? Note: reference the software
section to access the log file.
2. After turning it off, does it power on?
3. Is there an error immediately upon powering it up?
4. Does it communicate through Ethernet Utility or GSIOC Utility?
5. Will it respond to a command using the Ethernet Utility or GSIOC Utility without
an error? Note: reference the user guide GSIOC section to send commands using
the GSIOC Utility; reference the Ethernet Utility command list within the software to
send commands to Ethernet components.
6. Will it run through the same Trilution method without an error?
Maintenance
A routine maintenance program on Gilsons hardware systems is intended to keep your laboratory
operational without needing to keep track of instrument maintenance. Gilson, Inc. has programs
to optimize instrumentation life and optimize performance.
The purpose of a preventive maintenance program is to schedule and replace wearable
instrument parts just prior to the end of their life cycle, allowing Gilson instrumentation to
continue running optimally and without unscheduled disruption to your laboratory operations.
Depending on your Gilson HPLC system components and rate of use, maintenance is scheduled
every six months to one year.
Two other options are available to assist you with maintenance on your Gilson system:
Extended warranties lengthen the maintenance period of your Gilson system from the
initial purchase.
Rebuilt exchange may be available for some system components, allowing a rebuilt
item to be available at a reduced cost when service is required.
Contact Gilsons Customer Service Department or your local Gilson representative for additional
information on Gilsons maintenance options.
System
System problems can result from flow path and plumbing
mistakes, pressure issues, and column chromatography
problems. Isolating and identifying system problems can be
accomplished by understanding the flow path and plumbing
333 PUMP
on the system, running some basic system checks, and
UV/VIS-151 334 PUMP
understanding some basic column chromatography problems
that are inherent to HPLC.
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 Flow Path & Plumbing Connections System Initialization

Gilsons HPLC systems are installed for optimal performance in the laboratory. Verifying that
the system is still optimal for the purposes of injection or injection and fraction collection is an
important step. In addition, verifying that there havent been any changes within the system
is also important to note. By visually tracing through the flow path in front of the Gilson HPLC
system, any potential areas where tubing is disconnected, crimped, or in disrepair can be easily
identified.
Proper system care prior to operation is referred to as system initialization. This process ensures
that the system is free of air, that all liquid lines are properly filled for accurate transfer, that the
system fluid lines are put under pressure at the operating flow rate.
The Trilution software package should be used to initialize the system. This can be accomplished
through a dedicated Method or through Manual Control.

Verification of the pumping system Initialization considerations:

Pump shows no red error light or indication of error
Inlet tubing is free of air and is free of crimps Homing Liquid Handler and Pump
Mobile phase bottles have sufficient liquid Verifies that instrument is over a designated rinse location in order to eliminate
Mobile phase is clear and inlet filter in mobile phase container is any remaining volume in the syringe or solvent pump
free of residue
321 PUMP

Pump heads are not leaking Priming

Outlet tubing is properly connected to outlet filter
Verification of the liquid handler and injector
Injector shows no indication of error
Liquid handler shows no error code and no indication
of error
Verify LED is lit on GX Solvent System
Injection valve is not leaking (area around injection
valve has no residual sample/solvent that has dried)
Injection valve plumbing is in tact and tubing is free of
crimps
Injection port is free of liquid and free of damage from probe; remove probe and
verify probe is undamaged and tip is free of burrs
Transfer tubing is clean and free of air
Syringe piston seal is clean and syringe is free of particulates
Reservoir transfer tubing is clean and reservoir solvent is clear with inlet filter free
of residue
Verification of other valves in system
Valve(s) shows no indication of error
Injection valve plumbing is in tact and tubing is free of crimps
Verification of Detector
Detector shows no red error light or indication of error
Plumbing through column is in tact and tubing is free of
crimps
Flow cell area is not leaking
Aspirates and dispenses reservoir solvent to fill the transfer tubing from the
syringe or solvent pump to the probe
Rinsing
Inside Rinsing-Cleaning of the inside of the probe
Outside Rinsing-Cleaning of the outside of the probe
Injection Port Rinsing-Cleaning of one or more injection port locations on the
liquid handler bed
Mobile Phase Flow Rate Ramping
Ramping of the flow from the system mobile phase pumps over a certain period
of time until final flow rate is achieved (prevents pressure shock to the column)
Pressure
Both low and high pressure situations are
inherent in any HPLC system, and proper
monitoring and reaction of these conditions
VALVEMATE II
VALVE ACTUATOR will eliminate severe problems when
operating samples. All Gilson HPLC systems
should have error handling conditions built
into the Trilution Method. Be cautious when establishing low pressure error handling conditions
within a Start Method or Stop Method as a result of flow rates not being at their normal operation
rate within these methods.
Low pressure conditions should be set for a pressure level below the normal operating column
UV/VIS-151

pressure level, as recommended by the column manufacturer; a level that would indicate the
Outlet tubing is connected to a union and not a back system is not functioning properly. The pressure level that error handling should trigger at should
pressure regulator for flows > 10 mL/min be >20 psi or equivalent.

Verification of Fraction Collector

Collection tubing is extending about 2 mm below the foot Note: in both cases, the Error Method linking to the low pressure and high pressure
Tubing connected from detector outlet union to the fraction levels should be a simple Method designed to turn off the flow or lower the flow
collection valve is long enough for the fraction collector arm significantly to the mobile phase pumps.
to freely move across the full bed space
Waste tubing is of a larger diameter than the collection
tubing

