TROUBLESHOOTING GUIDE
Contents
1. HPLC TROUBLESHOOTING GUIDE J phenomenex breaking with tradition al a CALL PHENOMENEX FOR HPLC Columns capillary to preparative SEC Columns Aqueous GFC and non Aqueous GPC Amino Acid Analysis HPLC Specialty Columns for Analysis of Basic acidic and amphoteric drugs High Low pH separations pH 1 12 Proteins Peptides by reversed phase Biopolymers Proteins and Nucleic Acids by GFC SEC Synthetic polymers Foods and Beverages Environmental Samples Drugs in biological fluids m HPLC Bulk Media m HPLC Accessories such as Sample and Solvent Filters SecurityGuard Column Protection Syringe Filters Syringes and Vials Column Heater Column Chiller Heater HPLC Injection Valves Tubings and Fittings Solvent Degassers Column Selectors Fluid Processors GC Columns SPE Tubes and 96 Well plates Application Development and Validation Support Outstanding Technical Service Vi VII TABLE OF CONTENTS IMROGUCTON Ba fsctivaadentatieceettvvewesta cuted el weustlathcnecderswenitdchececdersenstancieenee 1 Abnormal PreSSure ci cc cidsase Seendect chee aaa aeii i 2 LC AKS E AEE T AAEE E E E A EE EET 4 Problems with the Chromatogram ssssssssiesssrssssrrssrnnsssrnnserens 6 Problems with the InjeCtor ccccccceeeeeeesseceeeeeeeeeeeneeeeeeeeeeeeeaaeees 14 Problems Detected by Smell Sight or Sound cceeeeeeeees 15 Key Problem Areas and Preventive Maintenance2. From injector gt 2 contaminants compounds of interest Holder HPLC column UNIVERSAL FIT A G With the patented design SecurityGuard can adjust to fit virtually any manufacturer s B 4 i f FN female inverted endfitting guarantee If you are not completely satisfied with the performance of SecurityGuard simply contact Phenomenex within 45 days and keep SecurityGuard Patented Design for FREE Feature applies to analytical sized guard system only and SecurityGuard is a trademark of does not apply to SemiPrep or PREP guard cartridges Phenomenex Inc 19 Phenex Syringe Filters For Sample and Solvent Filtration Prior to Chromatography e Less system down time e Consistent reproducible results e Increased column lifetime Phenex Offers Low absorption Maximum chemical compatibility Minimum extractables Excellent flow rate High total throughput Low hold up volume Certified quality 100 integrity tested Easy pore identification Bi directional use Membrane Types RC NY Regenerated Cellulose Nylon PTFE CA Polytetrafluoroethylene Cellulose Acetate PES GF Polyethersulfone Glass Fiber Above syringe filters are non sterile Housing is made of medical grade polypropylene PP Tip Try a Sample Pack The best way to determine if a specific Phenex membrane is suitable fo
3. Problem LOSS OF RESOLUTION 12 IV PROBLEMS WITH THE CHROMATOGRAM continued Q All peaks too small POSSIBLE CAUSE SOLUTION 1 Detector attenuation too high 1 Reduce attenuation 2 Detector time constant too large 2 Use smaller time constant 3 Injection size too small 3 a Increase sample concentration b Increase injection volume if column size allows 4 Improper recorder connection 4 Use correct connection R All peaks too large POSSIBLE CAUSE SOLUTION 1 Detector attenuation too low 1 Use larger attenuation 2 Injection size too large 2 a Reduce sample concentration b Decrease injection volume use a smaller sample loop or use partial loop filling 3 Improper recorder connection 3 Use correct connection 13 V PROBLEMS WITH THE INJECTOR These problems are usually detected while you are using the injection valve Leaky injection valves are discussed in Section Ill Leaks A Manual injector hard to turn POSSIBLE CAUSE SOLUTION 1 Damaged rotor seal 1 Rebuild or replace valve 2 Rotor too tight 2 Adjust rotor tension B Manual injector hard to load POSSIBLE CAUSE SOLUTION 1 Valve misaligned 1 Adjust alignment 2 Blocked loop 2 Replace loop 3 Dirty syringe 3 Clean or replace syringe 4 Blocked lines 4 Clear or replace lines C Autoinjector won t turn POSSIBLE CAUSE SOLUTION 1 No air pressure or power 1 Sup
