HPLC Troubleshooting Guide - Sigma
Contents
1. Flush cell with methanol or other strong solvent If necessary clean cell with 1N HNO never with HCI and never use nitric acid with PEEK tubing or fittings Unplug or replace line Refer to detector manual to replace window Correct composition flow rate To avoid problem routinely monitor composition and flow rate Flush column with intermediate strength solvent run 10 20 column volumes of new mobile phase through column before analysis Check make up of mobile phase page 2 Use guard column If necessary flush column with strong solvent between injections or periodically during analysis Change wavelength to UV absor bance maximum Problem No 13 Baseline Noise regular Normal 794 0748 Problem 794 0765 Air in mobile phase detector cell or pump Pump pulsations Incomplete mobile phase mixing Temperature effect column at high temperature detector unheated Other electronic equipment on same line Leak Degas mobile phase Flush system to remove air from detector cell or pump Incorporate pulse damper into system Mix mobile phase by hand or use less viscous solvent Reduce differential or add heat exchanger Isolate LC detector recorder to determine if source of problem is external Correct as necessary Check system for loose fittings Check pump for leaks salt buildup unu2. P000550 Description Cat No Valco Precolumn Filters Frit Filter 58420 U Screen Filter 58279 U Frits pk of 10 0 5 um pores 59037 2 0 um pores 59129 Screens pk of 10 58284 3Frits and screens should not be interchanged in these filters Isolation Technologies Precolumn Filter In line installation O T High capacity inlet fil ter minimizes dead volume and band broadening to prevent loss of column efficiency while protecting your column Frit porosity 0 5 um Complete as shown Description Cat No Isolation Technologies Precolumn Filter with 3 mm frit 4 6 mm columns 57675 U with 1 5 mm frit 2 1 mm columns 57676 U Frits ok of 5 3mm 57677 1 5mm 57678 Upchurch Precolumn Filter In line installation Stainless steel body with inert polyetherether ketone PEEK endfittings and a 0 5 um or 2 um PEEK frit in one endfitting di Description Cat No Upchurch Precolumn Filter 0 5 um frit 55079 2 um frit 55078 Frits pk of 10 0 5 um 55080 U 2 um 55081 S IGMA ALDRICH Rheodyne Model 7725 and 77251 Injectors The Rheodyne Model 7725 injector allows you to inject 1 uL 5 mL samples with accuracy and precision The rugged easily maintained design offers many advanced features e Patented continuous flow design see figure flow is uninterrupted when you switch trom LOAD to INJECT e Easy seal adjustment using pressure screw on front of injector e Wide port angle 30 for easy acce
3. Probable Cause Remedy Comments Problem No 10 Fronting Peaks Normal 794 0760 Problem 794 0761 Column overloaded Sample solvent incompatible with mobile phase Shoulder or gradual baseline rise before a main peak may be another sample component 1 Inject smaller volume e g 10 uL vs 100 uL Dilute the sample 1 10 or 1 100 fold in case of mass overload 2 Adjust solvent Whenever possible inject samples in mobile phase Flush polar bonded phase column with 50 column volumes HPLC grade ethyl acetate at 2 3 times the Standard flow rate then with intermediate polarity solvent prior to analysis 3 Increase efficiency or change selectivity of system to improve resolution Try another column type if necessary e g switch from nonpolar C18 to polar cyano phase Problem No 11 Rounded Peaks Normal 794 0762 Problem 794 0763 U N Detector operating outside linear dynamic range Recorder gain set too low Column overloaded Sample column interaction Detector and or recorder time constants are set too high 1 Reduce sample volume and or concentration 2 Adjust gain 3 Inject smaller volume e g 10 uL vs 100 uL or 1 10 or 1 100 dilution of sample 4 Change buffer strength pH or mobile phase composition If necessary raise column temperature or change column type Analysis of solute structure may help predict
4. 50 mL methylene chloride 3 50 mL hexane 4 25 mL isopropanol 5 25 mL mobile phase Evaluate column performance Polar Bonded Phase Column amino cyano or diol column or Pirkle type chiral columns For a column used in the reversed phase mode e g organic solvent aqueous buffer mobile phase follow the same cleanup procedure as for silica based reversed phase columns For a column used with nonaqueous mobile phases use the following scheme Flush with the following 50 mL chloroform 50 mL methanol 50 mL acetonitrile 25 mL methylene chloride 25 mL methanol 25 mL mobile phase Evaluate column performance DUE TE Wa C83 C18 etc Note Volumes listed in Table 2 are for 25 cm x 4 6 mm I D columns which have a column volume of 4 15 mL When restoring a 4 6 mm I D column shorter or longer than 25 cm multiply all volumes in Table 2 by the ratio of the column length to 25 e g fora 15 cm column 15 25 or 0 6 times the volumes in Table 2 When restoring a column of internal diameter other than 4 6 mm multiply all volumes in Table 2 by the ratio of the square of the column I D to 4 6 e g for a 3 2 mm I D column 3 2 A4 6 10 24 21 16 0 48 times the values in Table 2 SUPELCOSIL LC PCN Column A To Remove Protein Flush with 10 column volumes of acetonitrile water 50 50 containing 0 1 trifluoroacetic acid B To Remove TCA Flush with 10 column volumes of distilled water adjust pH to 2 5 with
5. H PO then with 10 column volumes each of 1 water to remove salts 2 methanol to remove water 3 methanol methylene chloride 50 50 a general clean up solution 4 methanol If column performance still is not acceptable prepare the mobile phase buffer at 10X the concentration used for the analysis and recycle through the column overnight Reequilibrate the column with mobile phase at the normal buffer concentration and reevaluate Use caution with some buffer types and or concentrations a 10 fold increase in concentration can cause precipitation Silica Based lon Exchange Columns strong or weak anion or cation exchange Most analyses involving ion exchange systems use ionic mobile phases Compounds that may affect column performance are usually insoluble or only slightly soluble in water The following procedure should be sufficient to remove these compounds Flush with the following 50 mL hot 40 60 distilled water 50 mL methanol 50 mL acetonitrile 25 mL methylene chloride 25 mL methanol 25 mL mobile phase Evaluate column performance NUBRWN gt Silica Based Columns for RPLC of Proteins and Peptides Follow the protocol for silica based reversed phase columns Alternatively make one or more 100 uL injections of trifluoroethanol determine the number of injections by evaluating column performance after each injection Evaluate column performance S IGMA ALDAICFH Table 3 Properties