11
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13
 High pressure conditions should be set for a pressure level that is above the normal operating
column pressure level, as recommended by the column manufacturer; a level that would indicate
the system is not functioning properly AND a level that is safe from tubing failure.
Fraction Collection/Purification
Verification of fraction collection delay volume and optimizing fraction collection are two
common procedures implemented to ensure that compound recovery for your application
is optimal. Gilsons expertise provides guidance on each of these procedures. There are many
simple methods, including visual verification that will accomplish the same verification of delay
volume. Gilson allows for a wide selection of fraction collection modes that can optimize fraction
collection. Reference Gilsons Trilution LC Technical Notes Handbook series for full details on these
procedures (see the back cover of this guide).
Fraction Collection Delay Volume
Verifying that the compound of interest collects into the fraction tube according to the Trilution
LC fraction tracking is part of standard Gilson installation. Post installation, any changing of
hardware components, tubing, flow rate, fraction collection peak width, and detector mode may
all impact how fraction collection is performed. Running a simple visual test can quickly and
effectively verify proper fraction collection.
Example: Acridine Orange, hydrochloride hydrate, 98%, Sigma-Aldrich
Fraction collection at 200 mL/min with Gilson 215 Preparative LC System
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Optimizing Fraction Collection
There are many modes available for isolating the compound(s) of interest. Optimal isolation or
purification is performed when a combination of the following occurs:
Collect as much compound as possible in the least amount of column eluent as
possible
Collect into the fewest number of fraction tubes per injection as possible
Collect the desired compound(s) into separate tubes
Collect to recover as much of the eluting compound as possible
Collect from as many complimentary data or detector sources for the compound of
interest as possible (see image below of complimentary fraction collection using UV
detection - blue trace - and prepELS detection - orange trace).
Chromatography
Chromatography can vary based on the type of analysis being performed;
analytical, semi-preparative, and preparative. Depending on the sample
load, the type of column used, the solubility of the compound(s) in solution,
sample matrix effect, etc. you may experience HPLC issues visible in the
chromatography over the course of time. Changes in chromatography during
routine analysis or purification can cost valuable time in the laboratory, and troubleshooting the
most common chromatography problems can be challenging even for the most experienced
chemist. In this section, we discuss four very common problems visible in chromatography and
discuss potential areas to troubleshoot and investigate.
Some of the most common problems visible in chromatography are:
1. Shifting retention time
2. No peaks
3. Reduced peak size
4. Poor peak shape
15
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 Use the examples below to assist you with understanding common solutions to visible Shifting Retention Times Reference:
chromatography problems. Within each example is a checklist that you can use to assist you with
the troubleshooting process. In general, chromatography can be a very helpful troubleshooting
tool because it displays potential problems when the chromatography changes, allowing us to Potential Causes Potential Solution
visibly troubleshoot solutions. The key to finding the solution is often in our understanding of
how and where to begin troubleshooting. Can the time of the planned temperature change be
Temperature in the laboratory may impact
modified in the thermostat?
the chromatography at the same time each
1. Shifting Retention Times day. Verify time retention time shifts for a daily
If the temperature shift varies and is not predictable, then
pattern. Correlate this to a planned thermostat
try using a column heater or water jacket to stabilize the
temperature change in the laboratory.
Retention times of consecutive injections should be fairly consistent for samples with similar chromatography elution.
concentrations and injections onto the same column using the same mobile phase gradient. Measure the flow rate with a setting of 100% gradient on
Some variation is acceptable, but why do significant retention time shifts occur from injection Verify the flow rate has not changed and is
each pump head; testing each pump head separately.
to injection? What is the cause of these shifts in order to reduce or eliminate their occurrence? Also check the flow rate with a setting of 50% on each
accurate for your Method.
pump head. If the flow rate is not accurate, preventive
Please see the reference table on the next page. maintenance may be required.
A new column may need additional time to equilibrate
Verify if the retention time shift is the result of
with mobile phase and injections of reference standard.
using a new column, a new lot of column, or a
new manufacturer. Verify if the same column lot
Column performance varies from lot to lot. Try another
or manufacturer was used for prior analysis.
column with the same lot as the original lot.
Mobile phase that requires preparation (i.e. pH
Prepare new mobile phase and allow sufficient
adjustment, modifiers) may need additonal time
equilibration time prior to injecting 3-5 standards for
for adequate mixing before use.
retention time observation.
Verify the correct mobile phase is being used on
Correlate the solvents to the Trilution LC software Method.
pump A and on pump B.
Perform a full preventive maintenance on each pump
and pump head. Verify proper replacement of the inlet
and outlet check valve, piston seal (appropriate type for
Proper and regular preventive maintenance on
3.25 3.50 mobile phase pumps is essential to accurate
mobile phase solvent used), bellows (if required), and
outlet filter. Also check if inlet filters need to be replaced.
Minutes flow rates and proper pump operation.
Verify mobile flow rate on each pump head is constant
and consistent at the gradient flow rate used in the
software Method.

Trilution LC software Methods require linking Compare injection Method with Method Skeleton
of synchronize tasks to injection tasks. These document to verify there is a synchronize task prior to
synchronizes allow for efficient Method data collection and after data collection (if doing fraction
operation and reduction in retention time shifts. collection).

15
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16
 2. No Peaks No Peaks Reference:

Injections of sample or standard with known concentrations of compounds should display

chromatographic peaks where expected over the course of the injection. When an injection Potential Causes Potential Solution
shows no visible chromatographic peaks, it may be a combination of potential problems. These
can be both chemistry-related and hardware component related. The next page provides a quick Correlate the solvents to the Trilution LC software Method. Switching
Check all system solvent lines,
solvent lines can cause compounds to stay retained on the HPLC
reference table to help you identify where to identify potential problem areas on your Gilson including mobile phase and
column.
system. reservoir for adequate volume.
Check the mobile phase solvents on
If the solvents were switched on pump A and pump B, start pumping
both pump A and pump B to make
using the correct solvents on each pump and perform a series of blank
certain the correct solvent is used
or solvent injections to elute the compound retained on the HPLC
-10.0 on each pump.
column before continuing with further analysis of samples.
-12.0
Verify that the correct Select the Bed Layout tab in the Application Run or Sample List to
mVolts

-14.0
Trilution LC software Method and verify the sample well number location on the rack. Compare the
-16.0 sample well number was used in Method name for that sample in the Sample List to verify the correct
-18.0 the Application Run or Sample List. one was run.
-20.0
Capped vials containing septa may produce a vacuum effect if a
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Confirm that the probe is not grooved probe is not used to pierce the septa, causing variation in the
Minutes
clogged and that sample was aspiration volume used for injection.
aspirated from the vial. Pick up
the vial and verify that sample was Fill a vial with blank or solvent and perform an injection. Watch the
3. Reduced Peak Size removed from the vial. HPLC system during the injection to confirm sample is aspirated and
properly injected onto the system.

Two consecutive injections of the same sample were overlapped in this example to show the Fill a vial with blank or solvent and perform an injection. Watch the
HPLC system during the injection to confirm sample is aspirated and
reduction in peak size for two compunds. Known concentrations of standard and replicate
properly injected onto the system.
injections of the same sample should generate similar responses in chromatographic peak height.
Wide variances in peak size could be related to a single hardware problem. Please see page 19 for If using direct injection with an injection port, verify that sample is not
an excellent reference table to help your identify the potential problem on your Gilson system. Verify that sample was injected bubbling over the top of the injection port. Improper sample injection
onto the system. Two types of may be the result of a worn injection port seal or crimped waste tubing
injections can be made onto Gilson on the injection valve. Disconnect the waste tubing and perform
systems direct injection with another injection and/or verify the preventive maintenance on the
an injection port and z-injection injection port seal.
1.0
without an injection port (sample
0.0 pulled into loop via the probe). If using z-injection without an injection port, verify that sample is
mVolts

-1.0 pulled completely into the sample loop prior to the valve switch.
-2.0 Improper pull volumes used to inject sample will cause underfilling
or overfilling of the sample loop showing reduced chromatographic
-3.0
response. Calculate the pull volume for your HPLC system and/or
-4.0 contact your Gilson Customer Service Representative for assistance.
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Minutes Verify that the lamp is lit on the detector, verify the detector has no
Detector lamps have a maximum
flashing error lights, verify the preventive maintenance on the detector
number of hours that they can be
for the last lamp change, and verify that the proper wavelength was
used before requiring replacement.
used for the Trilution LC software Method.