4. 17 SecurityGuard Universal HPLC Guard Cartridge System 19 Phenex Syringe Filters ccccceeesceeeeeeneeeeeeeeeeeeeeeeeeeseaeeeeseeeeeeeea 20 2008 Phenomenex Inc All rights reserved No part of this booklet may be copied without prior written permission from Phenomenex Inc USA While every attempt has been made to ensure the accuracy of the information contained in this guide Phenomenex assumes no responsibility for its use We welcome any additions or corrections for incorporation into future editions Il INTRODUCTION LOCATING AND CORRECTING THE PROBLEM A systematic approach to identifying the problem is the best approach to trou bleshooting your HPLC system This guide is organized by five major categories of symptoms to help you quickly identify the source of the problem s you are encountering e pressure abnormalities e leaks e problems with the chromatogram e injector problems e other problems detected by the senses of smell sight and sound When you have corrected the problem record the incident in the system record book to help with future problems PREVENTION Many LC problems can be prevented with routine preventive maintenance For example replacing pump seals at regular intervals should eliminate pump seal failure and its associated problems Section VII lists the most common problem areas for each LC module and preventive maintenance practices that will re duce their frequency The
5. detector too long or ID too large 0 010 in ID tubing as practical d Recorder response time too high d Reduce response time Normal Problem BROAD PEAKS 11 IV PROBLEMS WITH THE CHROMATOGRAM ccontinued O Broad peaks continued POSSIBLE CAUSE SOLUTION 6 Buffer concentration too low 6 Increase concentration 7 Guard column 7 Replace guard column contaminated worn out 8 Column contaminated worn out 8 Replace column with new one Low plate number of same type 9 Void at column inlet 9 Open inlet end and fill void or replace column 10 Peak represents two or more poorly 10 Change column type to improve resolved compounds separation 11 Column temperature too low 11 Increase temperature Do not exceed 60 C unless higher temperatures are acceptable to column manufacturer 12 Detector time constant too large 12 Use smaller time constant P Loss of resolution POSSIBLE CAUSE SOLUTION 1 Mobile phase contaminated 1 Prepare new mobile phase deteriorated causing retention time to change 2 Obstructed guard or 2 Remove guard column and analytical column attempt analysis Replace guard if necessary If analytical column is obstructed reverse and flush If problem persists column may be fouled with strongly retained contaminants Use appropriate restoration procedure If problem persists inlet is probably plugged Change frit or replace column Normal
6. pH 4 Adjust pH For basic compounds lower pH usually provides more symmetric peaks 5 Sample reacting with active sites Normal Problem PEAK TAILING B Peak fronting POSSIBLE CAUSE 5 a Add ion pair reagent or volatile basic modifier b Change column SOLUTION 1 Low temperature 1 Increase column temperature 2 Wrong sample solvent 2 Use mobile phase for injection solvent 3 Sample overload 3 Decrease sample concentration 4 Bad column C Split peaks POSSIBLE CAUSE 4 See A 1 and A 2 SOLUTION 1 Contamination on guard or analytical column inlet 1 Remove guard column and attempt analysis Replace guard continued on next page Check manufacturer s column warranty first Removal of end fittings may void column warranty 6 IV PROBLEMS WITH THE CHROMATOGRAM continued C Split peaks continued POSSIBLE CAUSE SOLUTION Problem UL SPLIT PEAKS Normal if necessary If analytical column is obstructed reverse and flush If problem persists column may be fouled with strongly retained contaminants Use appropriate restoration procedure If problem persists inlet is probably plugged Change frit or replace column 2 Sample solvent incompatible with mobile phase D Distortion of larger peaks POSSIBLE CAUSE 2 Change solvent Whenever possible inject samples in mobile phase SOLUTION 1 Sam