6. Same analytical column after replacing Supelguard column Analytical column plus Supelguard column after 100 serum extract injections Trimipramine int std Doxepin 3 Amitriptyline 1 Imipramine Desmethyldoxepin Nortriptyline Desipramine 4 Protriptyline int std 8 56 CONDUBRWN gt 794 0809 794 0810 Getting the Most from Your Analytical Column Regardless of whether the column contains a bonded reversed or normal phase ion exchange affinity hydrophobic interaction size exclusion or resin silica based packing the most common problem associated with analytical columns is deterioration Symptoms of deterioration are poor peak shape split peaks shoulders loss of resolution decreased retention times and high back pressure These symptoms indicate contaminants have accumulated on the frit or column inlet or there are voids channels or a depression in the packing bed Deterioration is more evident in higher efficiency columns For example a 3 micron packing retained by 0 5 micron frits is more susceptible to plugging than a 5 or 10 micron packing retained by 2 micron or larger frits Proper column protection and sample preparation are essential to getting the most from each column Overloading a column can cause poor peak shapes and other problems Column capacity depends on many factors but typical values for total amounts of analytes on a column are Analytical column 25 cm x 4 6
7. The Autoclean valve is especially useful if you are using a single pump with mobile phases containing buffer or other salts The Autoclean valve installs between the mobile phase reservoir and Threshold the pump It has two inlet lines one for the mobile phase and one for the wash solvent The valve can be factory installed or you can 0 1 2 3 A 5 6 order it separately and install it yourself Min E Economy Priced Unit The economically priced SRS 1000 includes the same solvent saving features as the SRS 3000 unit A simpler display and no advanced features no validation output Autoclean option or Description Cat No method storage memory allow us to keep the price substantially lower SRS 3000 Solvent Recovery System 57431 Both Systems are Ready to Use Both systems include a control SRS 3000 System with Autoclean 57432 unit with switching valve a power cord a 2 lead signal cable Switching valve assembly for SRS 3000 unit 97435 I l SRS 1000 Solvent Recovery System Teflon tubing and fittings and an instruction manual 110 VAC 506125 In addition to these components the SRS 3000 system with the 220 VAC 506133 Autoclean valve has the wash valve additional tubing and fittings CE approved a wash start cable and a pump remote stop cable The SRS 3000 and SRS 1000 units meet all CE requirements The SRS 1000 units also meet UL and CSA requirements World Headquarters Order Customer Service 800 325 3010 e F
8. 0771 Problem 794 0772 Check make up of mobile phase page 2 2 Confirm identity of column packing For reproducible analyses use same column type Establish whether change took place gradu ally If so bonded phase may have stripped Column activity may have changed or column may be contaminated 3 Adjust solvent Whenever possible inject sample in mobile phase 4 Adjust temperature If needed use column oven to maintain constant temperature S IGMA ALDRICH Problem Probable Cause Remedy Comments Problem No 18 Negative Peak s Normal 1 Recorder leads reversed 1 Check polarity 2 Refractive index of solute less than 2 Use mobile phase with lower that of mobile phase RI detector refractive index or reverse recorder leads 3 Sample solvent and mobile phase 3 Adjust or change sample solvent differ greatly in composition vacancy Dilute sample in mobile phase peaks whenever possible 4 Mobile phase more absorptive than 4 a Change polarity when using 794 0747 sample components to UV wavelength indirect UV detection or b Change UV wavelength or use mobile phase that does not adsorb chosen wavelength Problem 794 0773 Problem No 19 Ghost Peak Previous 1 Contamination in injector or column Flush injector between analyses a Sample good routine practice If necessary run strong solvent through column to remove late eluters Incl
9. If problem still persists contact system manufacturer S IGMA ALDAICH Problem Probable Cause Remedy Comments Problem No 3 No Pressure Pressure Lower Than Usual Normal 794 0750 Problem Ul 794 0751 CO N Leak Mobile phase flow interrupted obstructed Air trapped in pump head Revealed by pressure fluctuations Leak at column inlet end fitting Air trapped elsewhere in system Worn pump seal causing leaks around pump head Faulty check valve Faulty pump seals Ul oo N Check system for loose fittings Check pump for leaks salt buildup unusual noises Change pump seals if necessary Check mobile phase level in reservoir s Check flow throughout system Examine sample loop for obstruction or air lock Make sure mobile phase components are miscible and mobile phase is properly degassed Disconnect tubing at guard column if present or analytical column inlet Check for flow Purge pump at high flow rate e g 10 mL min prime system if necessary Prime each pump head separately If system has check valve loosen valve to allow air to escape Reconnect column and pump solvent at double the flow rate If pressure is still low check for leaks at inlet fitting or column end fitting Disconnect guard and analytical column and purge system Recon nect column s If problem persists flush system w
10. SRS 1000 Data System Solvent Recovery System 1 Connect SRS 3000 or SRS 1000 unit to detector signal output cable is included 2 Connect SRS 3000 or SRS 1000 unit to mobile phase and waste reservoirs and directs the solvent to the waste reservoir only when a peak is detected detector Teflon tubing is included When the baseline falls below the threshold you select the uncon 3 Set the threshold value and begin saving time and money ET taminated solvent is directed back to the mobile phase reservoir In a typical isocratic analysis 80 90 of the mobile phase is uncontami nated and can be recycled Settings for threshold detection range Recover 80 or More of the Mobile Phase and delay time enable you to precisely control the switching valve Used In an Isocratic Analysis In addition to the basic features mentioned above the SRS 3000 3 unit offers validation output included an Autoclean option see below and storage for up to 10 method files The validation output me ONCE REC CIcO 1 Uracil provides a continuous auditable data trail of the solvent recycling a ea or TTA valve position for GMP GLP or ISO 9000 protocols The valve A N N Diethyl m toluamide position is recorded by superimposing tick marks over a separate 3 o ee copy of the chromatographic signal Autoclean Valve The SRS 3000 system also is available with a valve that enables you to select a different solvent to flush the HPLC system
