Cromatografia líquida moderna

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Cromatografia líquida moderna

  1. 1. O que é cromatografia líquida?
  2. 2. •http://chemkeys.com/br/2000/07/18/cromatografia-a-gas-curso-em-diapositivos/ CROMATOGRAFIA Histórico M. TSWEET (1903): Separação de misturas de pigmentos vegetais em colunas recheadas com adsorventes sólidos e solventes variados. éter de petróleo CaCO3 mistura de pigmentos pigmentos separados Cromatografia = kroma [cor] + graph [escrever] (grego) http://chemkeys.com/br/2000/07/18/cromatografia-a-gas-curso-em-diapositivos/
  3. 3. “Xpомофиллы в растительном и животном мире” (Chromophils in plant and animal world) Doctor of Science dissertation,Warsaw, 1910, 380 pp. Reprinted from Chromatographic adsorption analysis, selected works of M. S. Tswet by Academy of Sciences of the USSR, 1946.
  4. 4. CROMATOGRAFIA Princípio Básico Separação de misturas por interação diferencial dos seus componentes entre uma FASE ESTACIONÁRIA (líquido ou sólido) e uma FASE MÓVEL (líquido ou gás).
  5. 5. CROMATOGRAFIA Modalidades e Classificação FM = Líquido FM = Gás Cromatografia Líquida Cromatografia Gasosa (CG) Em CG a FE pode ser: Sólida Líquida Cromatografia Gás-Sólido (CGS) Cromatografia Gás-Líquido (CGL)
  6. 6. O equipamento
  7. 7. http://www.pharmainfo.net/reviews/introduction-analytical-method-development-pharmaceutical- formulations
  8. 8. O injetor
  9. 9. http://community.asdlib.org/imageandvideoexchangeforum/2013/08/02/loop-injector-for-hplc/
  10. 10. Parâmetros cromatográficos que você deve conhecer, quando trabalha com cromatografia.
  11. 11. Cromatograma com as medidas relacionadas à determinação de parâmetros cromatográficos C.H. Collins, G.L. Braga, P.S. Bonato, Fundamentos de Cromatografia, 4ª edição .,:Editora da Unicamp, Campinas, 2006. R M t t R M M t t t k '    2 1 2    1 ' R t ' R t k k      t t R R 2 1 2 1,177   s w w   R R 2 1              1 2 1 2 h h b b t t w w R
  12. 12. Variação do volume de retenção (VM) da uracila e da tiouréia em função do tipo e da quantidade de modificador orgânico. Coluna: Restek Allure-C18 150 × 4,6 mm. VM = tM x F, onde F é a vazão da fase móvel Y. Kazakevich, R. Lobrutto, HPLC for Pharmaceutical Scientists, Wiley-Interscience Hoboken, 2005.
  13. 13. L N H  2 2     R R t t 545 , 5 16               h b w w N
  14. 14. Medida e cálculo do fator de assimetria do pico. A. Braithwaite, F.J.Smith, “Chromatographic Methods”, 4a. edição, Chapman and Hall, Londres, 1985.
  15. 15. Medida e cálculo do fator de alargamento do pico, segundo a USP
  16. 16. Efeito da retenção, da eficiência e da seletividade sobre a resolução (adaptado da referencia
  17. 17. C  B H  A   L M t   http://www.chromedia.org/chromedia?waxtrapp=yqegzCsHqnOxmOlIEcCbC&subNav=wnjedDs HqnOxmOlIEcCbCmF
  18. 18. R.M. Ornaf, M.W. Dong In S. Ahuja, M.W Dong (Eds) 6th Volume, Handbook of Pharmaceutical Analysis by HPLC, Elsevier Academic Press, Londres, 2005, pg 19-45.
