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Trends in development of stationary phases in chromatography

Separation Techniques, San Francisco, USA - 10 August 2015. Trends in development of stationary phases in chromatography. Petr Solich Charles University Czech Republic. CHARLES UNIVERSITY , FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ , CZECH REPUBLIC.

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Trends in development of stationary phases in chromatography

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  1. Separation Techniques, San Francisco, USA - 10 August 2015 Trends in development of stationary phasesin chromatography Petr Solich Charles University Czech Republic CHARLES UNIVERSITY, FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ, CZECH REPUBLIC

  2. Faculty of Pharmacy, Charles University, Hradec Kralove • Charles University in Prague • - founded1348 • - 48 000 students (6.000 foreign) • 17 faculties • located in - Prague (14) • Hradec Králové (2) • Pilsen (1) Faculty of Pharmacy in Hradec Králové – founded 1969 - 1500 students (100 foreign) - 11 Departments in 2 buildings - 23 research teams CHARLES UNIVERSITY, FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ, CZECH REPUBLIC

  3. High-performance liquid chromatography • High-pressure pump • Injector – Autosampler • Analytical column (stationary phase) • Detector • Chromatographic software software pump column detector autosampler

  4. Recent trends in developments of HPLC • sample preparation (automation, sample volume) • increased separation efficiency (columns) • increased selectivity of metods (detectors) • „Green chromatography“ (low consumption of organic phases) • miniaturization (columns, detectors, instruments) all to increased the laboratory throughput ! FAST CHROMATOGRAPHY

  5. Recent trends in developments of HPLC • sample preparation (automation, sample volume) • increased separation efficiency (columns) • increased selectivity of metods (detectors) • „Green chromatography“ (low consumption of organic phases) • miniaturization (columns, detectors, instruments) all to increased the laboratory throughput ! FAST CHROMATOGRAPHY

  6. Analytical columns – present situation Length - 10 - 250 mm Diameter - 1.0 – 4.6 mm Particlesize - 1 – 10 μm 100 mm

  7. Analytical columns – present situation Length - 10 - 250 mm Diameter - 1.0 – 4.6 mm Particlesize - 1 – 10 μm 100 mm 3.0 mm

  8. Analytical columns – present situation Length - 10 - 250 mm Diameter - 1.0 – 4.6 mm Particlesize - 1 – 10 μm 100 mm 3.0 mm 3.0 μm

  9. Analytical columns – present situation Length - 10 - 250 mm Diameter - 1.0 – 4.6 mm Particlesize - 1 – 10 μm 100 mm 3.0 mm 3.0 μm • 1. stationaryphasebased on silica (normal, reversed) • 2. stationaryphasefor HILIC (HydrophilicInteractionLiquidChromatography) • 3. stationaryphasesbased on zirconium oxide • 4. polymericstationaryphase • 5. monolithicstationaryphase • 6. hybrid stationaryphases (sub-2-micron phase) • 7. core-shellstationaryphase

  10. - smaller particles (2004)- higher operational pressure- low flow rate- low consumption of MP - short time of analysis- large sequences of samples ? Recent trends in chromatographic separation phases Monolithic columns Sub-2 m columns - monolithic rods (2001) • low operational pressure • high flow rate • relatively high consumption of MP - short time of analysis • large sequences of samples Low pressure separation (HPLC, SIA, …) High pressure for separation UHPLC Core-shell technology - porous shell on fused core (2009) - short time of analysis - low consumtion of MP - large sequences of samples ? Substantial decrease of analysis time !!!

  11. Stationary phases - comparison B - Monoliths - Porosity 80 % A - Particle based columns - Porosity 65 % Monolithic column- made of a highly porous material with two types of pore structure—macropores (size of2µm) and mesopores (size of 13 nm). This special porous character allows relatively high mobile phase flow rates (1–9 ml/min) while keeping the backpressure low.. J.Sep.Sci., 2009, 32 (15-16) – special issue devoted to Monoliths

  12. Core-shell (Porousshell) particlecolumns • - Comparablespeed and effectivityofanalysisto sub 2 μm particles • in UHPLC, but relativelylowback-pressure!! • - Shorterdiffusion path comparedto conventional • totalporousparticles • - RP-Amide, Phenyl-Hexyl, HILIC, F5, ES-Cyano • - pressurecca 600 psi, pH range2 – 9 Solid core Ascentis, Kinetex, Poroshell, Halo…