7. LEM PREVENTIVE MAINTENANCE 1 Blocked frit 1 a Filter mobile phase b Filter samples c Use in line filter and or guard column 2 Void at head of column 2 a Avoid mobile phase pH gt 8 Most silica based columns b Use guard column c Use precolumn saturator column 17 Vil KEY PROBLEM AREAS AND PREVENTIVE MAINTENANCE continued Detector PROBLEM PREVENTIVE MAINTENANCE 1 Lamp failure decreased detector 1 Replace 6 mo or keep response increased detector noise spare lamp 2 Bubbles in cell 2 a Keep cell clean b Use restrictor after cell c Degas mobile phase General PROBLEM PREVENTIVE MAINTENANCE 1 Corrosive abrasive damage 1 Flush buffer from LC and clean when not in use 18 WARNING CONTAMINANTS CAN CAUSE e High Backpressure e Split Peaks e Broad Peaks e Baseline Noise e Baseline Drift e Loss of Resolution e Irreversible Column Damage e System Damage PROTECT YOUR HPLC COLUMN AND RESULTS err o Additional information can be found at SecurityGuard www phenomenex com info securityguard A universal HPLC guard cartridge system designed to effectively protect your valuable analytical columns and results from the damaging effect of contaminants Trap contaminants without altering your chromatography Cutaway view showing Cartridge can be easily i A F HOW IT WORKS inspected for contaminants Universal Fingertight connection to HPLC column no wrenches required
8. apart and inspect for damage e g distorted ferrule or particles on the sealing surface damaged fittings should be discarded A Leaky fittings POSSIBLE CAUSE SOLUTION 1 Loose fitting 1 Tighten 2 Stripped fitting 2 Replace 3 Overtightened fitting 3 a Loosen and retighten b Replace 4 Dirty fitting 4 a Disassemble and clean b Replace 5 Mismatched parts B Leaks at pump POSSIBLE CAUSE 5 Use all parts from same brand SOLUTION 1 Loose check valves 1 a Tighten check valve do not overtighten b Replace check valve Loose fittings 2 Tighten fittings do not overtighten 3 Mixer seal failure 3 a Replace mixer seal b Replace mixer 4 Pump seal failure 4 Repair or replace 5 Pressure transducer failure 5 Repair or replace 6 Pulse damper failure 6 Replace pulse damper 7 Proportioning valve failure 7 a Check diaphragms replace if leaky b Check for fitting damage replace 8 Purge valve 8 Tighten valve of Replace purge valve Use fingertight end fittings to avoid sealing problems and the need for wrenches Ill LEAKS continued C Injector leaks POSSIBLE CAUSE SOLUTION 1 Rotor seal failure 1 Rebuild or replace injector Blocked loop Replace loop Loose injection port seal Adjust Improper syringe needle diameter Use correct syringe a A P Waste line siphoning a Po N Keep wast
9. e line above surface waste 6 Waste line blockage D Column leaks POSSIBLE CAUSE 6 Replace waste line SOLUTION 1 Loose endfitting 1 Tighten endfitting 2 Column packing in ferrule 2 Disassemble rinse ferrule reassemble 3 Improper frit thickness E Detector leaks POSSIBLE CAUSE 3 Use proper frit see chart below SOLUTION 1 Cell gasket failure 1 a Prevent excessive backpressure b Replace gasket Cracked cell window s Replace window s Leaky fittings Tighten or replace Blocked waste line Replace waste line 2 3 4 5 FRIT PORE SIZE SELECTION GUIDE When Particle Size of material is Blocked flow cell 5 20 um Q a j o Ny Rebuild or replace Frit Pore Size should be 0 5 um IV PROBLEMS WITH THE CHROMATOGRAM Many problems in the LC system show up as changes in the chromatogram Some of these can be solved by changes in the equipment however others require modification of the assay procedure Selecting the proper column type and mobile phase are keys to good chromatography A Peak tailing POSSIBLE CAUSE SOLUTION 1 Blocked frit 1 a Reverse flush column if allowed b Replace inlet frit c Replace column Column void Fill void Interfering peak 3 a Use longer column b Change mobile phase and or column selectivity 4 Wrong mobile phase