  19. 19. Eddy difusio
  20. 20. 푑푝 2휇 퐷푀 푣 = a) + 푐푣 b) 퐻 = 퐴푑푝 + 퐵퐷푀 휇 + 퐶 휇푑푝 퐷푚 ℎ = 퐻 푑푝 ℎ = 푎 + 푏 푣 a) Gráficos de van Deemter b) gráficos de Knox . Dm para a acetofenona é 1.2 × 10−9 m2/s . Fase móvel 30/70 acetonitrile/água temperatura: 40 ◦C, Colunas Acquity BEH C18, 1.7 m, 10 cm × 2.1mm ID; XBridge C18, 3.5m, 15 cm×4.6mm ID; XBridge C18, 5 m, 25 cm×4.6mm ID. de Villiers et al. / J. Chromatogr. A 1127 (2006) 60–69
  21. 21. Fekete et al., Journal of Chromatography A 1228 (2012) 57-71
  22. 22. ΔP = (ηFL)/(K0πr2dp2) onde K0 é a permeabilidade específica, η é a viscosidade da FM, F é a vazão da FM, r é o raio interno da coluna e dp é o diâmetro médio das partículas da fase estacionária (FE) que recheiam a coluna
  23. 23. Quais são os principais modos de cromatografia?
  24. 24. http://www.chromedia.org/chromedia? waxtrapp=yqegzCsHqnOxmOlIEcCbC&su bNav=yarwnEsHqnOxmOlIEcCzBYT Different mechanisms of retardation in liquid chromatography (J.F. Rubinson, K.A. Rubinson, Contemporary Chemical Analysis, Prentice Hall, Upper Saddle River, 1998).
  25. 25. Normal-phase LC NP-LC is performed by using silica sorbents which or chemically modified with polar and/or hydrophilic functional groups and in combination with non-polar eluents.
  26. 26. http://www.expertsmind.com/topic/packing-material-or-stationary-phase/ adsorption-chromatography-913002.aspx
  27. 27. Typical chromatogram of a reaction mixture collected during the course of reaction of phthalic anhydride with benzene in the presence of AlCl3, as catalyst. Peaks: 1, benzene; 2, anthraquinone; 3, phthalic anhydride; 4, maleic anhydride; 5, unknown. Chromatographic conditions: Column: Spherisob silica, 250 × 4.6mm, 10μm; mobile phase, n-heptane–ethanol chloroform–acetic acid (89 : 5 : 5 : 1, v/v/v/v); flow rate, 1mL/ min; detection, UV at 254 nm; temperature, 27°C.
  28. 28. Reversed phase chromatography
  29. 29. Viscosity as a function of organic/water composition
  30. 30. (A) 30% MeCN: 70% 20mM Phosphate, pH 7. (B) 50% MeCN: 50% 20mM Phosphate, pH 7. (C) 80% MeCN: 20% 20mM Phosphate, pH 7.
  31. 31. Retention of alkylbenzenes on Luna-C18 column from acetonitrile/water eluent.
  32. 32. %ACN tR(NB), min k(NB) tR(PP), min k(PP) α (PP/NB) 100 1,02 0,28 1,02 0,28 1 90 1,04 0,3 1,04 0,3 1 80 1,18 0,48 1,12 0,39 0,83 70 1,38 0,73 1,27 0,59 0,81 60 1,73 1,16 1,57 0,96 0,83 50 2,37 1,96 2,29 1,86 0,95 40 3,73 3,66 3,73 3,66 1 30 6,55 7,19 8,62 9,78 1,36 25 9,25 10,56 15,35 18,19 1,72 20 13,46 15,83 30,75 37,44 2,37 %MeOH tR(NB),min k(NB) tR(PP), min k(PP) α (PP/NB) 100 1,02 0,28 1,02 0,28 1 90 1,08 0,35 1,08 0,35 1 80 1,25 0,56 1,25 0,56 1 70 1,5 0,88 1,68 1,1 1,26 60 2,02 1,53 2,73 2,41 1,58 50 3,05 2,81 5,65 6,06 2,16 40 5,07 5,34 14,36 16,95 3,18 30 8,91 10,14 41 50,25 4,96 25 11,78 13,73 74 91,5 6,67 Tempo de retenção (tR), fator de retenção (k) e seletividade (α) do nitrobenzeno (NB) e do propilparabeno (PP) em função da percentagem e do tipo de modificador orgânico. Coluna: Symmetry C18, 3 μm, 75 × 4,6 mm, Waters. Vazão: 1mL/min. Temperatura 40 °C
  33. 33. Y. Kazakevich, R. Lobrutto, HPLC for Pharmaceutical Scientists, Wiley-Interscience Hoboken, 2005.