  13. 3- 5 µm w1/2 w1/2 Core-shellparticles – Mass transfer sub-2-microne porous shell – 20% w1/2

  14. Bioanalyticalapplications ofmonolithiccolumns

  15. Bioanalytical laboratory Charles University, Teaching Hospital, Hradec Kralove

  16. Biologically active compounds analysed in Bioanalytical laboratory in hospitalusing monolithic columns Retinoic acid in serum Neopterin in urine Retinyl esters = Vitamin A esters in serum Vitamin A, E, D in human serum in erythrocytes in lipoprotein fractions Application: • in clinical monitoring of patients with cardiovascular problems, - in monitoring of oncological treatment (side effects, nutritional status • in treatment of metabolic diseases (diabetes mellitus, pancreatitis) • clinical monitoring of elderly patients (level of antioxidants) - monitoring of patients with nephorological infections

  17. Bioanalyticalapplications ofmonolithiccolumns Determinationofretinoicacids (RA)

  18. Determination of retinoic acids (RA) Most important RA are 13-cis RA, all-trans RA For treatment of hemato-oncological deseases, a formulation Tretinoin is used (contain RA) tzv. Retinoidal syndrom – found in many patients (about 25 %) treated by Tretinoin Retinoidal syndrom: - high temperatures of body - acute pulmonary complications - hypotensis - eodems need for monitoring of RA in patients treated with formulation Tretinoin

  19. HPLC method for determination of RA, vitamin A (retinol) and vitamin E (tocopherols) in serum SPE Extraction: IS (20 µmol.L-1) 250 µl of serum Deproteination by ethanol (650 μl, 8 min, 4 ºC). centrifugation (2 000 ×g, 15 min, 4 ºC) supernatant placed on SPE colunmn Elution: by hexan (2 000 μl) Evaporation to dryiness - 35 ºC Addition of 250 μl of methanolu HPLC determination SP: monolithic column Chromolith Performance RP-18e, 100×4.6mm MP: ACN : 1% ammonium acetate 95:5 (v/v) 4 min flow rate1.5ml.min-1, 3 min flow rate3.2 ml.min-1(„flow rate“ gradient !!!) injection 20 μl DAD detection: of retinol, all-trans- and 13-cis RA 325 nm, alfa-, gama- tokoferol atocol 295 nm Time of analysis - 7 min

  20. Determination of retinoic acids and vitamins A and E in human serum Krčmová L, Urbánek L, Solichová D, Kašparová M., Vlčková L., Melichar B, Sobotka L., Solich P: J. Sep. Sci. 32 (15-16), 2009, 2804-2811 4 min - flow rate 1.5ml.min-1, 3 - min flow rate3.2 ml.min-1

  21. Determination of Vitamin D (+ vitamins A and E) Bioanalyticalapplications ofmonolithiccolumns

  22. Determination of Vitamin D Common name for a group of 9,10-secosteroids Most important – D3 – cholecalciferol – animal origin created in organism by UV radiation from provitamin D3 D2 –ergocalciferol – plant origin Both forms have the same activity in organism Vitamin D is transformed to active form by hydroxylation Influence on metabolism of phosphorus and calcium in the body Influence on calcium level in bones Prevention of osteoporosis

  23. Development of HPLC method for simultaneous determination of vitamines A, E and D Vitamin D: Ergocalciferol (D2) Cholecalciferol (D3) metabolite calcidiol - 25(OH)D3 metabolite calcitriol - 1,25(OH)D33 Vitamin A - retinol Vitamin E – α-tocopherol Concentration in serum 1,05 – 2,27 μmol/L Concentration in serum 19 – 35 μmol/L

  24. Development of HPLC method for A, E and D Chromolith RP-18e, 100×4.6mm + 50×4.6 mm standards

  25. Development of HPLC method for A, E and D Spiked serum Sklenářová H., Koblová P., Chocholouš P., Šatinský D., Krčmová L., Kašparová L., Solichová D., Solich P., Analytical Letters 44(1-3), 2011, p. 446-456.

