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A 14 Parameter Study of UHPLC’s for Method Development Transfer and Troubleshooting

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Abstract

Five ultra-high pressure liquid chromatography (UHPLC) instruments were compared to one another by examining the overall system performance and key functions of the system parts including: pump, auto-sampler, thermo-stated column compartment, and the detector. The five UHPLC systems used in this study were: ThermoFisher Vanquish, Agilent 1290 Infinity I, Agilent 1290 Infinity II, Waters Acquity I-Class, and Shimadzu Nexera X2. The identities of the systems were blinded in the results and discussion section to use this study for scientific purposes only rather than for competition and marketing. The following tests were performed to evaluate and compare the five UHPLC systems: injector linearity and precision, sample carryover, sample (autosampler) temperature accuracy, column temperature accuracy and precision, pressure ripple, pump mixing accuracy, flow rate accuracy, detector drift and noise, detector linearity, wavelength accuracy, extra-column volume, and dwell volume determination. This study presents an approach on how to test the performance of UHPLC systems along with potential problems that analysts may face when using the UHPLC systems, examples of such issues are: retention time irreproducibility, low sensitivity, method transfer failure, etc.

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References

  1. De VJ, Broeckhoven K, Eeltink S (2016) Advances in ultrahigh-pressure liquid chromatography technology and system design. Anal Chem 88:262–278

    Article  Google Scholar 

  2. Aasberg D, Lesko M, Samuelsson J, Kaczmarski K, Fornstedt T (2014) Method transfer from high-pressure liquid chromatography to ultra-high-pressure liquid chromatography. I. A thermodynamic perspective. J Chromatogr A 1362:206–217

    Article  CAS  Google Scholar 

  3. McCalley DV (2014) The impact of pressure and frictional heating on retention, selectivity and efficiency in ultra-high-pressure liquid chromatography. TrAC. Trends Anal Chem 63:31–43

    Article  CAS  Google Scholar 

  4. Novakova L, Veuthey JL, Guillarme D (2011) Practical method transfer from high performance liquid chromatography to ultra-high performance liquid chromatography: the importance of frictional heating. J Chromatogr A 1218:7971–7981

    Article  CAS  Google Scholar 

  5. Boswell PG, Schellenberg JR, Carr PW, Cohen JD, Hegeman AD (2011) Easy and accurate high-performance liquid chromatography retention prediction with different gradients, flow rates, and instruments by back-calculation of gradient and flow rate profiles. J Chromatogr A 1218:6742–6749

    Article  CAS  Google Scholar 

  6. Dolan JW (2006) Dwell volume revisited. LCGC North Am 24:458–466

    CAS  Google Scholar 

  7. Schellinger AP, Carr PW (2005) A practical approach to transferring linear gradient elution methods. J Chromatogr A 1077:110–119

    Article  CAS  Google Scholar 

  8. Snyder LR, Dolan JW (2006) Method development. High-performance gradient elution. Wiley, New York, pp 74–132

    Chapter  Google Scholar 

  9. Gritti F, Felinger A, Guiochon G (2006) Influence of the errors made in the measurement of the extra-column volume on the accuracies of estimates of the column efficiency and the mass transfer kinetics parameters. J Chromatogr A 1136:57–72

    Article  CAS  Google Scholar 

  10. Gritti F, Guiochon G (2010) On the extra-column band-broadening contributions of modern, very high pressure liquid chromatographs using 2.1 mm I.D. columns packed with sub-2 μm particles. J Chromatogr A 1217:7677–7689

    Article  CAS  Google Scholar 

  11. Gritti F, Guiochon G (2014) Accurate measurements of the true column efficiency and of the instrument band broadening contributions in the presence of a chromatographic column. J Chromatogr A 1327:49–56

    Article  CAS  Google Scholar 

  12. Gritti F, McDonald T, Gilar M (2015) Impact of the column hardware volume on resolution in very high pressure liquid chromatography non-invasive investigations. J Chromatogr A 1420:54–65

    Article  CAS  Google Scholar 

  13. Walter TH, Andrews RW (2014) Recent innovations in UHPLC columns and instrumentation. TrAC Trends Anal Chem 63:14–20

    Article  CAS  Google Scholar 

  14. Gritti F, Guiochon G (2011) On the minimization of the band-broadening contributions of a modern, very high pressure liquid chromatograph. J Chromatogr A 1218:4632–4648

    Article  CAS  Google Scholar 

  15. Buckenmaier S, Miller CA, van de Goor T, Dittmann MM (2015) Instrument contributions to resolution and sensitivity in ultra high performance liquid chromatography using small bore columns: comparison of diode array and triple quadrupole mass spectrometry detection. J Chromatogr A 1377:64–74

    Article  CAS  Google Scholar 

  16. Fekete S, Kohler I, Rudaz S, Guillarme D (2014) Importance of instrumentation for fast liquid chromatography in pharmaceutical analysis. J Pharm Biomed Anal 87:105–119

    Article  CAS  Google Scholar 

  17. Dong MW, Zhang K (2014) Ultra-high-pressure liquid chromatography (UHPLC) in method development. TrAC. Trends Anal Chem 63:21–30

    Article  CAS  Google Scholar 

  18. Wahab MF, Dasgupta PK, Kadjo AF, Armstrong DW (2016) Sampling frequency, response times and embedded signal filtration in fast, high efficiency liquid chromatography: a tutorial. Anal Chim Acta 907:31–44

    Article  CAS  Google Scholar 

  19. Colgate SO, Berger TA (2015) On axial temperature gradients due to large pressure drops in dense fluid chromatography. J Chromatogr A 1385:94–102