10. e of curve UV absorbance maximum IV PROBLEMS WITH THE CHROMATOGRAM ccontinued M Baseline noise regular POSSIBLE CAUSE SOLUTION 1 Air in mobile phase detector cell or pump 1 Degas mobile phase Flush system to remove air from detector cell or pump 2 Leak Normal k paesan siiin N Problem BASELINE NOISE See section Ill Check system for loose fittings Check pump for leaks salt build up unusual noises Change pump seals if necessary 3 Incomplete mobile phase mixing less viscous solvent Mix mobile phase by hand or use 4 Temperature effect column at high temperature detector unheated Reduce differential or add heat exchanger 5 Other electronic equipment on same line Isolate LC detector or recorder to determine if source of problem is external Correct as neccessary 6 Pump pulsations N Baseline noise irregular POSSIBLE CAUSE Incorporate pulse dampener into system SOLUTION 1 Leak m Normal Problem BASELINE NOISE 1 See section Ill Check for loose fittings Check pump for leaks salt build up unusual noises Change seals if neccessary Check for detector cell leak 2 Mobile phase contaminated deteriorated or prepared from low quality materials Check make up of mobile phase 3 Mobile phase solvents immiscible Select and use only miscible solvents 4 Detector recorder electronics Isolate de
11. ex com Germany Zeppelinstr 5 63741 Aschaffenburg Germany 06021 58830 0 06021 58830 11 anfrage phenomenex com New Zealand P O Box 31 601 Milford 0741 North Shore City New Zealand 09 4780951 09 4780952 nzinfo phenomenex com Belgium Maarssenbroeksedijk 13D 3542 DL Utrecht Netherlands 31 0 30 2418700 31 0 30 2383749 beinfo phenomenex com Ireland Queens Avenue Hurdsfield Ind Est Macclesfield Cheshire SK10 2BN UK 01 247 5405 44 1625 501796 eireinfo phenomenex com Puerto Rico 273 Sierra Morena Suite 104 San Juan Puerto Rico 00926 800 541 HPLC 3810 328 7768 info phenomenex com Canada 411 Madrid Ave Torrance CA 90501 1430 USA 800 543 3681 310 328 7768 info phenomenex com Italy Via M Serenari 15 D 40013 Castel Maggiore BO Italy 051 6327511 051 6327555 italiainfo phenomenex com United Kingdom Queens Avenue Hurdsfield Ind Est Macclesfield Cheshire SK10 2BN UK 01625 501367 01625 501796 ukinfo phenomenex com Denmark Gydevang 39 41 3450 Aller d Denmark 4824 8048 4810 6265 dkinfo phenomenex com Luxembourg Maarssenbroeksedijk 13D 3542 DL Utrecht Netherlands 31 0 30 2418700 31 0 30 2383749 nlinfo phenomenex com USA 411 Madrid Ave Torrance CA 90501 1430 USA 810 212 0555 310 328 7768 info phenomenex com 6116_L
12. ine filter Check manufacturer s column warranty first Removal of end fittings may void column warranty 2 ll ABNORMAL PRESSURE continued D Steady low pressure POSSIBLE CAUSE SOLUTION 1 Flow set too low 1 Adjust flow rate 2 Leak in system 2 Locate and correct 3 Improper column 3 Use proper column 4 Column temperature too high 4 Lower temperature 5 Controller malfunction 5 Repair or replace controller E Pressure climbing POSSIBLE CAUSE SOLUTION 1 See section C 1 See section C F Pressure dropping to zero POSSIBLE CAUSE SOLUTION 1 See sections A and B 1 See sections A and B G Pressure dropping but not to zero POSSIBLE CAUSE SOLUTION 1 See section D 1 See section D H Pressure cycling POSSIBLE CAUSE SOLUTION 1 Airin pump 1 a Degas solvent b Bleed air from pump Faulty check valve s 2 Replace check valve s Pump seal failure 3 Replace pump seal Insufficient degassing 4 a Degas solvent b Change degassing methods use Degassex on line degasser Leak in system 5 Locate and correct Using gradient elution 6 Pressure cycling is normal due to viscosity changes lil LEAKS Leaks are usually stopped by tightening or replacing a fitting Be aware howev er that overtightened metal compression fittings can leak and plastic fingertights can wear out If a fitting leak does not stop when the fitting is tightened a little take the fitting
