HPLC Troubleshooting NOE Ce Guide ) ae tl i aoe eT CMa ene ere Desc as | ACE HPLC Columns 1 HPLC Columns John DolanYour decision has lasting effects. ACE HPLC Columns Ultra Inert Base-Deactivated HPLC Columns For Performance, Sletvity and Guwrantesd Reprodcbity AC E performance guarantee ACE dons not upetrthecann you sre caer ug, ‘inp corn oor ord an hp bw ACE ke FREE or coe. 1d 23004 John Dolan is best known for his manthly LC Troubleshooting columa in ECGC and LGC Europe. in addition to over 250 installments of is coluran, De Dolan has published mote than 100 papers related to IIPLC. His research interests are method development, column characterization and yradient lution. He has worked in all aspects of HPC from instrument design, t0 ‘writing software, fo managing a contract laboratory and to teaching HPLC techniques. Currently John is a principal in LC Resourecs, a company dedicated to training chromatographers and providing consultation for chromatographic problems. He sharcs some of his expertise with usin this guide for HPLC troubleshooting. Further information on courses taught by John may be found on the LC Resources weh site: www LCResources.com8. Solvent Miscibility Chart pana 4 ce TOCTEE t t ‘Sooners “niece tthe ne io ‘Sete ‘nage ‘ro deste “tunes ‘eto ‘or. ace-nple.com HPLC Troubleshooting Guide Contents “Troubleshooting Tables Peak Shape Retention Variation Ghost Peaks Colum Backpressure Introduction 1. Beak Shape LL Peak ailing 1.2 Insufficient buffer or additive 1 Peale tailing or distortion 14 Poorly resolved peaks 5. Fronting peaks 2, Retention Variation 2.1. Mobile phase composition changes 22. Column chemistry changes 23, Column temperaire changes 2.4, Fae rate problems 2.5, Proportoning-valve failures 5. Ghost Peaks 3.1 Late elation 3.2 Ghost peaks ingredient uns 333 Negative peaks 4. Columa Backpressure 4.1, Locating pressure problems 42. laine filters 43, Guard cartridges 44, Buer precipitation 5. Column Care Si. Equilibration 52. Column fishing 53. Column storage 6. Sumnary 7, References 8. Solvent Miseiility Chart .ace-hpecom u n 1 R R 6 6 7 uv 18 18 18 18 20 Py 20 2 BQuick Reference Tables I you have a problem you need to solve now, use these Troubleshooting Tables. Locate the table for the type of problem you have, fi the possible cause and use the short description ofthe Soltior or the eoss-eference to the main body ofthe guide to help you identity and selve your problem quickly Problems with Peak Shape Possible Cause Prevention/Solution Peak Tailing Traction with cve sanale T, Ove lias figh party silica based satiny phase (ee 1.1). 2, Adi basic mobile phase alive (ep, TEA) ot needed with lta igh panty pics ‘Cicltion with mata Tons Th SONA Pe Ae above ‘Weovg mobile phase pil ‘Decrease mobive pase pT To Rape silonl ionisation (ee 12) Tnerease bul concentration (eee 12) Blocked it ‘Reverse fla te column (ee 5) Use inline ier (ee 1.3, 4.2), Cama void ‘Revere lash the coluran fee 32) Replace the cola Taser ded vara ‘Minis nuriber of eonncotans ‘Use shortce connection bing Check ll fitiags ae tight Split Peaks ‘Gontaination on guard or analical clam inlet T Remove guard earge and cay oot analysis ~ replace guard if necessary 2. Revers hush analytical column (sce $2) 3. Foc strony retained contaminants, ry regeneration procedure (see 52} Replace clamn Blocked Hie Reverse fash the coun (ee 52) ‘Saample solvent macompaibie with mabie lace Tact sample in mobo pave ‘Simulaneows elution of sscond eomponsut ‘Use sample cleas-up prior to ijection ‘Change soloctiviry by changing mobile ‘ave or caumn phase 4 T 2, Use inne ier (86 13,42) T T 2 Cofamn verona T-Use higher capacity saiionary Pane 2 Increase column diameter 3. Decrease sample amount ‘wu. ace-ple.com 7. References 1 . Hawkins and JW, Dolan, LGC 21 (2003) 1134-1138, RD. Mossison and J.W. Dolan, LCGC 23 (2005) 566-574 JEW, Dolan, LEGC 14 (1996) 294-299, JU, Gilroy and JW. Dolan, LCGC 22 (2004) 982-988. DH. Marchand, P-L. Zhu, and JW. Dolan, LCGC 14 (1996) 1028-1033. EW. Dolan, LEGC 7 (1989) 822-826, LW. Dolan, IR. Kern, and T Calles, LOGC 14 (1996) 202-208 ww ace-e.com5.2.7. Size Exclusion Columas for Proteins For weakly retained proteins 4. 