  34. 34. Selectivity for steroids as a function of organic mobile-phase component. Chromatograms showing the elution order of all eight congeners as a function of organic modifier with 0.1% formic acid as the buffer phase on YMC ODS-AQ column at ambient temperature. (Top panel) 25% acetonitrile, 1.5-mL/min flow rate; (middle panel) 45% methanol, 1.2-mL/min flow rate; (bottom panel) 20% tetrahydrofuran, 1.5- mL/min flow rate. (Reprinted from reference 51, with permission.) P. Zhuang, R. Thompson, and T. O’Brien, J. Liq. Chrom. Rel. Technol. 28 (2005), 1345–1356.
  35. 35. Time (min) A (%) B (%) 0 100 0 4 55 45 40 0 100 45 0 100 48 100 0 A. Stafiej et al. / J. Biochem. Biophys. Methods 69 (2006) 15–24
  36. 36. Y. Kazakevich, R. Lobrutto, HPLC for Pharmaceutical Scientists, Wiley-Interscience Hoboken, 2005.
  37. 37. Theoretical retention versus pH profiles for acidic and a zwitterionic component. Chromatographic conditions: Column: 15-cm × 0.46-cm Phenoemenex Luna C18(2), 5μm; 70% 15mM K2HPO4 adjusted pH 2–9 with phosphoric acid; 30% MeCN; flow rate, 1mL/min; temperature, 25°C. R. LoBrutto, A. Jones, Y. V. Kazakevich, and H. M. McNair, J. Chromatogr. A 913 (2001), 173–187
  38. 38. Y. Kazakevich, R. Lobrutto, HPLC for Pharmaceutical Scientists, Wiley-Interscience Hoboken, 2005.
  39. 39. R. LoBrutto, A. Jones, Y. V. Kazakevich, and H. M. McNair, J. Chromatogr. A 913 (2001), 173–187
  40. 40. Y. Kazakevich, R. Lobrutto, HPLC for Pharmaceutical Scientists, Wiley-Interscience Hoboken, 2005.
  41. 41. http://pubs.acs.org/cen/coverstory/86/8617cover.html acessada em 2 de fevereiro de 2011.
  42. 42. Diferentes seletividades obtidas para uma mistura de compostos básicos com fases estacionárias preparadas com diferentes tipos de ligantes químicos sobre a mesma sílica. Colunas: 250 x 4,6 mm, dp 5 μm, todas da ACE. Fase móvel: metanol:tampão fosfato (pH 6; 25 mmol/L) 80:20 (v/v). Vazão: 1.5 mL/min. Identificação dos solutos: 1 = norefedrina; 2 = nortriptilina; 3 = tolueno; 4 = amitriptilina; 5 imipramina Dolan, J. W.; LCGC North Am. 2007, 25, 1014
  43. 43. J. Layne / J. Chromatogr. A 957 (2002) 149–164
  44. 44. H. Engelhardt, R. Grüner, M. Schererv Chromatographia, 53 (2001), pp. S154–S161 Separation of tricyclic antidepressants on an (a) Prontosil C18 H and (b) Prontosil C 18 ACE/EPS column (150 mm × 4.6 mm, dp 5 μm). Mobile phase 65:35 (v/v) methanol:phosphate buffer (20 mM, pH 7). Peaks: 1 = uracil, 2 = protriptyline, 3 = nortriptyline, 4 = doxepine, 5 = imipramine, 6 = amitryptyline, 7 = trimipramine, 8 = clomipramine.
  45. 45. O que acontece quando usamos partículas com diâmetro reduzido?
  46. 46. R.M. Ornaf, M.W. Dong In S. Ahuja, M.W Dong (Eds) 6th Volume, Handbook of Pharmaceutical Analysis by HPLC, Elsevier Academic Press, Londres, 2005, pg 19-45.