13. ity salts and additives Degas rather than cyclic pattern from mobile phase before use sparge temperature fluctuation with helium during use 3 Contaminant or air buildup in 3 Flush cell with methanol or other detector cell strong solvent If necessary clean cell with 1N HNO never with HCl 4 Plugged outlet line after detector 4 Unplug or replace line Refer High pressure cracks cell window to detector manual to replace producing noisy baseline window 5 Mobile phase mixing problem or 5 Correct composition flow rate change in flow rate To avoid routinely monitor composition and flow rate 6 Slow column equililbration 6 Flush with intermediate strength especially when changing solvent run 10 20 column mobile phase volumes of new mobile phase before analysis 7 Mobile phase contaminated 7 Check make up of mobile phase deteriorated or prepared from Use highest grade chemicals and low quality materials HPLC solvents 8 Strongly retained materials in 8 Use guard column If necessary sample high k can elute as flush column with strong solvent very broad peaks and appear between injections or periodically to be a rising baseline Gradient during analysis analyses canaggravate problem 9 Mobile phase recycled but 9 Reset baseline Use new mobile detector not adjusted phase when dynamic range of detector is exceeded 10 Detector UV not set at 10 Change wavelength to absorbance maximum but at slop
14. m Choose the category below that best fits the symptoms that you observe and follow the suggestions to correct the problem A No pressure reading no flow POSSIBLE CAUSE SOLUTION 1 Power off 1 Turn on power 2 Fuse blown 2 Replace fuse 3 Controller setting or failure 3 a Verify proper settings b Repair or replace controller 4 Broken piston 4 Replace piston 5 Air trapped in pump head 5 Degas solvents bleed air from pump prime pump 6 Insufficient mobile phase 6 a Replenish reservoir b Replace inlet frit if blocked 7 Faulty check valve s 7 Replace check valve s 8 Major leak 8 Tighten or replace fittings B No pressure reading flow is normal POSSIBLE CAUSE SOLUTION 1 Faulty meter 1 Replace meter 2 Faulty pressure transducer 2 Replace transducer C Steady high pressure POSSIBLE CAUSE SOLUTION 1 Flow rate set too high 1 Adjust setting 2 Blocked column frit 2 Backflush column if permitted Replace frit Replace column 3 Improper mobile phase 3 precipitated buffer Use correct mobile phase Wash column Dv oom Improper column Use proper column Injector blockage Clear blockage or replace injector Column temperature too low Raise temperature Controller malfunction Repair or replace controller Blocked guard column Remove replace guard column N 9 oO S oj N 9 o S Blocked in line filter Remove replace in l
15. nents in sample 1 Normal 2 Late eluting peak from 2 a Increase run time previous injection or gradient slope b Increase flow rate 3 Vacancy or ghost peaks 3 a Check purity of mobile phase b Use mobile phase as injection solvent c Reduce injection volume 4 Contamination 4 Filter sample J Retention time drifts POSSIBLE CAUSE SOLUTION 1 Poor temperature control 1 Thermostat column 2 Mobile phase changing 2 Prevent change evaporation reaction etc 3 Poor column equilibration 3 Allow more time for column equilibration between runs K Abrupt retention time changes POSSIBLE CAUSE SOLUTION 1 Flow rate change 1 Reset flowrate 2 Air bubble in pump 2 Bleed air from pump 3 Improper mobile phase 3 a Replace with proper mobile phase b Set proper mobile phase mixture on controller IV PROBLEMS WITH THE CHROMATOGRAM continued L Baseline drift POSSIBLE CAUSE SOLUTION 1 Column temperature fluctuation Even small changes cause cyclic baseline rise and fall Most often affects refractive index and conductivity detectors or UV detectors at high sensitivity or in direct photometric mode 1 Control column and mobile phase temperature use heat exchanger NORMAL before detector PROBLEM BASELINE DRIFT 2 Nonhomogenous mobile phase 2 Use HPLC grade solvents high Drift usually to higher absorbance pur