    Article  CAS  Google Scholar 

  20. de Villiers A, Lauer H, Szucs R, Goodall S, Sandra P (2006) Influence of frictional heating on temperature gradients in ultra-high-pressure liquid chromatography on 2.1 mm I.D. columns. J Chromatogr A 1113:84–91

    Article  Google Scholar 

  21. Fallas MM, Hadley MR, McCalley DV (2009) Practical assessment of frictional heating effects and thermostat design on the performance of conventional (3 μm and 5 μm) columns in reversed-phase high-performance liquid chromatography. J Chromatogr A 1216:3961–3969

    Article  CAS  Google Scholar 

  22. Fekete S, Guillarme D (2015) Estimation of pressure-, temperature- and frictional heating-related effects on proteins’ retention under ultra-high-pressure liquid chromatographic conditions. J Chromatogr A 1393:73–80

    Article  CAS  Google Scholar 

  23. Fekete S, Guillarme D, Fekete J (2014) Estimation of the effects of longitudinal temperature gradients caused by frictional heating on the solute retention using fully porous and superficially porous sub-2 μm materials. J Chromatogr A 1359:124–130

    Article  CAS  Google Scholar 

  24. Fekete S, Veuthey J-L, Guillarme D (2015) Comparison of the most recent chromatographic approaches applied for fast and high resolution separations: theory and practice. J Chromatogr A 1408:1–14

    Article  CAS  Google Scholar 

  25. Fekete S, Veuthey J-L, McCalley DV, Guillarme D (2012) The effect of pressure and mobile phase velocity on the retention properties of small analytes and large biomolecules in ultra-high pressure liquid chromatography. J Chromatogr A 1270:127–138

    Article  CAS  Google Scholar 

  26. Grinias JP, Keil DS, Jorgenson JW (2014) Observation of enhanced heat dissipation in columns packed with superficially porous particles. J Chromatogr A 1371:261–264

    Article  CAS  Google Scholar 

  27. Nguyen DTT, Guillarme D, Rudaz S, Veuthey J-L (2006) Fast analysis in liquid chromatography using small particle size and high pressure. J Sep Sci 29:1836–1848

    Article  CAS  Google Scholar 

  28. Camenzuli M, Ritchie HJ, Shalliker RA (2013) Improving HPLC separation performance using parallel segmented flow chromatography. Microchem J 111:3–7

    Article  CAS  Google Scholar 

  29. Shalliker RA, Ritchie H (2014) Segmented flow and curtain flow chromatography: overcoming the wall effect and heterogeneous bed structures. J Chromatogr A 1335:122–135

    Article  CAS  Google Scholar 

  30. Soliven A, Foley D, Pereira L, Hua S, Edge T, Ritchie H, Dennis GR, Andrew Shalliker R (2014) Improving the performance of narrow-bore HPLC columns using active flow technology. Microchem J 116:230–234

    Article  CAS  Google Scholar 

  31. Karger BL, Martin M, Guiochon G (1974) Role of column parameters and injection volume on detection limits in liquid chromatography. Anal Chem 46:1640–1647

    Article  CAS  Google Scholar 

  32. Neue UD, Kele M (2007) Performance of idealized column structures under high pressure. J Chromatogr A 1149:236–244

    Article  CAS  Google Scholar 

  33. Haidar Ahmad IA (2017) Necessary analytical skills and knowledge for identifying, understanding, and performing HPLC troubleshooting. Chromatographia 80:705–730

    Article  CAS  Google Scholar 

  34. Abate-Pella D, Freund DM, Ma Y, Simón-Manso Y, Hollender J, Broeckling CD, Huhman DV, Krokhin OV, Stoll DR, Hegeman AD, Kind T, Fiehn O, Schymanski EL, Prenni JE, Sumner LW, Boswell PG (2015) Retention projection enables accurate calculation of liquid chromatographic retention times across labs and methods. J Chromatogr A 1412:43–51

    Article  CAS  Google Scholar 

  35. Thompson JW, Kaiser TJ, Jorgenson JW (2006) Viscosity measurements of methanol–water and acetonitrile–water mixtures at pressures up to 3500 bar using a novel capillary time-of-flight viscometer. J Chromatogr A 1134:201–209

    Article  CAS  Google Scholar 

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Acknowledgements

The authors would like to thank the Vendors (Agilent, Shimadzu, ThermoFisher, and Waters) for the provision of loan instruments and scientific advice.

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Authors and Affiliations

  1. Novartis Pharmaceuticals Corporation, Technical Research and Development, PHAD, San Carlos, CA, USA

    Imad A. Haidar Ahmad, Frank Hrovat, Arianne Soliven, Thomas Tarara & Andrei Blasko

  2. Novartis Pharma AG, Chemical and Analytical Development, CHAD, Basel, Switzerland

    Adrian Clarke

  3. Department of Horticultural Science, University of Minnesota, St. Paul, MN, USA

    Paul Boswell

Authors
  1. Imad A. Haidar Ahmad
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  2. Frank Hrovat
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  3. Arianne Soliven
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  4. Adrian Clarke
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  5. Paul Boswell
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  6. Thomas Tarara
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  7. Andrei Blasko
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Correspondence to Imad A. Haidar Ahmad or Andrei Blasko.

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This article does not contain any studies with human participants or animals performed by the authors.

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Imad A. Haidar Ahmad and Frank Hrovat are first authors.

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Ahmad, I.A.H., Hrovat, F., Soliven, A. et al. A 14 Parameter Study of UHPLC’s for Method Development Transfer and Troubleshooting. Chromatographia 80, 1143–1159 (2017). https://doi.org/10.1007/s10337-017-3337-8

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  • DOI: https://doi.org/10.1007/s10337-017-3337-8

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