  26. Bioanalyticalapplications ofmonolithiccolumns Determination of neopterin in urine

  27. Neopterin = a diagnostic marker reflect the stage of activation of the cellular immune system and pathogenesis and progression of various diseases:- in viral infections (HIV…)- in autoimmune or inflammatory diseases- rejection episodes following allograft transplantation- in several malignant diseases- neopterin concentrations are very closely linked with the progression of these diseases !Therefore it is of interest for laboratory diagnosis to measure the degree of activation of the human immune system. This is possible in an easy but specific way by the determination of neopterin concentrations.

  28. Chromatogram of urineChromolith RP-18 e, 100 x 4.6 mm, flow rate 1,2 ml/min kreatinin Uric acid kynurenin neopterin

  29. Monolithicstationary phases - trends HR-monolithiccolumns(introduced2011) • Higher separation performance - increased by 50% (System pressure increased by 60% ) • Performance similar as 2.7 µm core-shell particle columns • Currently– increasingchoice (C18, C8, SILICA, NH2, CN, PHENYL) • Size- length 5 – 100 mm, diameter 2.0 - 4.6 mm • Working range of pH 2 – 7.5 (only)

  30. Applications of sub-2-micro columns Determinationofpharmaceuticalsby UHPLC

  31. Columns with 1,7 μm particles Particles 5 μm Human hair 60 μmParticles 1.7 μm hybrid sorbent X-Bridge (BEH-Bridged Ethylene Hybrid) - particles 1.7 µm - range pH - 1- 12 - pressure up to 1000 bar

  32. Ultra High Performance Liquid Chromatography (UHPLC) • working up to 100 MPa (15 000 Psi, 1000 bar) • pressure resistant connections • X-Bridge sorbents • particle size 1.7 μm • special injection system • acquisition rate 80 points/s • special optical cell (500 nl, 10 mm)

  33. Application of UHPLC – Diclophenac gel HPLC Classical C18 column 5 um, 125 x 4.6 mm Time of analysis: 16.3 min Solvent consumption: 11.4 ml Speed acceleration 7 x 0 4 8 12 16 min UPLC X-Bridge column 1.7 um, 50 x 2.1 mm Time of analysis: 2.3 min Solvent consumption 1.1 ml 0 0,5 1 1,5 2 min Nováková L, Solich P.: J.Sep.Sci. 29, 2006, 2433

  34. Bioanalyticalapplications of sub-2-micro columns Determinationofphenoliccompounds by UHPLC

  35. Phenolic acids Catechines Coumarins Flavonoids UHPLC separation of 30 phenolic compounds Acquity BEH C18 (100 x 2.1 mm/ 1.7 μm), gradient 0.1 % formic acid/MeOH/from 88:12 to 30:70 (v/v), f.rate 0.45 ml/min, UV - 280 nm, 25° C Nováková L., Spáčil Z., Seifrtová M., Solich P.: Talanta 80, 2010, 1970-1979

  36. Bioanalyticalapplications ofcore-shellcolumns Determinationof vitamin A and E

  37. Comparisonofcore-shell and HR monolithiccolumns – fordeterminationofvitamns A and E ! ! suitable for UHPLC system suitable for HPLC and UHPLC system suitable for HPLC system Kučerová B., Krčmová L., Solichová D., Plíšek J., Solich P.: Comparison of new high-resolution monolithic column with core-shell and fully porous columns for THE analysis of retinol and α-tocopherol in human serum and breast milk BY UHPLC, J. Sep. Sci. 23, 2013, p.2223-2230

  38. Comparison of chromatographic separation Determination of retinol and alpha-tocopherol in human serum Classical particulate C18 150 x 4.6 mm; 5 μm Monolithic column 100 x 4.6 mm UHPLC – BEH silica 100 x 2.1 mm; 1.7 μm

  39. Where is the future ??? X sub-2 m column Two opposite trends monolithic columns X Veryhigh pressure separation Low pressureseparation !? Combination of high pressure using monolithic design !? core-shell design X high-temperature LC Substantial decrease of analysis time !!! !? Combination of all features descibed !?

  40. Conclusions Applications of modern technologies (combination of modern HPLC/UHPLC method, new stationary phaseswith quick and easy sample preparation method) for (bioanalytical) applications allow: • Faster and robust analyses • Large sequences • Small volumes of samples • Small volumes of solvents • No environmental pollution • Cheap • Increase laboratory throughput

  41. Acknowledgement

  42. Thank you for your attention

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