16. ple overload E Distortion of early peaks POSSIBLE CAUSE 1 Reduce sample size SOLUTION 1 Wrong injection solvent 1 a Reduce injection volume b Use weaker injection solvent F Tailing early peaks more than later ones POSSIBLE CAUSE SOLUTION 1 Extra column effects 1 a Replumb system shorter narrower tubing b Use smaller volume detector cell G Increased tailing as k increases POSSIBLE CAUSE SOLUTION 1 Secondary retention effects 1 a Add triethylamine basic samples reversed phase mode b Add acetate acidic samples c Add salt or buffer ionic samples d Try a different column e See page 19 2 Secondary retention effects 2 a Add triethylamine normal phase mode basic compounds b Add acetic acid acidic compounds IV PROBLEMS WITH THE CHROMATOGRAM ccontinueg G Increased tailing as k increases continued POSSIBLE CAUSE SOLUTION 2 Secondary retention effects 2 c Add water normal phase mode poly functional compounds Only for normal phase methods which use water miscible solvents d Try a different LC method 3 Secondary retention effects ion pair 3 Add triethylamine basic samples H Acidic or basic peaks tail POSSIBLE CAUSE SOLUTION 1 Inadequate buffering 1 a Use 50 100 mM buffer concentration b Use buffer with pKa equal to pH of mobile phase c See page 19 I Extra peaks POSSIBLE CAUSE SOLUTION 1 Other compo
17. ply proper pressure power 2 Rotor too tight 2 Adjust 3 Valve misaligned 3 Adjust alignment D Autoinjector other problems POSSIBLE CAUSE SOLUTION 1 Blockage 1 Clear or replace blocked portion 2 Jammed mechanism 2 See service manual 3 Faulty controller 3 Repair or replace controller 14 Vi PROBLEMS DETECTED BY SMELL SIGHT OR SOUND You need to use all your senses to identify LC problems You should get in the habit of taking a few minutes each day to expose all of your senses except taste to the LC so that you can get a feel for how the LC performs normally This will help you to quickly locate problems For example often you can smell a leak before you see it The majority of problems are identified by sight most of these are included in the preceeding section A Solvent smell POSSIBLE CAUSE SOLUTION 1 Leak 1 See section Ill 2 Spill 2 a Check for overflowing waste container b Locate spill and clean up B Hot smell POSSIBLE CAUSE SOLUTION 1 Overheating module 1 a Check for proper ventilation adjust b Check temperature setting adjust c Shut module off see service manual C Abnormal meter readings POSSIBLE CAUSE SOLUTION 1 Pressure abnormality 1 See section Il 2 Column oven problem 2 a Check settings adjust b See service manual 3 Detector lamp failing 3 Replace lamp D Warning lamps POSSIBLE CAUSE SOLUTION 1 Pressu
18. r your application Request yours today by phone or visit www phenomenex com sample Please contact your local Phenomenex technical consultant or distributor for availability or assistance Larger quantity purchases at significant savings are available Phenex is a trademark of Phenomenex Inc d guarantee If Phenex Syringe Filters do not perform as well or better than your current syringe filter product of similar membrane diameter and pore size return the product with comparative data within 45 days for a FULL REFUND This publication is distributed free of charge Additional copies are available from www phenomenex com Phenomenex products are available worldwide For the distributor in your country contact Phenomenex USA International Department by telephone fax or email international phenomenex com J phenomenex breaking with tradition tel fax email tel fax email tel fax email Australia PO Box 4084 Lane Cove NSW 2066 Australia 02 9428 6444 02 9428 6445 auinfo phenomenex com France Parc des Grillons Bat 3 60 route de Sartrouville 78232 Le Pecq Cedex France 01 30 09 21 10 01 30 09 21 11 franceinfo phenomenex com Netherlands Maarssenbroeksedijk 13D 3542 DL Utrecht Netherlands 030 2418700 030 2383749 nlinfo phenomenex com Austria Zeppelinstr 5 63741 Aschaffenburg Germany 01 319 1301 01 319 1300 anfrage phenomen