11. growth by adding about 100 ppm of sodium azide to aqueous buffers Alternatively these buffers may also be mixed with 20 or more of an organic solvent such as ethanol or acetonitrile Figure A Components of an HPLC System Injector 3 Debubbler Pre injector Filter l l l Solvent Reservoir and Inlet Filters Column E Pulse Damper i i Precolumn gis EER T Filter Pump rT Column and Integrated Guard To prevent bubbles in the system degas the mobile phase Gener ally an in line degasser is a first choice but sparging with helium can be an alternative if the mobile phase does not contain any volatile components Use ion pair reagents carefully The optimum chain length and concentration of the reagent must be determined for each analysis Concentrations can be as low as 0 2 mM or as high as 150 mM or more In general increasing the concentration or chain length increases retention times High concentrations gt 50 of acetoni trile or some other organic solvents can precipitate ion pair re agents Also some salts of ion pair reagents are insoluble in water and will precipitate Avoid this by using sodium containing buffers in the presence of long chain sulfonic acids e g sodium dodecyl sulfate instead of potassium containing buffers Volatile basic and acidic modifiers such as triethylamine TEA and trifluoracetic acid TFA are useful when you wish to recove
12. 23 a Teflon AF membrane with NO OX fittings and tubing Description Cat No Supelco Mobile Phase Degassing System 55018 U Refer to the current Supelco catalog for many additional products and for prices 17 S IGMA ALDAICFH Filters A precolumn filter is essential for protecting HPLC columns against particulate matter which can accumulate on the column frit leading to split peaks and high back pressure Sources of particles include mobile phases especially when buffers are mixed with organic solvents pump and injector seals and samples Use a 2 0 um frit to protect columns containing 5 um or larger particles or a 0 5 um frit for columns with particles smaller than 5 um Supelco Filter Direct connect protects analytical and guard columns Our precolumn filter can be connected directly hand tight into any HPLC column or guard column listed in our current catalog or with any other column that has Valco compatible endfittings PEEK cap and body 2 um stainless steel frit For a metal free system order PEEK Teflon replacement frits ia Cat No 57430 U Description Cat No Supelco Precolumn Filter Z22732 3 Frits ok of 5 0 5 um pores Z29087 4 2 um pore Z22733 1 PEEK Teflon 2 um 57430 U Biocompatible metal free jaa ZA 4 s k JU EEE ii Jota j or E S a su 2 uw X P000548 SSI High Pressure Precolumn Filter In line installation The 316 stainless steel filter disc 0 5 um p
13. E 794 0756 1 Mobile phase contaminated deterio rated causing retention times and or selectivity to change 2 Obstructed guard or analytical column 1 Prepare fresh mobile phase page 2 Remove guard column if present and attempt analysis Replace guard column if necessary If analytical column is obstructed reverse and flush page 14 If problem persists column may be clogged with strongly retained contaminants Use appropriate restoration procedure Table 2 page 14 If problem still persists change inlet frit page 16 or replace column S IGMA ALDRICH Problem Probable Cause Remedy Comments Problem No 7 Split Peaks Normal 1 Contamination on guard or analytical column inlet l 794 0757 booo o oS Problem 2 Partially blocked frit 3 Small uneven void at column inlet 4 Sample solvent incompatible with mobile phase 794 0758 1 Remove guard column if present and attempt analysis Replace guard column if necessary If analytical column is obstructed reverse and flush page 14 If problem persists column may be clogged with strongly retained contaminants Use appropri ate restoration procedure Table 2 page 14 If problem still persists inlet frit is probably partially plugged Change frit page 16 or replace column 2 Replace frit see above 3 Repack top of column with pellicular particles of same bonded phase functionality Continue u
14. SIGMA ALDRICH ee 595 North Harrison Road Bellefonte PA 16823 0048 USA Tel 800 247 6628 814 359 3441 Fax 800 447 3044 814 359 3044 Bulletin 826E HPLC Troubleshooting Guide How to identify isolate and correct the most common HPLC problems Although HPLC method development has been im proved by advances in column technology and instru mentation problems still arise In this guide we offer you a systematic means of isolating identifying and correcting many typical problems The important segments of an HPLC system are the same whether you use a modular system or a more sophisticated unit Problems affecting overall system performance can arise in each component Some com mon problems are discussed here Solutions to these problems are presented in easy to use tables E000648 CONTENTS Isolating HPLC Problems How to Prevent Mobile Phase Problems Isolating Pump Problems Injector and Injection Solvents Column Protection Getting the Most from Your Analytical Column Solving Detector Problems Column Heater Recorder Keeping Accurate Records Problem Index HPLC Problems Causes and Remedies Restoring Your Column s Performance Preventing and Solving Common Fitting Problems A Selection of Column Protection Products sigma aldrich com SIGMA ALDAICH Isolating HPLC Problems In an HPLC system problems can arise from many sources First define the problem then isolate the source Use Table 1 pa
15. ax 800 325 5052 Accelerating Customers ne o Sr OU ME DI Technical Service 800 325 5832 sigma aldrich com techservice Success through Innovation and sigma aldrich com Development Custom Manufacturing Inquiries SAFC 800 244 1173 Leadership in Life Science High Technology and Service 2009 Sigma Aldrich Co All rights reserved SIGMA OF SAFC SAFC SIGMA ALDRICH ALDRICH O FLUKA a and SUPELCO are trademarks belonging to Sigma Aldrich Co and its affiliate Sigma Aldrich Biotechnology L P Sigma brand products are sold through Sigma Aldrich Inc Sigma Aldrich Inc warrants that its products conform to the information contained in this and other Sigma Aldrich publications Purchaser must determine the suitability of the product s for their particular use Additional terms and conditions may apply Please see reverse side of the invoice or packing slip T100826E AIV SIGMA ALDRICH