  47. 47. Experimental H–u plots of columns packed with 1.3, 1.7, 2.6 and 5 μm core–shell particles (peak widths were corrected for the extra-column band broadening). Test analyte: butylparaben. S. Fekete, D. Guillarme / J. Chromatogr. A 1308 (2013) 104– 113
  48. 48. Impurity profiling of α-estradiol. Peaks: α-estradiol (1), main impurity (2) and minor impurity (3). Columns: Phenomenex Kinetex 50 mm × 2.1 mm, 1.3, 1.7 and 2.6 μm. Mobile phase: water/ACN 60/40 (v/v), flow rate: 0.5 mL/min, temperature: 25 °C, Vinj: 0.5 μL (25 μg/mL estradiol), λ = 210 nm (80 Hz). S. Fekete, D. Guillarme / J. Chromatogr. A 1308 (2013) 104– 113
  49. 49. Fast separation of cashew nut extract. Columns: Phenomenex Kinetex 50 mm × 2.1 mm, 1.3, 1.7 and 2.6 μm. Mobile phase: water/ACN 14/86 (v/v), flow rate: 0.8 mL/min, temperature: 25 °C, Vinj: 0.3 μL, λ = 280 nm (80 Hz). S. Fekete, D. Guillarme / J. Chromatogr. A 1308 (2013) 104– 113
  50. 50. Transferência de método do HPLC para o UPLC, metodologia empregada para analisar uma formulação farmacêutica de composto 6 onde se encontram onze impurezas (a) método original para HPLC: coluna, XBridge C18 (150×4.6 mm, 5 μm); vazão: 1000 μL min−1; volume de injeção, 20 μL; tempo de gradiente, 45 min. (b) método UHPLC transferido para HPLC: coluna, Acquity BEH C18 (50×2.1 mm, 1.7 μm); vazão, 610 μL min−1; volume de injeção, 1.4 μL; tempo de gradiente, 5.1 min. (c) Otimização do método UHPLC: columa, Acquity BEH C18 (50×2.1 mm, 1.7 μm); vazão, 1000 μL min−1; volume de injeção, 1.4 μL; tempo total de gradiente, 3.1 min. Figura adaptada Guillarme et al. Anal Bioanal Chem (2010) 397:1069–1082.
  51. 51. Separation of a pharmaceutical formulation “Rapidocain” in isocratic mode. Experimental conditions: mobile phase containing ACN/phosphate buffer at pH 7.2 (40:60, v/v). A. Waters Xterra RP18, 150 × 4.6 mm, 5 μm, F = 1000 μl/min,Vinj = 20 μl. B. Waters Acquity Shield RP18, 50 × 2.1 mm, 1.7 μm, F = 613 μl/min, Vinj = 1.4 μl. C. Waters Acquity Shield RP18, 50 × 2.1 mm, 1.7 μm, F = 1000 μl/min, Vinj = 1.4 μl. 1, methylparaben; 2, 2,6- dimethylanalinine; 3, propylparaben; 4, lidocaine. (http://www.rsc.org/shop/books/ 2012/9781849733885.asp). S. Fekete et al. / Journal of Pharmaceutical and Biomedical Analysis 87 (2014) 105–119
  52. 52. Chromatograms for columns with different lengths. Conditions: 50, 100, and 150 mm BEH Shield C-18 columns packed with 1.7, 3.5, and 5.0 μm particles respectively; 2.1 mm internal diameter; flow rates for 50, 100, and 150 mm lengths were 0.36, 0.24, and 0.12 mL/min, respectively; the injection volumes for 50, 100, and 150 mm lengths were 1.0, 2.0, and 3 μL, respectively; the sample concentration was 0.1 mg/mL for each analyte;. Peak identifications: from left to right (1) uracil; (2) benzylalcohol; (3) acetophenone; (4) propiophenone; and (5) benzophenone N. Wu, A.C. Bradley / J. Chromatogr. A 1261 (2012) 113–120
  53. 53. Chromatograms for columns with different lengths. Conditions: 50, 100, and 150 mm BEH Shield C-18 columns packed with 1.7, 3.5, and 5.0 μm particles respectively; 2.1 mm internal diameter; flow rates for 50, 100, and 150 mm lengths were 0.36, 0.24, and 0.12 mL/min, respectively; the injection volumes for 50, 100, and 150 mm lengths were 1.0, 2.0, and 3 μL, respectively; the sample concentration was 0.1 mg/mL for each analyte;. Peak identifications: from left to right (1) uracil; (2) benzylalcohol; (3) acetophenone; (4) propiophenone; and (5) benzophenone.