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 Reduced Peak Size Reference:
Potential Causes
Verify that the correct Trilution LC software
Method and sample well number was
used in the Application Run or Sample List.
Confirm that sample was aspirated from
the vial. Pick up the vial and verify that
sample was removed from the vial.
Samples with particulates that require
filtration may have difficulty during
injection if filtering is not performed first.
Confirm that there was enough of the
sample in the vial to aspirate the amount
of volume required for injection.
Verify that sample was injected onto
the system. Two types of injections can
be made onto Gilson systems direct
injection with an injection port and
z-injection without an injection port
(sample aspirated into loop via the probe).
Detector lamps have a maximum number
of hours that they can be used before
requiring replacement.
19
20
Potential Solution
Select the bed layout tab in the Application Run or Sample List
to verify the sample well number location on the rack. Compare
the Method name for that sample in the Sample List to verify the
correct one was run.
Capped vials containing septa may produce a vacuum effect if a
grooved probe is not used to pierce the septa, causing variation
in the aspiration volume used for injection.
Confirm that sample in vial is clear or has been filtered prior to
injection.
Fill a vial with blank or solvent and perform an injection with the
same volume as the sample injection volume. Watch the HPLC
system during the injection to confirm sample is aspirated and
properly injected onto the system.
Fill a vial with blank or solvent and perform an injection. Watch
the HPLC system during the injection to confirm sample is
aspirated and properly injected onto the system.
If using direct injection with an injection port, verify that sample
is not bubbling over the top of the injection port. Improper
sample injection may be the result of a worn injection port seal
or crimped waste tubing on the injection valve. Also, verify the
waste line is above the waste port for a short distance to prevent
laminar flow. Disconnect the waste tubing and perform another
injection and/or verify the preventive maintenance on the
injection port seal.
If using z-injection without an injection port, verify that sample is
pulled completely into the sample loop prior to the valve switch
and at reasonable flow rates. Improper pull volumes used to
inject sample will cause underfilling or overfilling of the sample
loop showing reduced chromatographic response. Calculate the
pull volume for your HPLC system and/or contact your Gilson
Customer Service Representative for assistance.
Verify that the lamp is lit on the detector, verify the detector has
no flashing error lights, verify the preventive maintenance on
the detector for the last lamp change, and verify that the proper
wavelength was used for the Trilution LC software Method.
4. Poor Peak Shape
Generally speaking, poor peak shape is related to problems of chemistry; sample solubility,
column overloading, or column life. Below are some simple tips to improving peak shape for
subsequent injections onto your HPLC system.
80.0
PN:2
60.0
mVolts
40.0 PN:1
20.0
0.0
0.0 1.0 2.0 3.0 4.0 5.0 6.0
Minutes
Poor Peak Shape Tips
Sample solvent, rinse solvent, and mobile phase solvents should be
compatible. Solubility of sample with system solvents assures that efficient
liquid handling with minimal carryover is achieved.
Peak fronting may be caused by using the wrong sample solvent. Make certain
that the mobile phase, sample solvent, and reservoir solvent are similar in
chemistry. Sample overload may be causing the fronting appearance. Check
the load specifications listed for the HPLC column being used. Verify that
overloading is not causing the undesirable peak shape.
Peak tailing is commonly seen, indicating column-sample interaction issues.
Interfering peaks may be a source of tailing, and modifying the gradient
chemistry or mobile phase solvent may separate the peaks. Changing the
column chemistry may also eliminate the tailing. Tailing may also be reduced
by using a mobile phase additive, such as triethylamine (TEA) and/or by
changing the pH.
Column life may vary with the number of injections, the column lot, the type of
column packing, the routine maintenance and cleaning, etc. Column cleaning
can be an efficient means of extending column life. Column manufacturers
may have recommendations for column cleaning procedures. Integrating
column cleaning into your Gilson HPLC system is a simple and efficient way to
automate this process during analysis.
Use a back pressure regulator on the detector outlet tubing under analytical
flow rate conditions. Out gassing of mobile phase solvents may cause irregular
peak shape.
20
21
 Training
Sometimes HPLC problems are a direct result of not knowing how to run the system most
efficiently, how to make simple modifications to software Methods or how to optimize the use
of your system in the laboratory. Training can often solve these problems and answer additional
questions users have on their Gilson HPLC system.
Gilsons Knowledge Center offers a wide variety of complimentary training options, web-
based training options, and workshop/classroom training options. In addition, a basic system
familiarization is provided at system install with your Gilson representative. To inquire about
Gilsons education and training opportunities:
Visit our website: http://www.gilson.com to access our full education and training program
within our automated instrumentation section.
Tel: 608-836-1551 or 800-445-7661
training@gilson.com
3000 Parmenter Street
Middleton, WI 53562
Daily Use of System
General understanding of your Gilson HPLC system
and how it is programmed to operate can lead to its
efficient use in your laboratory for a few users or for
many users. A short list of five key topics is outlined
below. These topics are essential for every user to
understand for optimal daily use of the system.
Learning these basic topics doesnt take much time -
just a few short hours with a Gilson representative or with additional training.
1. Running samples using a pre-defined Sample List
2. Creating/Modifying a Sample List
3. Using the Sample List buttons to override the running Method
4. Operating the system using Manual Control
5. Basic Navigation within Trilution LC locating Methods and Chromatographic Data
Method Creation & Optimization
Users who are interested in optimizing their Gilson system will find additional training an efficient
means of learning how to adjust Trilution LC Methods. With an understanding of how your current
Methods operate, easy creation of additional Methods from these will allow you a chance to
optimize your system for current and future analysis. By focusing time on what you would like to
do in your application, training can focus on the tasks and capabilities within the Method.
Understanding how to use variables efficiently, how to create flexible mobile phase gradients and
flexible fraction collection, how to optimize fraction collection these are all topics that can assist
you to more efficiently use your Gilson HPLC system for your current and future applications.
21
22
Computer
Eliminating potential sources of HPLC problems also means taking a look at the computer
operating Gilsons Trilution LC Software. Verifying that the specifications match with
recommendations, verifying setup of the user rights/permissions on the computer, etc. are all
routine checks that can be done when problems related to system operation are in question.
Specifications
Computer specifications are listed for each version of Trilution LC software in the Installation
Guide within the Trilution LC media ( i.e. multi-CD software case). Specifications are also listed on
Gilsons public website to ensure optimal performance of the operating software.
Access
Installation of Trilution LC Software is documented in the
Installation Guide. Installation does require administrator
computer access. Separate user access levels can be set
within Trilution LC to limit software access to specific
functions based on group definition.
Automatic Updates
It is recommended that automatic updates be turned off
on computers operating Trilution LC Software. Disrupting
the collection of chromatographic data or operation of the
Gilson system with automatic operating system updates is not recommended.
Rebooting
Regular rebooting of the computer running Trilution LC Software is recommended on a 24-hour
basis to restore the computer RAM and achieve optimal daily performance.
Maintenance
Regular maintenance of Trilution LC Software is recommended for version 1.4 using the supplied
Trilution LC 1.4 Backup Utility. Maintenance of subsequent versions is accomplished by using the
Archive function within the Results screen. Use of the Archive function is described in the Help
menu within Trilution LC and on the Quick Start Tutorial CD-ROM by viewing the Import Export
document.
Laboratory/Facility
It is possible that some HPLC problems are related to issues within the laboratory or facility. While
this guide is not intended to solve these problems, it is recommended to at least understand that
problems could result from laboratory or facility conditions the Gilson HPLC is operating within.
23
22
 Some of these problems can be identified by verifying: Including a bi-directional flow-through rinse for the probe can reduce carryover significantly more
UNDERSTANDING CARRYOVER

than other standard rinse procedures. The standard rinse used in Gilsons Trilution LC Software has
Power the probe rinse from the reservoir, which uses flow in only one direction. There are hardware and
software options available to perform a bi-directional rinse using a flow-through rinse station or
Power surges may occur causing intermittent communication with hardware components. external rinse pump.