16. chromatography A sure sign of a leak is a buildup of salts at a pump connection Buffer salts should be flushed from the system daily with fresh deionized water To isolate and repair specific problems related to your apparatus use the troubleshooting and maintenance sections of the operation manual Pump seals require periodic replacement You should perform regular maintenance rather than waiting for a problem to occur Injector and Injection Solvents The injector rapidly introduces the sample into the system with minimal disruption of the solvent flow HPLC systems currently use variable loop fixed loop and syringe type injectors These are activated manually pneumatically or electrically Mechanical problems involving the injector e g leaks plugged capillary tubing worn seals are easy to spot and correct Use a pre column filter to prevent plugging of the column frit due to physical degradation of the injector seal Other problems such as irreproducible injections are more difficult to solve Variable peak heights split peaks and broad peaks can be caused by incompletely filled sample loops incompatibility of the injection solvent with the mobile phase or poor sample solubility Whenever possible dissolve and inject samples in mobile phase Otherwise be sure the injection solvent is of lower eluting strength than the mobile phase Table 3 Be aware that some autosamplers use separate syringe washing solutions Make sur
17. correct 4 Adjust settings 5 Extra column effects 5 a Inject smaller volume e g 10 uL 794 0767 Problem 794 0768 a Column overloaded b Detector response time or cell volume too large c Tubing between column and detector too long or I D too large d Recorder response time too high 6 Buffer concentration too low ooo N g Guard column contaminated worn out Column contaminated worn out Void at column inlet 10 Peak represents two or more poorly 11 resolved compounds Column temperature too low CON O 9 vs 100 uL or 1 10 and 1 100 dilutions of sample b Reduce response time or use smaller cell c Use as short a piece of 0 007 0 010 I D tubing as practical d Reduce response time Increase concentration Replace guard column Replace column with new one of same type If new column does not provide narrow peaks flush old column Table 2 page 14 then retest Replace column or open inlet end and fill void page 16 10 Change column type to improve separation 11 Increase temperature Do not exceed 75 C unless higher temperatures are acceptable to column manufacturer S IGMA ALDAICH Problem Probable Cause Remedy Comments Problem No 16 Change in Peak Height one or more peaks Normal 1 One or more sample components deteriorated or column activity changed 2 Leak especially between injection port and colum
18. e between detector and integrator or recorder 3 No mobile phase flow 4 No sample deteriorated sample wrong sample 5 Settings too high on detector or recorder Turn lamp on 2 Check electrical connections and cables 3 See No Flow Problem No 2 4 Be sure automatic sampler vials have sufficient liquid and no air bubbles in the sample Evaluate system performance with fresh standard to confirm sample as source of problem 5 Check attenuation or gain settings Check lamp status Auto zero if necessary Problem No 2 No Flow Normal 794 0747 Problem ESS 794 0748 1 Pump off 2 Flow interrupted obstructed 3 Leak 4 Air trapped in pump head Revealed by pressure fluctuations 1 Start pump 2 Check mobile phase level in reservoir s Check flow throughout system Examine sample loop for obstruction or air lock Make sure mobile phase components are miscible and mobile phase is properly degassed 3 Check system for loose fittings Check pump for leaks salt buildup unusual noises Change pump seals if necessary 4 Disconnect tubing at guard column if present or analytical column inlet Check for flow Purge pump at high flow rate e g 5 10 mL min prime system if necessary Prime each pump head separately If system has check valve loosen valve to allow air to escape If problem persists flush system with 100 methanol or isopropanol
19. e that the wash solution is compatible with and weaker than the mobile phase This is especially important when switching between reversed and normal phase analyses Column Protection Although not an integral part of most equipment mobile phase inlet filters pre injector and pre column filters and guard columns greatly reduce problems associated with complex separations We recommend that all samples be filtered through 0 45 um or 0 2 um syringe filters We strongly recommend the use of guard columns Filters and guard columns prevent particles and strongly retained compounds from accumulating on the analytical column The useful life of these disposable products depends on mobile phase composition sample purity pH etc As these devices become contaminated or plugged with particles pressure increases and peaks broaden or split As an example Figure B presents a clear case for the use of guard columns For more about column protection see the product pages of this guide and request Bulletin 781 Figure B Supelguard Columns Prolong the Lifespan of Your Analytical Columns SUPELCOSIL LC PCN 25 cm x 4 6 mm I D 5 um particles with Supelguard LC PCN guard column 58378 25 60 15 0 01 M potassium phosphate pH to 7 w 85 phosphoric acid acetronitrile methanol flow rate 2 mL min temp 30 C det UV 215 nm injection 100 uL reconstituted SPE eluant 20 ng mL each analyte and int std in serum column mobile phase
20. ed polar material Pump the solution through the affected column for 10 minutes at a rate of 4 mL minute then flush with mobile phase for 10 minutes at arate of 2 mL minute Evaluate column performance by using the test mixture for evaluating silica columns Cat No 58281 Perfor mance should be virtually the same as before the polar solvent was introduced Figure C3 Silica Column Regeneration Solution 200 mL 33175 Column Test Mixes Performance evaluation mixes for HPLC columns Well defined test mixes enable you to troubleshoot chromato graphic problems optimize system efficiency and evaluate col umns under conditions where their performance is understood We ship our test mixes in amber ampuls to prevent photodegradation and we include instructions for proper use and interpretation of results Choose from column specific or application specific mixes Refer to our catalog for our extensive selection of test mixes or call our Technical Service Department C1 2 propanol water Fig C2 99 4 0 5 0 1 Silica Column Regeneration Solution 4 mL min for 10 min then methylene chloride methanol water 2 mL min for 10 min Fig C3 flow rate 2 mL min temp ambient det UV 254 nm injection 10 uL Fig C3 obtained after equilibration 1 Benzene 2 Benzanilide C1 Properly Performing Column 3 Acetanilide 1 2 C2 Same Column Exposed to Water and Alcohol 1 C3 Same Column Treated 2 with Regeneration So