0.IM phosphate buffer, pH 3 For strongly retained proteins 18. Gradient of 100% water to 100% ACN in 60 min 5.3. Column storage practices will help extend the lifetime ofthe column. The simplest storage procedure is to remove any buffer from the columa (see 44.) then wash the coluuna with 10-20 colurin Volumes of strong mobile phase solvent (eg, MeOH or ACN for reversed-phase, a5 detailed in 5.2) to remove strongly retained material from dhe column. ‘Then flush the column with a further 10-20 ‘coluean votumes of the siorage mobile ahase specified by the manufacturer (this information should be detailed on the QC test chromatogram originally supplied ‘with the columa), Finally, cap the colurnn securely to prevent mobile phase ‘evaporation Except for specific cases for which the column manufacturer recommends otherwise, (€-2. some ion-exchange columns), do not store the columns with Ihuffer or less than about 25% oxganie solvent so 4s to avoid microbial growth. 6. Summary Several common causes of peak shape problems, retention time variation, ghost peaks and columa backpressure have been examined. Some ofthese problems ‘originate from the sample, others from the mobile phase and still others from the column or other instrument components. A few good habits will help to rinimize the eceurrence of such problems, + Usea new, Type-B, high-puriy«ilica-based column for cach new project and couple this with the highest quality HPLC-grade reagents. * Flush the HPLC system regularly to remove salts and buffers and service the system on a periodic basis te minimize check-valve and pump-seal problems. The more thorough the sample clean-up process, the cleaner the sample will be and the likeliood of sample-related problems will be lessened ‘+ At the completion ofa series of runs (or after every run, in some cases), stcong-solvent fash will help to remove strongly relained materials from, the column, minimize interferences in future tins and extend column lifetimes. CCofumns won't lat forever, but with proper eare, you should be able to get a ‘good return on your investment. 2 won. com Problems with Peak Shape (continued) Peuk Rronting. Tormaton of ehanaels in cola T. Replace the colamn 2. Operate within commenced p& Timi of column (se 15) oan overated TInjeat smaller volune ive tte sample solution 2, Use higher capacity stationary phase ‘Suiaple solve ncospatibe with mobile par T.Injeet sample in mobile pase Tow temperature Ty Tnerease volun temperate Problems with Retention Variation Possible Cause Prevention/Solution Decreasing Retention Times Tass of bonded statonary phase Replace cha 2 Operate at pH128 fr sia based RP colurans (ee 22) ative groups on Satnnary pase ‘Ue organi modifier ia mobile phase Iexess buf strength Tnreaing Now ate ‘Colum overloaded ‘Rede amount of ramp injostod 7 2 T. Check and adjust pamp Tow rate (ee 24) 7 2. Use column ih lager Increasing Retention Times ‘Changing mobile phase composition ‘Gover toventreersoin (eo 21) Propare fies mobile phase (82 2.1) Toi of Bonded aatonary pase Replace column Deeeasing How rte ‘Check and ado pannp flow rate Gee =A ‘Check for leaks i syste, including pump. seas (se 24) Bubbles i mobs pase ‘Check ow aie ad presse (eee 7A epas motile pase (see 2.4) Fluctuating Retention Times Trsafficient column equlibation ‘Elite column longer benween rans Condition the column with concentrated sample ‘Change inimabile phase Composiion T. Check make-up oF rable phase and mks ‘up now if necessary (ee 2.1) (Check proportioning valve sceuacy Se 2 Tosnficient batier capt ‘Use baffer concentatons =20mM Fluctuating colar temperate ‘Stabilse ambient tempertore eee 7) ace 2 T 1 2. Tharmaste the column (see 2.3) omProblems with Ghost Peaks Possible Cause Prevention/Solution Ghost Peaks ‘Contamination in col oF fjeoar Use only HPLC grade solvents Flush column o remove impuriis (re 5.2) Flush injecior between aalyace Tate eating peak rm previous jeton “Extend runtime (ee 3.1) Flush column with strone mobile phase tend of each rm (ee 3.1) For gradien rans, end at higher ccuncenteation Gee 3.1), Contaminated water RP HPLC Use HPLC grade water (ee 3.2) ‘Unknown inierfenences in sample Use sample clean-up (€, SPE) Negative Peaks "Retiaeive Index of colts Tower than that of mobile phase (RI detector) "Use mobile pave wit Tower refine nex Reverse dtecor polarity 1 obi postive eas “Absorption oF slate Tower han sorption cof mobile phase (UV detection) ‘Change OV wave ‘Use mubie pase with lover UV abuorption (S20 3.3) “Saniple sotvent and’ mobile phase differ composition [Change sample solvent Taio sample in mobile phase if posible Spikes i bubbles ia mobile phase Degas mobile phase Install back presure restrictor at detector ‘outlet Ensure al tings are ight Column sored without enieape Soe eoluains with endeaps (ee 53) Fash RP column with degassed methanol ww ace-tpte.com Follow this general procedure for column Hashing withthe specific solvents mentioned below for your ype of column Ic is aways good fo check the ‘munufyeturer’s recommendations prio to Hushing so that you don't damage the ‘column, 1. Disconnect and reverse the column 2. Connect the column tothe pump, Fut not the detector 3. Flush with 10-20 column volumes af solvent ata flow rate no higher than {hat used for the QC chromatogram, 4, altering the procedures belovy, be sure to use miscible solvents for each successive step (see table page 24) 5.2.1, Reversei-Phase Columns (C18, C8, C4, Phenyl, C 1. Mobile phase without buffer D. MeOH or ACN ‘if metal ions are thought to be causing contamination, flush with aqueous 0.05M EDTA, then water, followed by the above sequence. Columns which use Jon-pairing reagents should be dedicate to ion-pairing applications. 5.2.2, Reversed-Phase Protein/Peptide Columns a Mobile phase without buffer '. Gradient of 10-90% B; A = 0.1% TRA/water; B = 0.1% TEA/ACN. 5.2.3. Unbonded Silica Columns (SIL) a. IPA b. MeOH . Fithyl acetate 5.2.4, Bonded Nocmnal-Phase Columns (CH, NH, Diol) ‘Chloroform b TPA ‘c. Methylene chloride 4. Hexane 5.2.5, Anion-Exchange Columns (SAX, WAX) a Water b. Methanol Chloroform 4. Methanol © Water 5.2.6, Cation-Exchange Columns (SCX, WCX) 1a. Water (inject 4x 200 yi DMSO during fs) b THF “AQ? type) or ace hp.com a2 5.Ct mn Care Columns should be considered a consumable item and as such will have a Limited lifetime. For most applications, columas should fast $00-2000 injections, but this will vary with dhe cleanliness of the samples, the pH of dhe mobile phase and the use of guard cartridges. The practice listed hero should help to maximize the sefl life of silica-based colununs 5.1. Equilibration ofthe column when changing from one mobile phase o nother, or when recyeling a gradient, should take 10-20 column volusmes, The ‘column volume for vasious column dizensioas is shown below. The less drastic 2 change in solvent e.g, from 80% to 20% ACNiwater vs. from ACN to THF), the less volume should be required. The ecsiest way to chcek for equilibration isto ‘make two injections of sample — ifthe retention isthe same, the colurm was equilibrated adequately; ifretcntion shifts, inerease the equilibration volume and {ry again. Equilibration is related to the volume of solvent, not the time, so higher flow rates ean reduce equilibration tims Approximate Column Volumes (mL) ‘Calama ‘Column Feng id. 30mm | 150mm | 250mm 21 mm or 03 0S 32mm 03 oF, 12 46mm as 13 25 10.0 mm 2a 7A Tis 22 mm 6 37 378 5.2. Column flushing isa simple procedure tht ean extend column lifetime