Temperature/Humidity Rinsing with multiple solvents is another technique that can reduce carryover. The solvents
selected must be miscible and must not produce any reactions that will result in salt precipitation.
Large shifts may cause chromatography problems. Rinsing with two solvents requires 402 Dual Syringe Pump, or a low pressure VALVEMATE II
can be used to increase solvent selection option for rinsing. Please see Figure 1A and Figure
Waste Collection 1B. Alternatively, using a solvent system on a GX-281 or GX-271 Liquid Handler allows for easy
selection (in Trilution LC) of a different port for rinsing with a different solvent.
Verify that all waste fluid lines from the HPLC system run vertically to an appropriate collection
If highly concentrated samples are injected, the injection port may become contaminated,
container.
requiring extensive rinsing to clean the port. To clean a contaminated injection port, move the
probe to the rinse station, and repeat your standard rinse at least 10 times. Then move the probe
Areas where there is horizontal collection within the waste fluid lines cause irregular waste
to the injection port, and rinse the injection port 10 times, raising and lowering the probe after
collection and potential backups.
each rinse. It also may be necessary to rinse the injection port manually with another solvent to
completely clear the contamination.
System Cart
The following additional steps can be added to the injection procedure to further reduce
Enough space should be available to allow for proper waste collection and access to the HPLC carryover:
system.
After a sample is aspirated from the vial, dip the probe into a tube containing solvent or a flowing
The system cart should be level and be capable of accommodating the weight of the HPLC rinse station to remove sample from the outside of the probe. The outside of the probe is now
system components. clean and will not contaminate the injection port seal. This step requires only a few seconds and
can reduce carryover significantly in some applications.

FIGURE 1A
Understanding Carryover Dual-Solvent Hardware for Extra-Solvent Rinsing

How Solvent Selection Affects Carryover

Sample type, sample matrix and sample concentration all contribute to carryover. Select a sample
solvent in which the sample and matrix are readily soluble at the concentration you will be using.
Samples that are readily soluble are easily rinsed from the injection valve, injection probe and
tubing.
Tee connecting
If the sample concentration you are using is minimally soluble, extensive rinses of the probe, solvent lines to probe
transfer tubing, injection port, and injection valve are necessary to reduce carryover.

How Optimizing Rinse and Injection Procedures Reduces Carryover

Although the sample matrix cannot be changed, the rinse solvent can be optimized for solubility.
Rinse solvents must be compatible with the system mobile phase and should not affect or impact Solvent Solvent
chromatographic separation. For mass spectrometers, the rinse solvent cannot interfere with 1 2
ionization. Optimizing your system injection and rinse procedures, as described in the following
paragraphs, will help reduce the carryover in your system.

After the injection port has been rinsed, move the probe to the rinse station (optimally a flowing
rinse station) and rinse the probe by aspirating and dispensing 75% of the syringe or transfer
tubing volume three times at a speed of 10 mL/min.

23
24 24
25
 FIGURE 1B
0.1051
Low Pressure VALVEMATE II for Extra-Solvent Rinsing

MeOH blank 1
0.0929

Injection Following 980 ppb Anthracene Spike Injection

0.0345

0.0222

0.0206

0.0180
27067374
 (constricted,
beveled tip)
0.0253
27067373
 (constricted tip)

MeOH blank 2
0.0224
2507234
 (side-entry;
0.0063
septum-piercing tip)
0.0074
27067383
 (septum-
402402
Syringe
Syringe 0.0082 piercing, beveled tip)
Pump inlet
Pump inlet
0.0086 2507248
 (micro-volume;
conical tip)

0.0130 2507252
 (micro-volume;
septum-piercing,

MeOH blank 3
0.0063
constricted, beveled tip)
0.0000

How Probe Selection Affects Carryover 0.0046

0.0053
The internal and external diameter of the probe affects the amount of sample that adheres to
0.0059
the inside and outside of the probe after the sample is dispensed. Using probes with constant
internal diameters helps minimize the amount of sample that adheres to the inside of the probe.
Select a probe with the smallest possible internal and external diameter that is made from a

0.0000

0.0200

0.0400

0.0600

0.0800

0.1000

0.1200
material that does not have an affinity for your sample or sample matrix. Probes with constricted
tips can increase carryover under certain conditions. (See Figure 2 below.)
Average % Carryover

FIGURE 2 TABLE 1
Carryover Study Employing Various Probe Types
Constricted-Tip Probes
Area where test material can cling;
bi-directional rinsing can help
Sample
Using a bi-directional rinse may reduce carryover caused by using a constricted-tip probe. The
bi-directional rinse requires: aspirating and dispensing from a solvent-containing vessel on the
Flow bed, using a flow-through rinse station, or using an external rinse pump. Both procedures may
use the TRILUTION LC liquid handling tasks in combination with synchronize tasks. An example of an
injection procedure using rinse vessels is described.

Sample being pushed through probe,

and the affects of laminar flow

26
25 27
26
 Rinse Vial Procedure
1. Move arm to rinse vial location. FIGURE 3 FIGURE 4
Sychronize to end of task. Constricted-Tip Probe in Injection Port Injection Port Probe Heights
2. Move arm to proper rinse vial
z-height. Sychronize to end of task.
3. Aspirate 500 L of rinse vial solvent
at 6 mL/min. Sychronize to end of
task.
4. Move arm to proper rinse vial
z-height. Sychronize to end of task.
5. Dispense 500 L of rinse vial solvent
at 6 mL/min. Sychronize to end of
task.

Optimizing probe selection and probe depth

can reduce carryover. Select a probe that
provides the least amount of dead volume
for the injection port being used. Injection 3 mm total
ports are designed to seal around the largest port height Extra dead
diameter of the probe. Thus constricted-tip volume
probes produce more dead volume than
probes with a constant OD. See Figure 3.

A probe inserted at the incorrect height in

About 1 mm from bottom
the injection port can also increase carryover.
The correct probe height is 1 mm above the
bottom of the injection port. The total space
beneath the injection port seal to the bottom
of the injection port. For example, the total
space on a Gilson 819 Injection Module is
approximately 3 mm. A probe inserted just
below the seal (approximately 3 mm above
the bottom of the injection port) increases the
potential for carryover. The seal on sealed vials
wipes the probe when it is removed from the
vial, thereby decreasing carryover caused by
sample adhering to the outside of the probe.
Dipping the probe in solvent before moving to
the injection port can also help remove sample
that may be adhering to the outside of the Additional dead volume around
tip of constricted-tip probe
probe. See Figure 4.

If time is taken in choosing a probe that is

suitable for the application, and if injection parameters are optimized, carryover caused by the
probe can be reduced to a negligible level.