21. etc you can monitor your system s performance Records also help prevent mistakes such as introducing water into a silica column or precipitating buffer in the system by adding too much organic solvent Many analysts modify their HPLC systems in some way Reliable records are the best way to ensure that a modification does not introduce problems For problems relating to pumps detectors automatic samplers and data systems consult your instrument manual s troubleshooting guide Problem Index Problem Problem No Baseline drift 12 noise irregular 14 noise regular 13 Column back pressure higher than usual 4 lower than usual 3 Ghost peaks 19 Peak shapes incorrect broad 15 fronting 10 rounded 11 split 7 tailing 8 9 Peaks height change 16 missing 2 negative 18 no peaks 1 unresolved 6 Retention times variable 5 Selectivity change 17 Trademarks FPLC Amersham Pharmacia Biotech Iso Disc Pelliguard Sigma Aldrich Supelco SUPELCOSIL Supelguard Trizma Sigma Aldrich Co LO Pulse Scientific Systems Inc Rheodyne Rheodyne Inc Swagelok Crawford Fitting Co Teflon E I du Pont de Nemours amp Co Inc S IGMA ALDRICH Table 1 Problem HPLC Problems Probable Causes and Remedies Probable Cause Remedy Comments Problem No 1 No Peaks Very Small Peaks Normal 794 0747 Problem 794 0748 Problem 794 0749 1 Detector lamp off 2 Loose broken wir
22. for a more stable baseline The SSI LO Pulse damper is a patented unit compatible with single piston reciprocating HPLC pumps Altex 110A Eldex pumps LDC Mini Pump VS SSI Models 200 and 300 etc At pressures from 500 psi to 6000 psi 35 420 kg cm it improves precision of quantitative analyses and detection limits for trace sample compo ject to wear will help you ki nents Fittings and instructions included avoid costly downtime Our extensive selection of Optimize Tech nologies check valves seals and pistons meet or exceed pump manufacturers specifications For the most up to date selection of Pulse Damper 58455 Description Cat No pump parts refer to the current Supelco catalog or call our Pulse Damper without Cabinet 58442 Technical Service Department S IGMA ALDRICH Supelco Solvent Recovery Systems Installation Is Simple SRS 3000 SRS 1000 Autoclean Valve option with SRS 3000 995 0148 397 0035 Flush Mobile f eres 27 Waste Recover and reuse clean mobile phase A san t PEE dispose of only contaminated mobile phase e Reduce solvent purchase and disposal costs e Save money mobile phase preparation time and the environment Supelco SRS 3000 and SRS 1000 Solvent Recovery Systems can Save money and time in any isocratic analysis A microprocessor controlled solvent switching valve monitors detector output and A N SRS 3000 or
23. ge 5 to determine which component s may be causing the trouble A process of elimination will usually enable you to pinpoint the specific cause and correct the problem How to Prevent Mobile Phase Problems Low sensitivity and rising baselines noise or spikes on the chro matogram can often be attributed to the mobile phase Contami nants in the mobile phase are especially troublesome in gradient elution The baseline may rise and spurious peaks can appear as the level of the contaminated component increases Water is the most common source of contamination in reversed phase analyses You should use only high purity distilled or deion ized water when formulating mobile phases However several common deionizers introduce organic contaminants into the water To remove these contaminants pass the deionized water through activated charcoal or a preparative C18 column Use only HPLC grade solvents salts ion pair reagents and base and acid modifiers Cleaning lower quality solvents is time consuming and trace levels of contaminants often remain These trace contami nants can cause problems when you use a high sensitivity ultraviolet or fluorescence detector Because many aqueous buffers promote the growth of bacteria or algae you should prepare these solutions fresh and filter them 0 2 um or 0 45 um filter before use Filtering also will remove particles that could produce a noisy baseline or plug the column Prevent microorganism
24. he detector and pump solvent through at twice the standard flow rate About 5 10 column volumes of solvent should be sufficient to dislodge small amounts of particulate material on the inlet frit Evaluate the performance of the cleaned column using a standard test mixture Replacing a Frit at the Column Inlet Sometimes neither solvent flushing see above nor restoration procedures see Table 2 restore a column s performance If you ve Figure D Typical HPLC Column Designs A Column with Conventional Endfittings 1 16 Ferrule 1 16 Nut isolated the column as the problem source and other restorative procedures have failed a void in the packing or a persistent obstruction on the inlet frit may exist Asa last resort open the inlet end of the column Caution opening the inlet end and more so opening the outlet end can permanently damage the packing bed Before opening columns consult the manufacturer s literature Never open either end of a resin filled column Use the following procedure to open a column 1 Disconnect the column from the system To prevent the packing from oozing out of the column perform subsequent steps as quickly as possible 2 Using a vise and wrench or two wrenches carefully remove the inlet end fitting see Figure D If the frit remains in the fitting dislodge it by tapping the fitting on a hard surface If the frit stays on the column slide it off rather than litt it off This will
25. help preserve the integrity of the packing bed Modular columns may require a special tool e g Cat No 55216 to remove the frit cap 3 Examine the old frit Compression of the frit against the Stainless steel tubing will leave a ring around the edge on the column side of a properly seated frit No ring can mean the ferrule is seated too near the tubing end The resulting loose connection can leak silica or act as a mixing chamber 4 Examine the packing bed If it is depressed or fractured you need a new column Replace the frit 6 Replace the end fitting Screw it down fingertight then tighten 1 4 turn with a wrench B Modular Column with Reusable Endfittings Column Packing 794 0785 Endfitting 794 0786 S IGMA ALDAICH A Selection of Column Protection Products Supelco Mobile Phase Filtration Apparatus Filtration Apparatus 1 connects to 1000 mL sidearm flask connect to aspiration line Includes 250 mL glass reservoir funnel base and stopper clamp Stainless steel holder and screen 10 Teflon gaskets 50 Nylon 66 filters 47 mm 0 45 um pores Filtration Apparatus 2 connects to aspiration line Includes 250 mL glass reservoir 34 45 tapered funnel base 34 45 tapered 1000 mL flask and glass cap clamp stainless steel holder and screen 10 Teflon gaskets 50 Nylon 66 filters 47 mm 0 45 um 250 mL Reservoir pores Description Cat No Supelco Mobile Phase Filtration Apparatus Funne