  54. 54. Loss in efficiency at various retention factors for columns with different lengths. Conditions: 0.1 mg/mL uracil, benzylalcohol, acetophenone, propiophenone; and benzophenone were used as analytes. Keys: (♦) 50 mm; (○) 100 mm; and (●) 150 mm.
  55. 55. Chromatograms obtained with a Kinetex® 100 × 2.1 mm, 2.6 μm column with A. a conventional HPLC system without modification, and B. after optimization. https://phenomenex.blob.c ore.windows.net/document s/ad6882fd-5ad1-478a-ab0a- 5ad746734529.pdf (06.02.2 013).
  56. 56. Graphical representation of A. pressure tolerance and type of pumping system, and B. standard system dwell volume of all the UHPLC systems (ΔP > 600 bar) commercially available. In A., light red expresses low pressure system volume while dark blue represents high pressure system volume. It is important to notice that the standard dwell volume reported in this figure can be modified on a few instruments either by bypassing the damper and mixer, or by changing the volume of the mixing chamber. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
  57. 57. Jul 1, 2012 By: Fabrice Gritti, Georges Guiochon LCGC North America Volume 7, Issue 30
  58. 58. Distribuição do tamanho de partícula. (A) Zorbax-XDB- 1.8 μm, (B) Acquity-BEH- 1.7 μm, (C) Kinetex-1.7 μm, and (D) EiS-150-1.7 μm. (E) Sobreposição de todas as figuras. Omamogho & Glennon; Anal. Chem. 2011, 83, 1547-1556
  59. 59. DA,B é o coeficiente de difusão de um soluto A em um solvente B, (cm2 s−1), MB é a massa molecular do solvente B (g mol−1), T é a temperatura absoluta em (K), ηB é a viscosidade do solvente B (cP) a uma temperatura T, VA é o volume molar do soluto A (cm3 g−1 mol−1) e ϕB, é o coeficiente de associação do soluto com a fase móvel B (adimensional) (Guillarme et al / Journal of Chromatography A, 1052 (2004) 39–51)
  60. 60. Curvas H–u (curvas de van Deenter) de colunas 1.7 μm core–shell (Kinetex C18, 5 cm×2.1mm) e 1.7 μm totalmente porosas (Waters BEH C18, 5 cm×2.1mm). Fase móvel: 440/560/1 (para proteínas de 18.8 kDa), 470/530/1 (para proteínas de 38.9 kDa) e 610/390/1 (para BSA, 66.3 kDa) acetonitrila/água/TFA, temperatura: 60 ◦C, injeção: 0.5 μl, analitos teste: proteínas. S. Fekete et al. / Journal of Pharmaceutical and Biomedical Analysis 54 (2011) 482–490.
  61. 61. Van Deemter plots das colunas Kinetex, Ascentis Express (2.7 μm), e sub-2 μm totalmente porosas. Fase móvel : (A) 48% acetonitrila–52% água para o estradiol e (B) 95% acetonitrila–5% água para a ivermectina. Temperatura: 35 °C, injeção: 0.5 μL, analito: (A) estradiol (B) ivermectina. (E. Olah et al. / J. Chromatogr. A 1217 (2010) 3642–3653)
  62. 62. Waters Acquity BEH 50 × 2.1 mm, 1.7 μm, C18 column. The mobile phase containing a mixture of 0.01% aqueous trifluroacetic acid and acetonitrile in the ratio of 75:25 (v/v) at a flow rate of 500 μl/min was used A Kromasil C18, 250 × 4.6 mm, 5 μm column was used for separation. Chromatographic separation was achieved in both the modes (isocratic and gradient). Mobile phase consisting of a mixture of A: 0.01% aqueous trifluroacetic acid and B: acetonitrile in the ratio 75:25 (v/v) for isocratic mode Journal of Pharmaceutical and Biomedical Analysis 46 (2008) 236–242 Chemical structures of primaquine phosphate and impurities. (A) primaquine phosphate (B) impurity I: 8-(4-amino-4-methylbutyl amino)-6 methoxyquinoline (C) impurity II: 8-nitro-6- methoxyquinoline.