27
28 28
29
 How Plumbing Affects Carryover
Sample also comes in contact with the transfer
tubing, which can potentially cause carryover.
Transfer tubing selection should be optimized
for the sample volumes being injected. (See
page 31 for transfer tubing recommendations.)
The further sample moves through the
transfer tubing, the greater the chance for
contamination. Sample should never travel
completely through the transfer tubing into
the syringe or solvent system. Once sample
has entered the syringe it is very difficult to
rinse out. Additionally, leaking syringes can
increase carryover and should be replaced.
Plumbing connections into the injection port
and injection valve have a significant influence
on carryover. Ferrules that are not seated
to the correct depth on the tubing will add
dead space and voids at the injection valve
and injection port connections which can
significantly influence carryover. Ferrules that
are not correctly seated onto the tubing from
the injection loop can increase carryover by a
factor of 10. Calibrated injection port tubing
(if required for the Gilson system) must also
be seated correctly to keep carryover to a
minimum. Please see Figures 5 and 6.
29
30
To reduce carryover caused by incorrectly
FIGURE 5 seated fittings, you need to first remove FIGURE 6
the nut and ferrule. If you are using
Calibrated Injection Port Tubing Incorrectly Seated Fittings
stainless tubing, you will need to remove
approximately 0.75 inches (19 mm) of
tubing. If you are using PEEK tubing,
remove approximately 0.25 in. (6 mm) of
tubing. When installing new ferrules, leave
approximately 0.16 in. (4 mm) of tubing
exposed from the end of the ferrule. Push
the tubing into the back of the valve port
while screwing in the nut. Care must be taken
to assure that all ferrules are seated to the
proper depth. See Figure 7.
The position of the waste lines out of the
injection valve can also influence carryover.
Make sure that the outlet of the waste tubing
is lower than the waste port of the injection
Dead volume
valve. Avoid any unnecessary bends in the
tubing. Be sure that the waste tubing is
positioned at a continuous downward slope.
Maintenance of the injection valve also
affects carryover. Old and worn rotor seals
may not leak, but they do increase the risk FIGURE 7
of carryover and higher % RSD. Worn or Correctly Seated Fittings
scratched rotor seals can increase carryover
Void; ferrule is by a factor of 10. Select a rotor seal made of
not properly a material that has no-to-minimal affinity for
seated on tubing your sample (PEEK, Tefzel and Vespel). This
may require some experimentation.
Carryover is not caused by one problem
alone. Observed carryover may be caused by
the sample, probe, injection port, plumbing
connections, or rinse solvent selection.
Negatively altering these conditions may
seem insignificant, but doing so can increase
carryover to unacceptable levels. Detailed
attention to all of the conditions described
here will help to reduce carryover. If your
carryover is greater than two percent, No dead volume
there is something significantly wrong with
your hardware or injection procedures.
Carryover does not have to be eliminated to ensure successful injection and analysis. However,
carryover must be reduced to the point where it does not interfere with the assay or is below the
minimum detection limit.
30
31
PROBE & TRANSFTER TUBING SELECTION
Carryover ranging from 0.1% to 0.01% is reasonable and should be achievable in your routine,
daily operations. With added precautions, carryover of less than 0.005% can be achieved. These FIGURE 8
added precautions may slightly increase time per injection, but the increase in time will not be Probe Types
significant if your chromatography separation time is greater than two minutes. Tip Design Description Features
Flat The tip is cut perpendicular to the length of Aspirates the last drop of liquid
This section does not address carryover caused by sample affinity for probes, tubing, injection the probe. Does not pierce septum
port seals or injector rotor seals. This phenomenon will require changing the material of one or No injection

more of your hardware components. The solution to reducing carryover is whatever works best
for the sample you are using.

Probe & Transfer Tubing Selection Constricted The tip of the probe has a smaller outer
diameter than the rest of the shaft.
Compatible with injection ports

Constricted-tip probes accommodate unique

Transfer Tubing Selection tip designs optimizing every application.

Choosing the correct volume of transfer tubing is based on the syringe size when using a Gilson
402 Syringe Pump. Choose transfer tubing that is at least as large as the syringe to prevent
Beveled The tip is cut at an angle, leaving a bevel. Sharp tip enables septum-piercing
sample from entering the syringe. Compatible with injection ports

When using a solvent system, Choose a volume of transfer tubing that is close to the largest
volume to be regularly aspirated and dispensed.

Spring Loaded Low volume, touch-off dispensing. Ideal for MALDI spotting applications
Gilson Probe Product Number Identification Spring loaded probe removes any potential of
damaging spotting surface
Gilson probes are laser engraved with their product number on the collar of each probe
providing a quick and effortless identification reference for re-ordering and support.

Vented Two styles: Sharp-tip enables septum-piercing

1. A shallow groove cut in the probe shaft, Vent allows pressures inside and outside
Probes above the tip (shown).
2. Two probes are assembled concentrically,
a sealed vessel to equilibrate as liquid is
dispensed or aspirated
creating two separate channels, the inner
Gilson offers many different types of probes, probe tips and coatings to meet any and all channel for liquid and the outer channel
application needs providing solutions for all liquid handling requirements. See Figure 8 for more for gas.
Vented probes may have different tip designs.
details.
Side-entry The liquid flow path opens on the side of the Recommended for thick septa
probe, not at the tip. Prevents coring of septa
Compatible with injection ports
Not recommended with limited volume

Deflected Based on the beveled tip design, the probe tip Sharp-tip enables septum-piercing
is curved and very sharp. Tip pushes septa out of the way as it
pierces to prevent coring

Conical Designed for PEEK injection ports. Capable of piercing thin septa
Aspirates the last drop of liquid

32
31 32
33
 The injection rinse prcedure needs
Troubleshooting Carryover refinement and is not optimized. FIGURE 9
Dip the probe in solvent before
Injection Port Probe Height
1. Is carryover greater than 0.02 %? moving to the injection port. Increase
the probe rinse volume and the
The sample is not soluble enough in the rinse solvent. dispense speed. Try rinsing with a
TROUBLESHOOTING

Switch to a solvent in which the sample is readily soluble, or try using a dual-solvent second solvent using a 402 Syringe
rinse. (See Figure 1) Pump or VALVEMATE II solvent
selector. See Figure 1B
The sample has affinity for the proble tubing injection port or rotor seal.
If possible try switching to a Vespel Tefzel or PEEK rotor seal. However, if the system has 3. Is carryover greater than 5%?
been working with this sample, dont change the rotor seal type.
The Probe is plugged.
The injector rotor seal may be scratched or damaged. Unplug or replace the probe.
When in doubt, replace the rotor seal.
The calibrated tubing and/or
Ferrules may not be seated correctly in the injection valve. injection valve are plugged, or the
The tubing connections into the injection valve from the sample loop and from fittings are not seated correctly.
the injection valve to the column must be perfect. If unsure, remove and replace all Unplug or replace the calibrated
fittings. Push the tubing to the back of the port. Once the tubing is positioned, slide tubing and/or unplug the injection
the ferrule and nut into the injection port while holding the tubing firmly against the valve. Refer to the instructions on Probe manually
back of the port. (See Figures 57) how to properly seat fittings. lowered to
bottom of port Mark Z-arm height
The probe height into the injection port may not be set properly. The injection port seal is scrached or at either position
The probe height into the injection port must be approximately 1.0 mm (0.5 mm) not adjusted correctly.
above the bottom of the injection port. To check the probe height in the injection Remove and reinstall or replace the
port, power OFF the instrument, then manually lower the probe to the bottom of the injection port seal.
injection port. Mark the position of the probe holder in relation to the Z-arm. Power
the instrument ON, then move the probe to the injection port using the TRILUTION LC The probe height in the injection port Probe sent to injection port by
software. Is the position of the probe holder about 1 mm above your mark? Adjust the is too high. software (probe approximately
clamp height if it is not, or use the offset software utility for Gilson GX-271 and GX-281 The probe height in the injection port 1 mm above bottom of port)
liquid handlers . (See Figure 9) must be approximately 1.0 mm (0.5
mm) above the bottom of the injection
2. Is carryover greater than 1%? port. Adjust the probe to the correct
height as previously described.
The sample may not be, but must be soluble in the rinse solvent.
Sample is not (and must be) soluble
The sample may have, but cannot have affinity for the probe, transfer tubing, in the rinse solvent.
injection port or rotor seal.
The syringe pump valve is not
The probe ID may be too large for the volume of sample being aspirated. switching properly.
Use a probe with a smaller ID. Contact Gilson Customer Service.
The probe has constricted tip (i.e., sample is not being properly rinsed). The syringe probe connection is
Try replacing the probe with a more appropriate probe. (See Figure 8) loose, or the syringe is leaking.
The rotor seal may be scratched. Check and tighten all plumbing
When in doubt, change the rotor seal. However, if the system has been working in the connections (if necessary). Replace
past with this sample, dont change the rotor seal. any leaking syringes.