by ‘washing strongly retained material for the column. At the end ofeach day’s use of the column, remove any buffer (see 44), then flush the eoluran with 100% of | the strong solvent (generally ACN a: MeOH for reversed-phase methods). The _more extensive flushing procedures listed on the next page can be effective restoring column performance, but remember that the cole should be considered ‘a consumable item, so it should not be expected to last forever! Avoid fusing reversed-phase columns with 100% waier (except for embedded-polar-group or "AQ" columns), because phase dewetting will prevent good cleaning and column se-equilbration with mobile phase may bs very slow. wane acetple.com Problems with Column Backpressure Possible Cause Prevention/Solution High Backpressure Wrong punpseting Chek and sore ating [Normal for system Thereased backpeessure normal I 1. Switched to longer column 2. Changed to smaller pctles 3. Changed ro smaller fiameter 4 Increased flow rate ‘foo other changes made Prasaure higher dung middle of gaent 1. Normal Temperature 00 Tow 1. Adjust column oven emperatare ‘Color ageing T. Gradual inetease in pressure normal ‘over column lietime icske eam "Reverse lh the column (eee 32) Use inte fier (ee 13,42) Centifuge or filter samples Use guard caries (ce 4.3) ‘Blocked ine Tier Replace in-line Fier fit Gea 42) Centfage or Filter samples Pre-fitermabile phase ‘Bock guard eaniage TReplace guard caridge ware Teaony (eee 43) System Boeke Sytematicalymvestigat sytem to Find blockage (se 4.1) Baler pecipiation ‘Reverse fu he column wil waters 9 Review evaluation conditions (se 4.4) ‘Low Backpressure Teakin yatem Torte Teak and comet Column enperatir oo high Lower temperate Flow to fw T Toca flow ate won aee-hoe.comIntroduction ‘This toubleshooting guide contain examples of some ofthe most common problems observed in roversed-phase HPLC (RP-HPLC) separations. Four majae robles areas ae covered: peak shape, rezaton time changes, ghost peaks and problems relate to column backpreste. In addition, a section on column eae is included ~ procedures that will heip yeu get maximum lifaimes fom your columns. Within each section, several examples are given to ilustate various problems. A set of toubleshootng ables corzesponding o each section will help you quickly identify problem causes ad solutions. you ae in a hurry, you can a0 directly to the tables to help you solve an existing problem. Otherwise, we suggest that you read the enie ide sas to pick up some ideas that wil help you avoid problems inthe fare We bope that you find this guide weful iagnose problems and to gain an understanding 0° the underlying eases so that you can preven, or at east minimize, dei future occurrence 1. Peak Shape 1.1. Peak tailing has been the most common peak shape problem since the ezrly ‘ays of RP-HPLC. Most peak tailing is due to interaction with acidic or ionized fanol groups on the surface of the silica particles within the column, The low-purity silica (often called "Type-A* or acidic silica) has a high content of acidic silanol ($i-OH) groups and the presence of metal impurities especially iron and aluminum) further increases the ionization of these groups to ‘Which provides cation exchange sites. The PK these materials i region, meaning that at pE>6 most ofthe silanol groups. improve the purity and lower the acidity of silica led to higher purity silica particles ("Type-B") and since their inital troduction, the purity of these silica packings hus improved, High-purity silica has a pK. of >8, So there is minimal silanol ionization in the pH-stablerang> of 2 between 45% and 50% B. Unfortunately, his is where the peaks with arge rotention variations were eluted, ‘The HPLC system had a procedure for proportioning-valve adjustment and when this was performed, the steps beeame smenth and even and the rete {ell within specifications ‘woraace-tle.com "