The Column - June 2008 - (Page 21) June 2008 www.thecolumn.eu.com The Column Tips & Tricks: GPC/SEC HPLC Workshops Led by world renowned HPLC experts limit and the total penetration volume and is constant for the optimum separation range. If a polynomial fit function of 7th degree is chosen (compare Figure 3) the slope is not constant and local maxima and minima (that lack any physical significance) appear. This fit function should, therefore, be avoided because it can produce artefacts in the MMD (e.g., shoulders), that are not related to the sample characteristics.5 How often should I (re)calibrate? It depends. Many analytical laboratories calibrate their column sets before and after the samples have been measured. This ensures fast detection if the columns are still working properly after sample analysis. The time and effort required for this quality assurance strategy has been dramatically reduced by modern GPC/SEC software programs that offer automated calibration/re-calibration routines and the availability of convenient autosampler vials containing calibration mixtures ready to use after adding the solvent. Another approach is to run a validation sample with every sample queue. If the results of the validation sample are within a defined range, recalibration is not required. A good tool to provide long-term reproducibility when calibrants and samples are measured on different days/weeks/months is the use of an internal flow marker/standard. This is a low molar mass compound eluting at the end of the chromatogram. If calibration and sample runs are correlated to this internal reference highest accuracy and precision can be achieved with minimum effort. The rule of thumb is to increase the recalibration frequency when many different samples are run on the same columns and/or if the purity of samples is questionable (sample might contain impurities such as low molecular weight substances present from the manufacturing process). ■ John Dolan ■ David Carr ■ Mel Euerby ■ Tom Jupille HPLC Workshops HPL CW ork Led by world renowned HPLC experts Led by w John Dolan Mel Euerby John Dola orld Dr John Dolan is an acclaimed chromatography expert with over 20 year’s experience as one of the best HPLC trainers in the world. John has written more than 100 papers on HPLC and related topics and is co-author of ‘Troubleshooting HPLC Systems’ and of ‘High-Performance Gradient Elution’ which have become standard reference works in HPLC. He is perhaps best known for his monthly LC Troubleshooting column in the journal ‘LCGC’. Tom Jupille Tom Jupille has been a practising chromatographer for more than 30 years and he has written more than 30 papers on chromatography and related subjects. Tom has excellent experience in HPLC, GC, TLC and ion chromatography, is a consulting editor for the journal ‘LCGC’ and moderator for the online ‘Chromatography Forum’. His extensive practical experience ensures Tom always delivers high quality and relevant chromatography training. References 1. 2. 3. 4. 5. D. Held, The importance of molar mass distributions, The Column, 8 (2007). D. Held and G. Reinhold, A look at the importance of molar mass averages; The Column, 10 (2007). F. Gores and P. Kilz; Accuracy and precision in GPC/SEC; The Column, 2 (2008) ISO EN 13885, GPC in Tetrahydrofurane. P. Kilz and D. Held; Qualification of GPC/GFC/SEC Data and Results in Quantification in LC and GC — A Practical Guide To Good Chromatography Data by S. Kromidas, H-J. Kuss (Eds.), Wiley-VCH, Weinheim, in press (2008). An Intro The Analysis of Protein duct HPLCTherapeutic Drugs by HPLC ion to HP Trou HPLC LC bles HILIC Chromatography hoot Meth HPLC Method Developmentcr In ing od easi Deve ng Validating HPLC Methods Th The lopm Adva Sam Increasing Sample Throughput erap Analys en ple nced eutic is Optimising HPLC tSeparations Thro of Meth Drug Prot Advanced Method Development LC-M ughp HI od s by ein S fo DryLab Chromatography Method LIC Ch ut Deve HPLC r Ch rom Valid lopm LC-MS for Chromatographers Development atog rom atin ent atog raph g HP Optim raph y LC Spon ising ers CHRO Meth DryL M HPLC ods Hich sored ab Sponsored by by Chro Sepa rom m ratio Lim CHROM Hichrom Limited Deve atog ited ns lopmraphy ent Meth od An Introduction to HPLC HPLC Troubleshooting Dr Joh Professor Mel Euerby has had a chro distinguished career in thenfield of Dol yea matogr an 20 separation science spanning over is an r’s aph acc HPL years. 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Tom ograph re than ion nce cou mittee Uni chro several courses for understanding in HPL rse. vers for y has edit the ity and or mat C, of exc and the basic principlesGCHPLC and the mod for theograph elle nt era aspects of , TLC ‘Ch important journaly, is areversed-phase Dav con is and an rom tor for id ‘LC the atog exp method development andsultrated HPL Carr GC onli erie ’ and ing and excellent hy For ne by all. David has extensive expertise nce rap instructor ack C sinc has bee rele theens e now invan analysis and characterisation of proteins and um’ ure bee ledg 1971. n invo . t chr s lved n ed peptides.oma Tom His exte ove teaching HPL He is an in r togr always nsive sev 30 yea HPL C exp aph deli eral rs. He C clas ert and y train ver practic the ing. s high al ses has bas courses has imp ic for qua orta principl for und develop lity met nt hod aspectses of erstand ed exc HPLC ing elle dev of and in the nt instelopmen reve rsed the pep analysi ructor t and -ph tide s and by all. is rate ase s. Dav d an cha id ract eris has exte atio nsiv n of protein e exp s and ertise ren own n ed HPL Mel sho Ce xpe Euer by rts ps David Carr Tom Jupi lle Davi d Ca rr Contact us for your free 2008 training brochure Tel: +44 (0) 118 9303 660 seminars@hichrom.co.uk Daniela Held studied chemistry in Mainz, Germany and is working in the PSS software and instrument department. She is also responsible for education and customer training. Hichrom Limited Website: www.polymer.de CHROM sales@hichrom.co.uk www.hichrom.co.uk Tel: +44(0)118 9303 660 21 http://www.thecolumn.eu.com http://www.hichrom.co.uk http://www.hichrom.co.uk http://www.polymer.de
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