The probe height into the injecton port must be about 1.0mm (0.5 mm) above the
bottom of the injection port.
Adjust the probe to the correct height as previously described.
The tubing connections into the injection valve from the sample loop and from the
injector to the column must be perfect.
If not, re-seat them as previously described.
System fluids have become contaminated.
Replace all solvents, and flush the system.
The injection rinse prcedure needs refinement and is not optimized.
Dip the probe in solvent before moving to the injection port. Increase the probe rinse
volume and the dispense speed. Try rinsing with a second solvent using a 402 Syringe
Pump or VALVEMATE II solvent selector.

34
33 34
35
TROUBLESHOOTING HARDWARE COMPONENTS

4. Do you want carryover to be less than 0.01%? Can you accurately measure Valves
this?
VALVEMATE II
VALVE ACTUATOR

You may need to dip the probe into a flowing rinse before and after moving to the
injection port.
Dual-solvent rinsing may be necessary. Install a 402 Dual Syringe Pump or a Identified Problem Troubleshooting Procedures
VALVEMATE II solvent selector.
Change the valve rotor seal (according to the replacement part
The sample must be soluble in the rinse solvent. instructions supplied with the valve)
All fittings into the valve must be seated to the proper depth. Rheodyne valve leaking Correctly mount the stator on the valve. The gap distance should be
the same all around the valve (according to the replacement part
instructions supplied with the valve)
Slightly loosen the injection port seal, then slowly retighten just until
Rheodyne valve leaking from
the leaking stops or replace the seal
the top of the injection port
Replace the calibrated tubing
Loosen the adjusting screws until the valve leaks, then tighten just until
Rheodyne valve making strange
valve stops leaking (refer to the Rheodyne instructions supplied with
Troubleshooting Hardware Components sounds (straining to rotate)
valve to ensure proper stator alignment)

Valco valve making an erratic Make certain that valve has been properly installed in its home position
Troubleshooting often refers to a standard set of checks that are performed on a specific noise while moving (refer to the users guide on installing the valve in the home position)
hardware component identified and isolated with the problem. The problem is often related to
Make certain that valve is plumbed properly
unexpected performance. This section details specific troubleshooting that can be accomplished Valco valve leaking Replace rotor seal
on each main component in a Gilson HPLC system. Refer to this section often once you have Contact service@gilson.com to discuss possible replacement of unit
identified a hardware problem exists.

Pumps

321 PUMP

Identified Problem Troubleshooting Procedures

Air bubbles in pump
inlet tubing
No flow from pumping
system
Pump flow does not stop
at the end of the running
software Method
Carefully reconnect inlet tubing to assure there is no cross-threading of fittings
Pump head may be dry; make take up to 15 minutes to prime
Check for a plugged inlet filter in the solvent bottle
If using a priming tee and syringe, you may need to replace the priming tee
and/or flange fittings on inlet tubing
May need to replace check valves
Check for power and for blown fuses
Re-prime pump heads; may take up to 15 minutes
Check that mobile phase bottles have solvent
Check flow rate for each pump head separately
May need to replace check valves
Check that the software Method being run has a 0 ml/min flow rate as the last
task
Verify that the error handling software Method being run has a 0 ml/min flow
rate as the last task
Verify that the pump head piston seals do not require replacement
Verify that the pump still has communication through Trilution LC

