HPLC Troubleshooting 2.0 - How the HPLC
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4. CHROMATOGRAPHY HPLC Troubleshooting 2 0 How the HPLC Troubleshooting App Saves on Time and Frustration Holger Franz Thermo Fisher Scientific Germering Germany pnig ese Introduction he During the development of a new HPLC method there were 0 unexpected results all peaks except for the injection peak were negative Figure 1 Even with advances in chromatography z to eliminate problems something unexpected is bound to happen How would you go about finding out what s wrong 300 Although the web is full of HPLC troubleshooting guides Nue s many of these are dozens of pages long They can be helpful in teaching systematic approaches to troubleshooting but when you need an answer fast digging through these Figure 1 Negative peaks during method development experiments resources to find what you need can be slow and tedious System Conditions An alternative is using a poster such as the Thermo Scientific e Thermo Scientific Dionex UltiMate HPLC Troubleshooting Guide poster where all troubleshooting 3000 x2 Dual System including Column ence a suggestions are available at a glance and a useful suggestion SRD 3600 Solvent Rack with six i sue m ien ees degasser channels Standard Caffeine anthracene An alternative for people with smart phones is the Thermo DGP 3600RS Dual Gradient Pump and phenanthrene i Scientific HPLC Troubleshooting App for iPhone Android WPS 3000TRS Thermostatted We
5. LC Troubleshooting Guide Back HPLC Troubleshooting Guide Back HPLC Troubleshooting la Thermo Select Topic Select Topic gt Peaks gt Select Symptom opic gt Peaks gt Select Symptom gt No peaks SCIENTIFIC Select solutions and share with a friend Peaks LI No peaks Ll Begin Troubleshooting I3 Peak Area Precision Peak tailing Pressure Peak fronting Possible cause LJ Instrument failure wrong elution conditions Solution Does the detector show typical baseline noise If the detector output is just a flat line the detector or the data transfer failed Inject known test substance without column and check detector response A See our products Changing Retention Times Broad peaks 4 Recommend this app Baseline Ghost peaks Rate this app Split or double peaks gt gt gt a Contact a specialist gt Possible cause LJ No injection not enough sample volume d v wv wv wv wv v v v Negative peaks Too low signal to noise Solution Figure 3 Generating probable cause using the Thermo Scientific HPLC Troubleshooting App Troubleshooting Case Study Discussion of Possible Causes and Solutions A Few Clicks to Find the Probable Cause Negative peaks can be caused by the detector or by how the Using the problem of negative peaks described above the HPLC detection signal is processed Consequently the different causes Troubleshooting App finds a
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7. V Vis reference wavelengths and bandwidths Conclusion A solution is just a few taps away select your problem and the Troubleshooting App guides you to the answer Comprehensive in its inclusion of specific technologies such as fluorescence detection charged aerosol detection electrochemical detection and nano LC this powerful free tool effectively diagnoses various potential issues with HPLC analysis A Share function even lets you send the solution to your team Simply check the troubleshooting solution of interest and select share and enter the email address from your contacts list Did You Know How Reference and Detection Wavelengths Work Reference Wavelength The reference wavelength is intended to correct for subtract absorption that can occur from changes in conditions during analysis Those changes can sometimes be significant such as if there is a reduction in the lamp energy lamp drift The reference wavelength selected therefore must be in a quiet area of the spectrum where little to no absorption of the sample analytes occurs Because the signal from the reference wavelength is subtracted from the signal received from the detection wavelength it has a direct impact on results Reference Bandwidth Reference bandwidth serves to average several photodiode signals above and below the reference wavelength A reference bandwidth should be selected as wide as possible for example 30 100 nm but narrow enough not to in
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9. elect Symptom gt Negative peaks Select solutions and share with a friend Possible cause LI Inappropriate reference wavelength setting DAD Category Peaks Symptom Negative peaks Possible Cause Inappropriate reference wavelength setting DAD Solution The sample must not absorb Solution in the range of the reference wavelength If possible use a method without reference wavelength The sample must not absorb in the range of the reference wavelength If possible use a method without reference wavelength More information on how to leverage the advantages of UHPLC can be found at Possible cause Drainage spiking CAD This information derives from the free of charge Thermo Scientific HPLC Somn Troubleshooting App available for Figure 4 The App showing probable cause Inappropriate reference wavelength setting DAD with the solution Also note the App has the ability to share the solution with a colleague and links if you want to learn more 100 lt L 0 0 5 10 Minutes Figure 5 Obtaining the chromatogram without reference Reference Off displays the peaks in a normal way and removes side effects such as baseline drift and injection peak In a UV Vis DAD you can select both a reference and a detection wavelength When used correctly the reference signal corrects for changing conditions such as baseline drift If the reference wavelength is not chosen correctly and absorbs in the ran