  63. 63. The LOD and LOQ demonstrated for HPLC (isocratic mode) and UPLC.
  64. 64. O efeito da pressão na retenção.
  65. 65. Effect of pressure (generated by restrictor tubes at column outlet) on the retention of small analytes. Column: Acquity BEHC18 (50 mm × 2.1 mm), mobile phase: water (0.1% TFA) + acetonitrile (0.1% TFA): 70 + 30 (v/v), flow-rate: 100 μL/min, temperature: 30 °C, injected volume: 0.5 μL, detection: 210 nm. Peaks: lidocaine (1), salicylic acid (2), bupivacaine (3), propranolol (4), propylparaben (5) and testosterone (6). S. Fekete et al. / J. Chromatogr. A 1270 (2012) 127– 138
  66. 66. Effect of pressure (generated by restrictor tubes at column outlet) on the retention of related peptides (1–1.3 kDa). Column: Acquity BEH300 C18 (50 mm × 2.1 mm), mobile phase: water (0.1% TFA) + acetonitrile (0.1% TFA): 73 + 27 (v/v), flow-rate: 100 μL/min, temperature: 40 °C, injected volume: 1 μL, detection: fluorescence ex.: 280 nm, em.: 360 nm. Peaks: P-868 (1), P-866 (2), P-870 (3), and P-869 (4).
  67. 67. Effect of pressure (generated by restrictor tubes at column outlet) on the retention of insulin and related proteins. Column: Acquity BEH300 C18 (50 mm × 2.1 mm), mobile phase: water (0.1% TFA) + acetonitrile (0.1% TFA): 69 + 31 (v/v), flow-rate: 100 μL/min, temperature: 50 °C, injected volume: 1 μL, detection: fluorescence ex.: 280 nm, em.: 360 nm. Peaks: insulin (1), related proteins (2–4). Please note that time scale is normalized to the last eluted peak.
  68. 68. Effect of pressure (A) and the combination of pressure and mobile phase velocitiy (B) on the retention (relative change in k) of different size analytes (1.1 kDa peptide, 5.7 kDa insulin, 12.3 kDa cytochrome C and 17 kDa myoglobin). Column: Acquity BEH300 C18 (50 mm × 2.1 mm), mobile phase A: water (0.1% TFA), mobile phase B: acetonitrile (0.1% TFA). Detection: fluorescence ex.: 280 nm, em.: 360 nm. Investigated pressure range: 100– 1100 bar.
  69. 69. Effect of very high pressure (generated by restrictor tubes at column outlet) on the retention (A) and peak shape (A and B) of cytochrome C. Column: Acquity BEH300 C18 (50 mm × 2.1 mm), mobile phase: water (0.1% TFA) + acetonitrile (0.1% TFA): 70.5 + 29.5 (v/v), flow-rate: 200 μL/min, temperature: 65 °C, injected volume: 1 μL, detection: fluorescence ex.: 280 nm, em.: 360 nm.