36
35 36
37
 Detectors Continuation of Detector Troubleshooting
UV/VIS-151
Identified Problem Troubleshooting Procedures
Check that detector has power
Check for blown fuses
Detector lamp does not ignite
Check for a loose lamp connector (refer to the maintenance section of
or lamp fails while illuminated
the user guide)
Replace the lamp (refer to the maintenance section of the user guide) Rounded peaks
The lamp may need more warm up time (see user guide)
Install a back pressure regulator in line after the detector flow cell outlet
tubing to rid the system of air bubbles for flow rates <10 mL/min
Noisy baseline Flow cell may contain air bubbles - slowly increase the flow rate and
carefully apply a back pressure regulator to the outlet tubing. Reduce the
flow rate when the air bubble has been eliminated
Replace lamp if necessary after checking lamp hours
Degas the mobile phase to remove bubbles associated with the flow cell
Prime the mobile phase inlet tubing lines
Tighten or replace any leaking fittings (be careful to only hand-tighten Decreased response
Noisy baseline ONLY while
inlet tubing)
pumps are running
Re-equilibrate the column by running with the column manufacturer
specified mobile phase
Verify the pump solvents are not absorbing at the set wavelength
Monitor the manometric output when there is no flow for pulsations for
any electrical problems. The system pressure should be at zero psi
Noisy baseline during pump
Check electrical grounds on all system components. Eliminate ground
operation and after pumps are
loops by connecting the detector and 506C interface module to the
turned off
same power outlet
Contact service@gilson.com to discuss problem further
Allow additional time for lamp to warm per the user guide
Check for a column that may have residual sample matrix causing the
drift. Clean the column per the manufacturer instructions and/or replace
Drifting baseline the column
Check that the mobile phase solvents are fresh and degassed
Check the flow rate accuracy on both pump heads
Check for a leaky flow cell and replace any worn inlet and outlet fittings
Stop the system flow
If the spikes disappear, the problem is probably flow-related
If the spikes continue, the problem is probably electrical Peak resolution is
Check for leaks from the column outlet to the detector. deteriorating
Degas the mobile phase solvents
Sharp spikes in baseline Install a back pressure regulator if the flow is < 10 mL/min.
The detector may need to be plugged into a different electrical circuit
Inspect all connections and plugs to be sure all are secure
Inspect the detector lamps to be sure they are securely in place
Check electrical grounds on all system components; eliminate ground
loops by connecting detector and interface module to the same outlet
Flat-topped peaks The detector sensitivity may not be set correctly (see user guide)
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Identified Problem Troubleshooting Procedures
You may be operating beyond the linear dynamic range of the detector and/
or you may be overloading the column
Reduce the injection volume
Dilute the sample
The flow cell may be dirty (see user guide for cleaning the flow cell)
Absorption could be occurring between the sample and the column packing
Increase the temperature of the column
Change the pH of the mobile phase
Switch to another column chemistry
If using an analog signal into your interface module, your signal polarity from
Negative peaks the detector may be reversed.
Switch the polarity of the detector signal wires into the interface module
If using analog signal, inspect the physical connections to the interface
module to assure all are secure
Verify the sample concentration calculation
to known sample Verify the injection volume and the injection procedure
concentration Verify the in-line guard column being used prior to the column is not clogged
or defective
Verify the detector lamp hours are within normal operating range
Check the injector to be sure that the entire sample was injected. Clean or
replace the syringe or injection valve
Check that the injection method is using the correct injection volume and
No peaks or detector
that all synchronizes are being used correctly
response
Check that the correct rack and z-value into the sample location are correct to
aspirate the sample
Check that the correct detector settings are being used
An insufficient amount of sample has been injected, so watch to make certain
the correct sample amount is being aspirated by the probe
Low sensitivity The sample may not be fully eluting from the column
Verify the sample chemistry is compatible with column and mobile phase
Verify the detector lamp hours are within normal operating range
Tailing could result from overloading of the column. Reduce sample mass by
diluting the sample or injecting a smaller sample amount. Volume overload
may also cause poor peak shape. In both cases, refer to the table in
Appendix B for recommendations
Verify that the correct column and mobile phase are being used
The column life may be declining or column may need regeneration, so clean
column if possible per manufacturer instruction
Column may need to be replaced
There may be bubbles in the flow cell.
The mobile phase could be contaminated. Replace with fresh, mobile phase
Detector signal will not
Inspect the flow cell for contamination and leaks
zero
The system may not be fully equilibrated. Continue to flush the system at the
operating flow rate
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 Automated Liquid Handlers/Injectors
Identified Problem
GX Solvent System not
aspirating and dispensing
liquids
Leaking injection valve on
liquid handler
Rheodyne valve making
strange sounds (straining to
rotate)
215 Liquid Handler or XL
Sampler Y-arm (horizontal)
wont move
Liquid coming out of overflow
port on 402 dilutor valve
z-arm vibrates rapidly
z-arm continues to access
incorrect bed positions and
bends probes
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39
Troubleshooting Procedures
Verify plumbing connections are correct and per the user guide, with the
first port being connected to a waste bottle
Verify liquid lines are primed with solvent and free of air
Stop the system flow
If any of the fittings are leaking, carefully tighten them just until the leak
stops, being careful not to over tighten. Start the flow, and if the leak
continues, replace the entire fitting
If the valve is leaking around its circumference, replace the rotor seal (refer
to the user guide or the instructions supplied with the valve)
If liquid is leaking around the probe during loading of the sample loop, the
loop or the connection between the loop and the valve is plugged
If calibrated tubing is being used between the injection port and the valve,
clean this elbow per the user guide
If you are using a manual injection valve, clean or replace the injection
loop and fittings
Loosen the adjusting screws until the valve leaks, then tighten just until
the valve stops leaking (refer to the Rheodyne instructions supplied with
valve to ensure proper stator alignment
The arm bracket or locking screw is still in place. For an XL sampler, remove
the locking screw from the right side of the sampler using a Phillips
screwdriver. For a 215 remove the arm bracket from the horizontal arm by
loosening the allen screws
Observe the liquid handler and sampler for any interferences within the
fluid path or tubing that is restricting or catching on the tubing
Check for errors on 215
Check to make sure nothing is impeding movement (tubing)
There is a plug between the dilutor outlet and the injection valve. Isolate
the location of the plug, and then:
1. Clean the line from the dilutor to the probe to eliminate possible high
back pressure
2. Replace or clean the probe to eliminate any blockage
3. Clean the injection port or replace
4. Clean the calibrated tubing or replace (if present)
5. Clean the valve by switching back and forth from load to inject
positions every 30 seconds while the mobile phase is flowing
6. Sonicate the dilutor valve ceramic parts and re-assemble the valve
Contact Gilson Customer Service for support
For GX-2XX Liquid Handlers, use the Offset Utility software supplied and
instructions in the user guide to correctly offset the instrument with a new
probe(s)
For 215 Liquid Handlers, contact Gilsons Customer Service Department
for support
Fraction Collectors Prep FC
Identified
Troubleshooting Procedures
Problem
Collector is inoperative Check that the collector is plugged in and powered on
and unresponsive Check for blown fuses
The stepper motor and microprocessors must establish a reference point
Collector makes a loud (home position left rear) each time the collector is powered on. It is normal
noise when powered on for the collector to make a vibrating noise for a few seconds while the motors
(FC203B, FC204) are homing. This noise should occur only after the collector is powered on or
when power is restored after a power failure
Collector makes a loud
Check for an obstruction to the X or Y movement by turning power off and
noise when changing
manually moving the drop head and horizontal arm. They should move the full
X or Y position during
range of their travel smoothly and easily
a run
If the collector is running from a keypad, verify that each rack code matches the
installed rack
If the collector is running via prepFC Control Software or Trilution LC Software,
verify that the bed layout has the proper tray and racks selected
Inspect the positioning of the racks to be certain they are properly seated on
the tray
Check for a crimp in the inlet tubing from the detector. Replace any crimped or
damaged tubing
Drops are missing the Check that the tubing from the 3-way valve or drop detector is not bent of
collection tubes pushed up above the foot. Replace bent tubing. Replace any tubing that is too
short and does not fall ~2 mm below the foot
The collector may be out of alignment (FC203B, FC204, prepFC). Check the
horizontal arm assembly by powering the collector off then on. Contact Gilsons
Customer Service Department if the unit is still out of alignment
The collector may need to be offset using the offset utility software (GX-281,
GX-271). Follow the instructions in the user guide
The collector may be out of alignment (215). Contact Gilsons Customer Service
Department if the unit is still out of alignment after powering off then on
Check that the tubing in the drop detector is not bent and is properly gauged
(refer to FC203B, FC204, prepFC fraction collector user guide)
Verify flow from the pumps is accurate and mobile phase lines are free of air.
Ensure that the inlet tubing fitting is completely tightened (the tubing should
not move up and down as the drop detector moves)
The tubing in the drop detector may be too short or too long
The drop detector is The drop detector tubing may have particulates on the tip. Clean the drop
not counting drops or is detector collar with a wetted cotton swab
counting erratically The drop detector assembly may be defective; contact Gilsons Customer
Service Department for assistance with replacement
Ensure that the tubing coming from the detector source is not protruding more
than 1 mm past the nut/ferrule. Tubing that protrudes too far into the 3-way
valve can damage the valves ability to open and close
Verify settings in keypad or control software to make certain the proper volume
is set for collection into the tubes
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 Continuation of Fraction Collector Troubleshooting
Identified Problem Troubleshooting Procedures
If the collector is being controlled by prepFC Control Software, verify the
parameters are appropriate for the peak shape and peak height of the signal
If the collector is being controlled by Trilution LC Software, use the Fraction
Collection Simulation function in the Method to try different peak collection
parameters (see Help menu for assistance). Verify the parameters are
appropriate for the peak shape and peak height of the signal. Verify the
proper data signal is being used to trigger collection (right-click on the data
channel and make sure that primary channel is selected with a check mark)
If the collector is operating in a stand-alone mode, check the peak
Collection problems in
parameters in the keypad:
peak mode
When collecting peaks by slope, the peak height should be the mV height
of the smallest peak
When collecting by level, the peak level should be the mV value above
which to collect peaks
The peak width should be the average width of the peaks at half height
Eliminate any leaks. Install a back pressure regulator in the system if the flow
is < 10 mL/min
Verify that the delay volume or delay time has been accurately calculated and
is used for collection (for flows < 10mL/min)
Check that the detector input voltage is less than 100 mV full scale
Check that the peak parameters set in the software match the size and shape
of the peaks to be collected
Check the polarity of the detector signal connections into the collector or
into the 506C (if using analog signal) to make certain the connections are not
reversed
Confirm that the fraction collector analog input or 506C is correctly zeroed:
1. Disconnect the analog signal wires from the detector input connection on
the barrier strip at the back of the collector or 506C
2. Short the detector input by connecting a piece of wire between the
positive (+) and negative (-) connections
3. If using the keypad, press EDIT 3 on the collector keypad to access the
Not recognizing peaks in Service Menu. Press the Next softkey until the collector display reads
peak mode Detector A/D: xxxx uV. Watch the display for a few minutes to see that the
analog input stays zeroed (the actual reading may be +/- 20 uV). Press the
Zero softkey to zero the analog input if it does not read 0 uV. Press the
Quit softkey to return to the main screen
4. If using the 506C and Trilution LC Software, access the Manual Control
screen and send an Autozero task to the 506C. Verify the signal displays
0 mV
5. Disconnect the short from the detector input on the barrier strip and
reconnect the signal wires from the detector
Confirm that your Trilution LC Software Method is sending a contact closure
pulse to autozero the data signal through the 506C output and not using the
autozero task on the 506C (prevents future problems with incorrect zeroing
on the 506C)
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Continuation of Fraction Collector Troubleshooting
Identified Problem Troubleshooting Procedures
Turn off power to the collector and check that the connection to the socket
and/or barrier strip is secure and no wires are loose
Check that the connector is fully installed
Ensure that the tubing coming from the detector source is not protruding
3-way valve is not
more than 1 mm past the nut/ferrule. Tubing that protrudes too far into the
functioning or is
3-way valve can damage the valves ability to open and close
functioning incorrectly
Verify the flows are appropriate for the type of valve being used on the
collector (reference specifications in user guide)
The 3-way valve may be defective; contact Gilsons Customer Service
Department
Ensure that the wiring connections into the terminal block connector or
barrier strip are secure, and that the connector is completely seated into the
back of the fraction collector
Verify the correct contact closure inputs or outputs are being used on the
barrier strip and that these match with either the keypad or control software
program
Check the connections to the device providing input or output contact
closure to verify these are secure and the correct pins are used. Verify wires
Contact closure input or are not reversed
output functions are not To check the input contact closure, short the collector input. If this
operating successfully trips the input, then check the output from the external device.
If this does not trip the input, check the barrier strip connector, multipurpose
Input/Output port, or contact Gilsons Customer Service Department
To check the input contact closure when using control software, verify that
manual control can properly trigger the contact closure
Check the polarity of the input connection. Inputs should be a contact
closure or TTL level (logic 0 activates this on a FC203B or FC204)
The output signals from the collector are contact closures. This must be
compatible with the external device
42
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 Putting it All Together Appendix A: PEEK Tubing Pressure vs. Flow Rate
This guide discussed both system and component troubleshooting with Gilson HPLC Systems. As
PUTTING IT ALL TOGETHER