26. interaction 5 Reduce settings to lowest values or values at which no further improve ments are seen S IGMA ALDAICH Problem Probable Cause Remedy Comments Problem No 12 Baseline Drift Normal a 794 0748 Problem oo 794 0764 Column temperature fluctuation Even small changes cause cyclic baseline rise and fall Most often affects refractive index and conductivity detectors UV detectors at high sensitivity or in indirect photometric mode Nonhomogeneous mobile phase Drift usually to higher absorbance rather than cyclic pattern from temperature fluctuation Contaminant or air buildup in detector cell Plugged outlet line after detector High pressure cracks cell window producing noisy baseline Mobile phase mixing problem or change in flow rate Slow column equilibration especially when changing mobile phase Mobile phase contaminated dete riorated or not prepared from high quality chemicals Strongly retained materials in sample high k can elute as very broad peaks and appear to be a rising baseline Gradient analyses can aggravate problem Detector UV not set at absorbance maximum but at slope of curve Control column and mobile phase temperature use heat exchanger before detector Use HPLC grade solvents high purity salts and additives Degas mobile phase before use sparge with helium during use
27. ith 100 methanol or isopropanol Replace seal If problem persists replace piston and seal Rebuild or replace valve Replace seals Problem No 4 Pressure Higher Than Usual Normal 2 794 0750 Problem 794 0752 Problem in pump injector in line filter or tubing Obstructed guard column or analytical column Remove guard column and analytical column from system Replace with unions and 0 010 D or larger tubing to reconnect injector to detector Run pump at 2 5 mL min If pressure is minimal see Cause 2 If not isolate cause by systematically eliminating system components starting with detector then in line filter and working back to pump Replace filter in pump if present Remove guard column if present and check pressure Replace guard column if necessary If analytical column is obstructed reverse and flush the column while disconnected from the detector page 14 If problem persists column may be clogged with strongly retained contaminants Use appropriate restoration procedure Table 2 page 14 If problem still persists change inlet frit page 16 or replace column S IGMA ALDAICFH Problem Probable Cause Remedy Comments Problem No 5 Variable Retention Times Normal 794 0753 Problem 794 0747 Problem 794 0754 1 Leak 2 Change in mobile phase composition Small changes ca
28. l Base Filtration Apparatus 1 58061 Filtration Apparatus 2 58062 U Replacement Glass Parts For Filtration Apparatus 1 Reservoir 250 mL 58063 Reservoir 500 mL 58074 Funnel Base and Stopper 58064 For Filtration Apparatus 2 Tapered Funnel Base 58068 Tapered Flask 1000 mL for Apparatus 2 58070 U Tapered Flask 2000 mL for Apparatus 2 58075 Cap for Flask for Apparatus 2 58071 Replacement Filter Parts for Both Apparatus Filter Holder and Screen SS 58065 en Gaskets Teflon pk of 10 58066 Protect your instrument and columns by removing particles and Filter Nylon 66 47 mm pk of 50 gases from solvents and other mobile phase components Nylon 66 0 45 um pores 58067 membrane filters are compatible with all solvents commonly used 0 20 um pores 38060 U in HPLC Clamp Spring Action 58053 Supelco Mobile Phase Degassing System e 4 Channel e 0 5 mL min Flow Range Validation output satisfies system compliance e Smart sensor detects leaks and communicates with vacuum pump e LED indicates degassing status e Unprecedented 4 year warranty The Supelco Mobile Phase Degassing system with its smart sensor not only detects and alerts you to leaks it also communicates with the vacuum pump If a change in vacuum is detected due to mobile phase flow rate changes the pump can compensate by changing its speed The validation output records vacuum level to satisfy system pmu compliance and validation requirements The degassing system has E0009
29. lution 1 2 3 3 3 1 tt t tt tot 0 2 4 6 Min I ll O 2 Min 1t tt t tT 1 2 4 6 Min 794 0787 0788 0789 S IGMA ALDRICH Preventing and Solving Common Hardware Problems Preventing Leaks Leaks are a common problem in HPLC analyses To minimize leaks in your system avoid interchanging hardware and fittings from ditferent manufacturers Incompatible fittings can be forced to fit initially but the separation may show problems and repeated connections may eventually cause the fitting to leak If interchang ing is absolutely necessary use appropriate adapters and check all connections for leaks before proceeding Highly concentrated salts gt 0 2 M and caustic mobile phases can reduce pump Seal efficiency The lifetime of injector rotor seals also depends on mobile phase conditions particularly operation at high OH In some cases prolonged use of ion pair reagents has a lubricating effect on pump pistons that may produce small leaks at the seal Some seals do not perform well with certain solvents Before using a pump under adverse conditions read the instrument manufacturer s specifications To replace seals refer to the mainte nance section of the pump manual Unclogging the Column Frit A clogged column frit is another common HPLC problem To minimize this problem trom the start use a precolumn filter and guard column To clean the inlet first disconnect and reverse the column Connect it to the pump but not to t
30. mm lt 500 ug Semi preparative column 25 cm x 10 mm lt 100 mg Preparative column 25 cm x 21 mm lt 500 mg S IGMA ALDAICH Solving Detector Problems Detector problems fall into two categories electrical and me chanical optical For electrical problems you should contact the instrument manufacturer Mechanical or optical problems usually can be traced to the flow cell Detector related problems include leaks air bubbles and cell contamination These usually produce spikes or baseline noise on the chromatograms or low sensitivity Some cells especially those used in refractive index detectors are sensitive to pressure Flow rates or back pressures that exceed the manufacturer s recommendation will break the cell window Old or defective lamps as well as incorrect detector rise time gain or attenuation will reduce sensitivity and peak height Faulty or reversed cable connections can also be the source of problems Column Heater Recorder These components seldom cause problems with the system They will be discussed in the troubleshooting table Table 1 Keeping Accurate Records Most problems don t occur overnight but develop gradually Accurate record keeping then is vital to detecting and solving many problems Evaluate every column you receive when you receive it and at regular intervals thereafter By keeping a written history of column efficiency mobile phases used lamp current pump performance