10. ette coa 1 0c Pe umant ante wad Se umeris Bpa Dns n qus creda PARSER UE F Da ten i fe ema Diye Aaa Fam i pec teens Lte s PRI sars cw joy LA prn LIII 1a e raa te Nees lemmik Dee dul age Sa eo mi aeng s mmen amam III UHPLC a rra ceno ocused 3 T m mang UH PLC tsc ha ribgy malia tro to at Acton ssa Weer usert ali laborzt anat ard Maral oth For a copy of the troubleshooting poster please request it from your Thermo Fisher Scientific sales representative with the part number 6040 0050 www thermoscientific com dionex 2013 Thermo Fisher Scientific Inc All rights reserved ISO is a trademark of the International Standards Organization Android is a trademark of Google Inc iPhone is a registered trademark of Apple Corporation Windows is a registered trademark of Microsoft Corporation All other trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries Specifications terms and pricing are subject to change Not all products are available in all countries Please consult your local Thermo Fisher Scientific Sunnyvale CA A USA is ISO 9001 2008 Certified Certified System ISO 9001 sales representative for details ns Th r Japan 81 6 6885 1213 Switzerland 41 62 205 9966 e i i i D Australia 61 3 9757 4486 Denmark 45 70 23 62 60 Austria 43 1 333 50 34 0 France 33 160924800 Korea 82 2 3420 8600 Taiwan 886 2 8751 6655 SCIENTIFIC Belgium 32 53 73 42 41 Germany 49 6126 991
11. ge of your analyte then you may get a negative response See the sidebar Did You Know for more information In this case study the reference and detection wavelengths for the analysis of butylparabene were selected incorrectly e With a reference wavelength of 240 nm and a bandwidth of 100 nm the reference absorption was taken from 190 nm to 290 nm e With a detection wavelength of 285 nm and a bandwidth of 4 nm detection absorption was taken from 283 nm to 287 nm The range of the reference indeed overlaps in the range of analyte detection making this the probable cause for negative peaks To verify that this is cause the reference wavelength can be switched to off Reference off is actually the recommended operation of Thermo Scientific Diode Array Detectors mainly to avoid implications with incorrect reference settings The resulting chromatogram Figure 5 shows that this change immediately solves the issue of negative peaks Note that the positive injection peak and the baseline drift are now gone Both were caused by the absorption of formic acid as part of the mobile phase affecting wavelengths below 260 nm The injection peak was positive as the sample diluent lacks formic acid and therefore absorbed less than the mobile phase With the initial improper reference wavelength settings the negative peak was inverted and shown as a positive peak The next step after finding this resolution would be to optimize U
12. llplate 0 05 mg mL in 70 ACN and Windows platforms This mobile application incorporates Sampler luents A 0 1 Formic acid all the Information from the HPLC troubleshooting poster and EE E TE Ne HIS ES re BI TRE Pu ET expands it to include electrochemical and nano LC solutions Compartment in acetonitrile In combination with solutions for charged aerosol detection OSS these additions make the troubleshooting app a comprehensive i DAD 3000RS Diode Array UV Vis O me mn eB tool for various system setups This mobile application is Detector with analytical flow cell a a available for free at http info1 thermoscientific com forms e Thermo Scientific Dionex Chromeleon 55 90 HPLC Troubleshooting App Chromatography Data System CDS 6 0 60 software version 7 1 SR1 10 0 60 e All modules were connected using 0 005 in Flow 1 mL min 0 13 mm i d Thermo Scientific Dionex Viper Fingertight Fitting System SUE Mp iUd UV wavelength 285 nm UV bandwidth 4 nm HPLC Troubleshooting Guide Thermo L Begin Troubleshooting A See our products Recommend this app xr Rate this app 2 Contact a specialist Figure 2 Thermo Scientific HPLC Troubleshooting App for iPhone Android hermo and Windows platforms SCIENTIFIC i Vodafonede 3G 09 39 EET m Vodafone de 3G 09 40 80 m Vodafone de 3G 09 40 80 gt m Vodafone de 3G 09 40 80 HPLC Troubleshooting Guide Back HP
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14. solution as follows are specific to the types of detectors In this example different causes in combination with UV detection variable wavelength MA PO S pee VWD or diode array detection DAD analog data acquisition fluorescence detection FLD charged aerosol detection CAD and e Because the issue is related to peaks select Peaks electrochemical detection ECD are listed and demonstrate the Figure 3b unique diversity of the Troubleshooting App P AOSE INEAN EA GUE In this example results were obtained digitally by DAD Possible e The App lists five Possible cause Solution causes 3 4 5 and 6 in Table 1 relate to ECD CAD analog data Figure 3d shown in Table 1 acquisition and FLD therefore do not apply This leaves two other possible causes Starting with cause 1 Inappropriate reference Table 1 List of Possible Causes Generated by Troubleshooting App wavelength setting DAD the Troubleshooting App shows that the Possible Causes related solution for an inappropriate reference wavelength is Figure 4 The sample must not absorb in the range of the reference wavelength If possible use a method without reference wavelength 4 Drainage spiking CAD 5 Fluorescence of the substance is quenched by matrix or mobile phase FLD 6 Possible reduction occurring at electrode ECD Back HPLC Troubleshoot 1G P These U HPLC troubleshooting solutions might be of interest to you Peaks gt S
15. terfere with the absorption spectrum of the analytes Figure 6 below visualizes appropriate reference settings for a butylparabene spectrum 55 0 Relative Absorption 96 Reference wavelength e g 350 nm Reference bandwidth e g 100 nm 190 200 220 240 260 280 300 320 340 360 380 402 Wavelenath nm Figure 6 Butylparabene spectrum with recommended reference wavelength settings UV Vis Detection Wavelength In contrast to the reference wavelength the UV Vis detection channel wavelength for recording the analyte absorption should be set to the absorbance maxima of the analytes of interest Download your Free HPLC Troubleshooting App The App is available for iPhone Android and Windows phones free at htto info1 thermoscientific com forms HPLC Troubleshooting A or scan this quick response QR code to download the App References 1 Successful HPLC Operation A Troubleshooting Guide Version 1 1 Thermo Electron Corporation 2004 2 HPLC Troubleshooting Guide Bulletin 826E Sigma Aldrich 2009 3 HPLC Troubleshooting Guide Phenomenex Inc 2008 4 Thermo Scientific HPLC Troubleshooting Guide Poster P N 6040 0050 2012 5 Thermo Scientific Dionex UltiMate 3000 Series Diode Array Detectors Operating Instructions Revision 1 3 http www dionex com en us webdocs 75683 Man LC U3000 DAD Operation Nov1 1 D0C4820 8250 1 3 pdf quickly resolve application or system issues SCIENTIFIC
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