you develop experience, you will be able to use this guide as a reference tool. Until then, keep this PEEK* Tubing Pressure vs. Flow Rate
guide handy in your library of essential tools to assist you daily in the laboratory.
Tubing ID (in)
Flow Rate (mL/min)
Photocopy the HPLC Troubleshooting Checklist on page 4 to assist you with easy and 0.02 Pressure (psi) 0.03 Pressure (psi) 0.04 Pressure (psi)
at your fingertips identification of system observations. 25 0 0 0
Photocopy the Software Troubleshooting Tips Checklist on page 6 to assist you with 50 43.5 0 0

APPENDIX A
software error identification. 75 101.5 0 0
Utilize the Identifying hardware Component Problems Checklist on page 10 to 100 203.1 14.5 0
troubleshoot when and what components in your system are the potential problem
125 333.6 43.5 0
source.
150 464.1 87 14.5
Utilize our additional publications (see the back cover of this guide) to assist you with
efficient and optimal use of your Gilson HPLC system. 175 623.7 101.5 14.5
200 797.7 145 29

Quality Policy
It is Gilson, Inc.s policy to promote a total commitment to quality by each individual throughout
the Gilson organization worldwide. It is Gilsons goal that this commitment to quality will result in
complete customer satisfaction with both Gilsons products and services.

ISO 9001 Certification

Gilson is committed to quality, and it shows in everything Gilson does from design to
manufacturing, technical support and training. Due to this commitment to quality and customer
satisfaction, Gilson has consistently been awarded with ISO 9001 certification. Gilson, Inc. and
Gilson S.A.S. are both ISO 9001 certified.

43
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 Appendix B: Column Sizes & Capacities Appendix C: Tubing Internal Diameters & Volumes

Internal Diameter (mm) Typical Injection Volume (L) Load Capacity (mg) Typical Flow Rates (mL/min) Tubing Internal Diameters & Volumes
4.6 15 1 0.5 2 Inch Millimeter Micron L/in L/mm

10 100 4.7 4 15 0.005 0.127 127 0.322 0.0127

21.2 400 19.5 10 50 0.01 0.254 254 1.287 0.0507

0.02 0.508 508 5.148 0.2027
APPENDIX B

APPENDIX C
30 1,000 42.5 40 100
50 2,000 118 100 300 0.03 0.762 762 11.583 0.456

100 10,000 473 400 1000 0.04 1.016 1016 20.593 0.8107

Temperature Conversion Formulas

C = F 32 1.8
C = K 273.15
C = degrees Celsius
F = 1.8 x C + 32 F = degrees Fahrenheit
F = R 459.67 K = degrees Kelvin
R = degrees Rankine
K = C + 273.15
R = F + 459.67

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47
 Appendix D: Pressure Conversion Chart Appendix E: Solvent Polarity Chart

*Unit of Measure Equivalent Measure

Solvent Polarity Chart
Pounds per square inch
Normal atmospheric pressure is 14.7 psi Relative Compound
(psi, PSI. lk/in2, lb/sq in) Group Representative Solvent Compounds
Polarity Formula
Atmosphere
APPENDIX D

APPENDIX E
Normal atmosphereic pressure = 1 atmosphere (atm) = 14.6956 psi = 760 torr Non-polar R-H Alkanes Petroleum Ethers, Ligroin, Hexanes
(atm)
Ar - H Aromatics Toluene, Benzene
Bar The bar is very similar to 1 atmosphere unit. 1 bar = 750.062 torr = 0.9869 atm =
(bar) 100,000 Pa R-O-R Ethers Diethyl Ether
1 pascal is equal to the force of 1 Newton per square meter. R-X Alkyl Halides Tetrachloromethane, Chloroform

Increasing Polarity
Pascal 1 Newton is the force required to accelerate 1 kg m/s2
R - COOR Esters Ethyl Acetate
(Pa) 1 pascal = 10 dyne/cm 2 = 0.01 mbar
1 atm = 101,325 Pascals = 760 mm Hg = 760 toor = 14.7 psi R - CO - R Aldehydes and Ketones Acetone, Methyl Ethyl, Ketone
1kilopascal = 1,000 Pa R - NH 2 Amines Pyridine, Triethylamine
Kilopascal
101.325 kPa = 1 atm = 760 torr
(kPa) R - OH Alcohols Methanol, Ethanol, Isopropanol, Butanol
100 kPa = 1 bar = 750 torr
R - COHN 2 Amides Dimethylformamide
Megapascal
1 megapascal = 1,000 kPa = 1,000,000 Pa R - COOH Carboxylic Acids Ethanoic Acid
(MPa)
Polar H - OH Water Water

*Info edited from: http://www.sengpielaudio.com/ConvPress.htm

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 Additional Publications from Gilson

2011Volume 1
Application Notes Handbook
Pipettes | Purification | Solid Phase Extraction

LC v2.1Vol 1
TriluTion LC Software v2.1
Quarterly Gilson Application Notes Handbook Technical Notes Handbook

TRILUTION
Trilution LC Technical Notes Handbook Series

Contact your local Gilson representative or contact training@gilson.com to obtain our quarterly
Gilson Application Notes Handbook and our series of Trilution LC Technical Notes Handbooks.

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