31. n inlet Retention also would change 794 0769 Problem 3 Inconsistent sample volume 4 Detector or recorder setting changed 5 Weak detector lamp 6 Contamination in detector cell 794 0770 1 Use fresh sample or standard to confirm sample as source of problem If some or all peaks are still smaller than expected replace column If new column improves analysis try to restore the old column following appropriate procedure Table 2 page 14 If performance does not improve discard old column Check system for loose fittings Check pump for leaks salt buildup unusual noises Change pump seals if necessary Be sure samples are consistent For fixed volume sample loop use 2 3 times loop volume to ensure loop is completely filled Be sure automatic sampler vials contain sufficient sample and no air bubbles Check syringe type injectors for air In systems with wash or flushing step be sure wash solution does not precipitate sample components Check settings Replace lamp Clean cell N UJ nus Problem No 17 Change in Selectivity Normal i Increase or decrease solvent ionic strength pH or additive concentration especially affects ionic solutes Column changed new column has different selectivity from that of old column UJ N Sample injected in incorrect solvent or excessive amount 100 200 uL of strong solvent 4 Column temperature change 794
32. n lead to large changes in retention times 3 Air trapped in pump Retention times increase and decrease at random times 4 Column temperature fluctuations especially evident in ion exchange systems 5 Column overloading Retention times usually decrease as mass of solute injected on column exceeds column capacity 6 Sample solvent incompatible with mobile phase 7 Column problem Not a common cause of erratic retention As a column ages retention times gradually decrease Check system for loose fittings Check pump for leaks salt buildup unusual noises Change pump seals if necessary Check make up of mobile phase If mobile phase is machine mixed using proportioning values hand mix and supply from one reservoir Purge air from pump head or check valves Change pump seals if necessary Be sure mobile phase is degassed Use reliable column oven Note higher column temperatures increase column efficiency For optimum results heat eluant before introducing it onto column Inject smaller volume e g 10 uL vs 100 uL or inject the same volume after 1 10 or 1 100 dilutions of sample Adjust solvent Whenever possible inject samples in mobile phase Substitute new column of same type to confirm column as cause Discard old column if restoration procedures fail see page 14 Problem No 6 Loss of Resolution Normal F 794 0755 Problem
33. of Organic Solvents Commonly Used in HPLC Miscible with Water UV Refractive Index Solvent Strength Viscosity Solvent Polarity Cutoff at 20 C e silica at 20 C cP Hexane nonpolar no 200 1 3750 0 00 0 33 lsooctane no 200 1 3910 0 01 0 50 Carbon tetrachloride no 263 1 4595 0 14 0 97 Chloroform no 245 1 4460 0 31 0 57 Methylene chloride no 235 1 4240 0 32 0 44 Tetrahydrofuran yes 215 1 4070 0 33 0 55 Diethyl ether no 215 1 3530 0 29 0 23 Acetone yes 330 1 3590 0 43 0 32 Ethyl acetate poorly 260 1 3720 0 45 0 45 Dioxane yes 215 1 4220 0 49 1 54 Acetonitrile yes 190 1 3440 0 50 0 37 2 Propanol yes 210 1 3770 0 63 2 30 Methanol yes 205 1 3290 0 73 0 60 Water yes 1 3328 gt 0 73 1 00 polar Typical values Nonbonded Silica Columns Exposed to Polar Solvent Figure C Regeneration Solution Restores Samples and mobile phases containing very strongly polar solvents Silica Column Performance such as water or alcohols can deactivate uncoated silica HPLC column SUPELCOSIL LC Si 15 cm x 4 6 mm I D 3 um packing 58981 aimn ET drastically aert Column performance particu mobile phase 50 50 methylene chloride methanol water 99 4 0 5 0 1 Fig larly solute retention and selectivity Figure C2 Even prolonged column flushing with a nonpolar solvent only partially restores column performance while wasting chemicals A silica regeneration solution quickly and inexpensively restores silica column performance by removing trapp
34. ores is easily replaced without removing the column end fitting Maxi mum operating pressure 15 000 psi 1054 kg cm For 1 16 tubing Description Cat No SSI High Pressure Precolumn Filter 10 32 Threads 59269 Waters Threads 59271 Filter Elements and Seals pk of 10 0 5 um pores 59273 2 um pores 59272 2 Most HPLC fittings except SSI and some Waters fittings have 10 32 threads SSI High Pressure Preinjector Filter Place between the pump se and injector to provide fi S y nal filtration for the mo SS bile phase Easily replaced A 316 stainless steel filter element 0 5 um pores Maximum operating pressure 15 000 psi 105MPa For 1 16 O D tubing 10 32 threads P000547 Description Cat No SSI High Pressure Preinjector Filter 59262 U Replacement Filter Elements and Seals pk of 2 0 5 um pores 59264 2 um pores 59265 Most HPLC fittings except SSI and some Waters fittings have 10 32 threads 18 Valco Precolumn Frit and Screen Filters In line installation Efficient low dead volume filters protect your columns from particles without reducing column per formance The replaceable 1 8 frit has 0 5 um pores to protect 3 um or 5 um column packings the replaceable screen has 2 um pores Choose the frit filter for higher filtration capacity most applications or the screen filter for less dead volume e g with microbore columns Use with 1 16 O D tubing 1 16 fittings included
35. r a compound for further analysis These modifiers also let you avoid problems associated with ion pair reagents They can be added to the buffer at concentrations of 0 1 to 1 0 TEA or 0 01 to 0 15 TFA Increasing the concentration may improve peak shape for certain compounds but can alter retention times Recycling the mobile phase used for isocratic separations has become more popular in recent years as a means of reducing the cost of solvents their disposal and mobile phase preparation time An apparatus such as the Supelco SRS 3000 or SRS 1000 Solvent Recovery System uses a microprocessor controlled switching valve to direct the solvent stream to waste when a peak Is detected When the baseline falls under the selected threshold uncontaminated solvent is directed back to the solvent reservoir Colac Solvent Oven Return zani Back Pressure Line a Regulator Detector od oo pn _ Waste Solvent Recovery System Data System la m e e e e eee eee e 794 0746 S IGMA ALDRICH Isolating Pump Problems The pump must deliver a constant flow of solvent to the column over a wide range of conditions Modern HPLC pumps incorpor ate single or dual piston syringe or diaphragm pump designs Pumping system problems are usually easy to spot and correct Some of the more common symptoms are erratic retention times noisy baselines or spikes in the chromatogram Leaks at pump fittings or seals will result in poor