19. re limit exceeded 1 a Check for blockage b Check limit setting adjust 2 Other warning lamps 2 See service manual 15 Vi PROBLEMS DETECTED BY SMELL SIGHT OR SOUND continued E Warning buzzers POSSIBLE CAUSE SOLUTION 1 Solvent leak spill 1 Locate and correct 2 Other warning buzzers 2 See service manual F Squeaks and squeals POSSIBLE CAUSE SOLUTION 1 Bearing failure 1 See service manual 2 Poor lubrication 2 Lubricate as necessary 3 Mechanical wear 3 See service manual 16 Vil KEY PROBLEM AREAS AND PREVENTIVE MAINTENANCE The chart below lists the most common problems that occur with each LC module In the right hand column are listed preventive maintenance practices that can reduce the failure rate The numbers in parentheses are suggested intervals between maintenance The operator s and service manuals for your LC may have additional suggestions for preventive maintenance of your model of LC Reservoir PROBLEM PREVENTIVE MAINTENANCE 1 Blocked inlet frit 1 a Replace 8 6 mo b Filter mobile phase 0 5p filter 2 Gas bubbles 2 Degas mobile phase Pump PROBLEM PREVENTIVE MAINTENANCE 1 Air bubbles 1 Degas mobile phase 2 Pump seal failure 2 Replace 3 mo 3 Check valve failure 3 Filter mobile phase use inlet line frit Keep spare Injector PROBLEM PREVENTIVE MAINTENANCE 1 Rotor seal wear 1 a Don t overtighten b Filter samples Column PROB
20. se suggestions should be modified to fit your particular model of LC and then made a regular part of your laboratory routine WHERE TO GET ADDITIONAL HELP e The operator s and service manuals for the instrument should be consulted These contain exploded diagrams troubleshooting procedures for specific models and part numbers to help you order replacement parts e Other people in the lab may have had experience solving a problem which is giving you trouble they can be a helpful resource e The manufacturer of your instrument can help you Most LC manufacturers offer free technical support to their customers e Phenomenex has experienced technical consultants who can assist you with almost any problem We welcome your phone calls faxes or emails e Phenomenex offers seminars as well as a complete line of reference books on HPLC e There are a number of reference sources that can give you guidance in problem solving J W Dolan and L R Snyder Troubleshooting LC Systems Humana Press NJ 1989 Phenomenex Order No AAO 1717 L R Snyder and J J Kirkland Introduction to Modern Liquid Chromatography 2nd ed Wiley NY 1979 Phenomenex Order No AAO 1700 D J Runser Maintaining and Troubleshooting HPLC Systems A User s Guide Wiley NY 1981 J W Dolan Troubleshooting LC GC Magazine This is a monthly column ll ABNORMAL PRESSURE A change in the operating pressure is a sign that there may be a proble
21. tector and recorder electronically Refer to instruction manual to correct problem 5 Air trapped in system Flush system with strong solvent 6 Air bubbles in detector 10 Purge detector Install back pressure device after detector continued on next page IV PROBLEMS WITH THE CHROMATOGRAM ccontinued N Baseline noise irregular continued POSSIBLE CAUSE SOLUTION 7 Detector cell contaminated even 7 Clean cell by flushing with small amounts of contaminants 1N HNO never with HCI can cause noise 8 Weak detector lamp 8 Replace lamp 9 Column leaking silica 9 Replace column or packing material 10 Mobil phase mixer inadequate 10 Repair or replace the mixer or malfunctioning or mix off line if isocratic O Broad peaks POSSIBLE CAUSE SOLUTION 1 Mobile phase composition changed 1 Prepare new mobile phase 2 Mobile phase flow rate too low 2 Adjust flow rate 3 Leaks especially between column 3 See section Ill Check for loose and detector fittings Check pump for leaks salt build up and unusual noises Change seals if necessary 4 Detector settings incorrect 4 Adjust settings 5 Extra column effects 5 a Inject smaller volume e g a Column overloaded 10 uL vs 100 uL 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 0 007
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