  70. 70. Effect of pressure on selectivity of separation of a diverse mixture of compounds Column: Ace Excel 5 C18, 5 cm × 0.21 cm I.D., 5 μm, mobile phase 25% ACN in 0.025 M phosphate buffer at pH 2.7. T = 30 °C, F = 0.3 mL/min, λ = 254 nm. Peak identities 1 = uracil, 2 = aniline, 3 = 2- naphthalenesulfonic acid, 4 = propranolol, 5 = prednisone, 6 = acetophenone, 7 = diphenhydramine, 8 = nitrobenzene. Results for this Figure obtained with the Acquity system. M.M. Fallas et al. / J. Chromatogr. A 1297 (2013) 37– 45
  71. 71. Análise de Macro-moléculas
  72. 72. Wide-pore fused-core particles for protein separations. Left: cartoon of particle dimensions. Right: scanning electron micrograph of particles. S.A. Schuster et al. / J. Chromatogr. A 1315 (2013) 118– 126
  73. 73. Focused ion beam scanning electron micrographs of wide-pore fused-core particles. Left: single particle showing porous outer shell. Right: particle cross-section showing shell thickness. S.A. Schuster et al. / J. Chromatogr. A 1315 (2013) 118– 126
  74. 74. Plate height comparison. Columns 100 mm × 2.1 mm; mobile phase: 1.7 μm totally porous – 40.5% acetonitrile/59.5% aqueous 0.1% (v/v) trifluoroacetic acid, = 3.4; 3.4 μm Halo 400 – 42.5% acetonitrile/57.5% aqueous 0.1% (v/v) trifluoroacetic acid, k = 3.6; solute: carbonic anhydrase (29 kDa), 0.1 mg/mL in 6 M urea/1.0% acetic acid; temperature: 60 °C
  75. 75. Reduced plate height comparison. Columns 100 mm × 2.1 mm; mobile phase: 1.7 μm totally porous – 40.5% acetonitrile/59.5% aqueous 0.1% (v/v) trifluoroacetic acid, = 3.4; 3.4 μm Halo 400 – 42.5% acetonitrile/57.5% aqueous 0.1% (v/v) trifluoroacetic acid, k = 3.6; solute: carbonic anhydrase (29 kDa), 0.1 mg/mL in 6 M urea/1.0% acetic acid; temperature: 60 °C
  76. 76. Effect of temperature on protein separations. Column: 100 mm × 2.1 mm Halo Protein C4; gradient: 28–58% B in 10.0 min; mobile phase A: aqueous 0.1% trifluoroacetic acid; mobile phase B: acetonitrile with 0.1% trifluoroacetic acid; flow rate: 0.45 mL/min; instrument: Agilent 1200 SL; injection volume, 2 μL; detection: 215 nm; temperatures as indicated; solutes, proteins as shown.
  77. 77. Effect of pore size on separation of small proteins. Columns: 100 mm×4.6 mm Halo C18 (90 Å pores) and 100 mm×4.6 mm Halo Peptide ES-C18 (160 Å pores); mobile phase: A=water/0.1% trifluoroacetic acid; B=acetonitrile/0.1% trifluoroacetic acid; gradient: 25–42% B in 10 min; flow rate: 1.5 mL/min; temperature: 30 °C; detection: 215 nm; and solutes in order of elution: (1) ribonuclease A, (2) bovine insulin, (3) human insulin, (4) cytochrome c, and (5) lysozyme. Peak widths in minutes above each peak. Journal of Pharmaceutical Analysis 2013;3(5):303–312
  78. 78. Fases estacionárias monolíticas
  79. 79. SEM photographs of monolithic silica rod columns prepared at different PEG concentrations in the starting reaction mixture. PEG: 9.4 g (a), 9.8 g (b), 10.2 g (c), and 10.4 g (d). Other starting reaction conditions: 45 mL TMOS, 100 mL of 0.01 M aqueous acetic acid solution. Concentration of NH4OH used for controlling the mesopore size: 0.01 M. The skeleton size and through-pore size are indicated by black and white arrows, respectively, in (a). Journal of Chromatography A, 1191 (2008) 231–252
  80. 80. Experimental Van Deemter plots of 2.6 μm shell-type (Kinetex), 2.7 μm shell-type (Ascentis Express), sub- 2 μm totally porous particles and a monolith column (peak widths were corrected for the extra-column broadening). Mobile phase: (A) 48% acetonitrile–52% water for estradiol and (B) 95% acetonitrile–5% water for ivermectin, temperature: 35 °C, injection: 0.5 μL, analyte: (A) estradiol and (B) ivermectin. Journal of Chromatography A, Volume 1217, Issue 23, 2010, 3642 - 3653
  81. 81. Structures and possible fragmentations of each analyte and IS. Yin Huang , Yuan Tian , Zunjian Zhang , Can Peng Journal of Chromatography B, Volume 905, 2012, 37 - 42
  82. 82. ZIC®-HILIC column (250 mm × 4.6 mm, 5 μm) from Merck (Darmstadt, Germany).The column temperature was maintained at 45 °C with an injection of 10 μL. Mobile phase A consisted of acetonitrile while mobile phase B onsisted of 10 mM ammonium acetate in redistilled water. The isocratic program was 70% A and 30% B at a flow rate of 0.5 mL/min. Y. Huang et al. / J. Chromatogr. B 905 (2012) 37–42.