36. sing the column in reverse flow direction 4 Adjust solvent Whenever possible inject samples in mobile phase Problem No 8 Peaks Tail on Initial and Later Injections Normal 1 Sample reacting with active sites 2 Wrong mobile phase pH 794 0747 3 Wrong column type Problem l 4 Small uneven void at column inlet 5 Wrong injection solvent 794 0759 1 First check column performance with standard column test mixture If results for test mix are good add ion pair reagent or competing base or acid modifier page 2 2 Adjust pH For basic compounds lower pH usually provides more symmetric peaks 3 Try another column type e g deactivated column for basic compounds 4 See Problem No 7 5 Peaks can tail when sample is injected in stronger solvent than mobile phase Dissolve sample in mobile phase Problem No 9 Tailing Peaks Guard or analytical column contamin Normal ated worn out Mobile phase contaminated deteriorated Interfering components in sample 794 0747 WU N Problem 794 0759 1 Remove guard column if present and attempt analysis Replace guard column if necessary If analytical column is source of problem use appropriate restoration procedure Table 2 page 14 If problem persists replace column 2 Check make up of mobile phase page 2 3 Check column performance with standards S IGMA ALDAICFH Problem
37. ss to fittings Injector includes a 20 uL sample loop and is supplied with a VESPEL rotor seal that can be replaced with a Tefzel rotor seal for operation at pH 0 14 Factory set at 5000 psi 345 bar adjustable to 7000 psi 483 bar Model 77251 has an internal position sensing switch A Model 7725 Injector Reduces Wear and Tear on Your Columns eee _ Model 7725 Injector 57620 U Model 77251 Injector 57621 2 kirom Pump Replacement Components MBB Passage VESPEL Rotor Seal 58830 U MBB Port Tefzel Rotor Seal 57633 To Column Stator Face Assembly 57634 Needle Port Cleaner 57635 Valve Angle Bracket for all metal Rneodyne valves 57636 RheBuild Kit for 7725 77251 7726 55049 Sample Loops wide angle ports fittings included eon 2 uL 57622 736 0423 10 uL 57624 A conventional HPLC valve momentarily interrupts flow during sample injection 20 uL 57625 subjecting your column to repetitive pressure shocks Rheodyne s patented MBB make 50 uL 57626 before break design makes the new connection before breaking the old one providing 100 uL 57627 uninterrupted flow 200 uL 57628 U 500 uL 57629 U 1 mL 57630 2 mL 57631 5 mL 57632 Note Use VESPEL seals to pH 10 Tefzel seals to pH 14 Supelco is an authorized Rheodyne dealer Optimize Technologies Pump Replacement Parts A preventative mainte nance program that in cludes routine replacement of pump parts that are sub SSI LO Pulse Damper A pulse damper controls pump pulsations
38. sual noises Change pump seals if necessary S IGMA ALDAICFH Problem Probable Cause Remedy Comments Problem No 14 Baseline Noise irregular Leak Check system for loose fittings Normal Check pump for leaks salt buildup unusual noises Change pump seals if necessary 2 Mobile phase contaminated deterio 2 Check make up of mobile phase 794 0748 rated or prepared from low quality page 2 materials Problem 3 Detector recorder electronics 3 Isolate detector and recorder electronically Refer to instruction manual to correct problem 4 Air trapped in system 4 Flush system with strong solvent 5 Air bubbles in detector 5 Purge detector Install back pressure regulator after detector 794 0766 Check the instrument manual particularly for RI detectors exces sive backpressure can cause the flow cell to crack 6 Detector cell contaminated Even 6 Clean cell small amounts of contaminants can cause noise 7 Weak detector lamp 7 Replace lamp 8 Column leaking silica or packing 8 Replace column and clean system material Problem No 15 Broad Peaks 1 Mobile phase composition changed 1 Prepare new mobile phase 2 Mobile phase flow rate too low 2 Adjust flow rate 3 Leak especially between column and 3 Check system for loose fittings detector Check pump for leaks salt buildup and unusual noises Change pump seals if necessary 4 Detector settings in
39. ude final wash step in gradient analyses to remove strongly retained compounds 2 Late eluting peak usually broad 2 a Check sample preparation present in sample b Include step gradient to quickly elute component 794 0760 Normal solvent injected after sample 794 0774 Problem solvent injected after sample 794 0775 Further Recommendations We also suggest referring to the maintenance and troubleshooting selectivity pages 14 15 suggestions on how to prevent and solve sections of your instrument manual Modern HPLC systems often column hardware problems page 16 and a selection of column have self diagnostic capabilities that help isolate the problem area protection products from the Supelco catalog Please refer to our within the instrument For persistent problems relating to the catalog for our complete line of accessories that prolong column life column or your particular analysis please contact Supelco s Technical and in general simplify or improve your HPLC or FPLC analysis service Department Finally phone us to request additional literature about our HPLC The remaining pages in this guide include procedures for restoring and FPLC products or use our ChromFax service for immediate column performance following loss in resolution retention or access to all our free technical literature S IGMA ALDAICH Restoring Your Column s Performance The following procedures should rejuvenate a col
40. umn whose performance has deteriorated due to sample contamination Disconnect and reverse the column Connect it to the pump but not the detector Follow the appropriate flushing procedure in this table using a flow rate that results in column back pressure of 1500 4500 psi but never higher than the maximum recommended pressure in the manufacturer s instruction manual If you have a SUPELCOSIL column analyze with the test mix and the conditions listed on the data sheet Efficiency symmetry and capacity should be within 10 15 of the test sheet values If not repack the column inlet page 16 or replace the column Table 2 Silica Column Flush with the following 1 50 mL hexane 2 50 mL methylene chloride 3 50 mL 2 propanol 4 50 mL methanol 5 25 mL methylene chloride 6 25 mL mobile phase Evaluate column performance according to conditions specified by the manufacturer Note See also the Silica Column Regeneration Solution listed on page 15 for rejuvenating a deactivated silica column Column Restoration Procedures Silica Based Reversed Phase Column alkyl phenyl or diphenyl column SUPELCOSIL LC PAH column A Water Soluble Samples Flush with the following Flush with warm 60 C distilled water 50 mL methanol 50 mL acetonitrile 25 mL methanol 25 mL mobile phase Evaluate column performance U e UN gt B Samples Not Soluble in Water Flush with the following 1 50 mL 2 propanol 2
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