  83. 83. Effect of water content on the retention of hydrazines on a ZIC HILIC column. Column temperature was at 30 °C with a flow rate of 0.4 mL/min using a splitter and CLND. The CLND system was set at 1050 °C combustion furnace, 50 mL/min argon, 280 mL/min oxygen, 75 mL/min makeup (argon), 30 mL/min ozone, 5 °C cooler, gain x1, 750 V on PMT. The analyte concentrations were about 30– 70 μg/mL in water/EtOH (20/80, v/v). Acetonitrile at 0.1% (v/v) level in EtOH was used as a void time marker. Injection volume was 10 μL. (A) Chromatographic separation of hydrazines as a function of water content in the mobile phase– TFA/water/ethyl alcohol (0.1/50–10/50– 90, v/v/v). 1: 1,2-Dimethylhydrazine, 2: 1,1-dimethylhydrazine, 3: methylhydrazine and 4: hydrazine. (B) Effect of water content on k′ in the isocratic condition with the mobile phases–TFA/water/ethyl alcohol (0.1/50– 10/50–90, v/v/v). Hydrazine (green), methylhydrazine (red), 1,1- dimethylhydrazine (pink) and 1,2- dimethylhydrazine (blue).
  84. 84. Chemical structures of SBD-F and thiols. (A) Structure of thiols: 1, Cys; 2, Hcy; 3, CA; 4, γ-GluCys; 5, CysGly; 6, GSH; 7, NAC; 8, MPG. (B) Reaction of SBD-F and thiols Analyst,2013, 138, 3802- 3808.
  85. 85. Effect of the type of alcohols on the retention of hydrazines on a ZIC HILIC column. For each separation, isocratic run was performed in the mobile phase of TFA/water/alcohol (0.1/10/90, v/v/v), column temperature at 30 °C, flow rate at 0.4 mL/min with a splitter and CLND. 1: 1,2- Dimethylhydrazine, 2: 1,1-dimethylhydrazine, 3: methylhydrazine and 4: hydrazine.
  86. 86. Effect of the type of acid modifiers on the retention of hydrazines on a ZIC HILIC column. For each separation, isocratic elution was performed with acid/water/ethyl alcohol (0.1/30/70, v/v/v) as a mobile phase, column temperature at 30 °C, flow rate at 0.4 mL/min with a splitter and CLND. 1: 1,2-Dimethylhydrazine, 2: 1,1-dimethylhydrazine, 3: methylhydrazine and 4: hydrazine.
  87. 87. The separation of hydrazines on different columns. Column temperature was at 30 °C; flow rate was 0.4 mL/min with a splitter and CLND. Mobile phase was formic acid/water/ethyl alcohol (0.5/20/80, v/v/v). 0.1% ACN (v/v) in ethyl alcohol was used as a void volume marker. 1: 1,2- Dimethylhydrazine, 2: 1,1-dimethylhydrazine, 3: methylhydrazine and 4: hydrazine. (a) Zorbax NH2, (b) Diol, (c) Amide-80 and (d) ZIC HILIC.
  88. 88. HILIC columns Functional group Chemical structure Inertsil SIL Bare silica Inertsil Amide Amide Inertsil Diol Diol TSKgel NH2-100 Amino PC HILIC Phosphorylcholine ZIC-HILIC Sulfobetaine
  89. 89. Retention time of SBD–thiols on five HILIC columns. Columns: Inertsil Diol, ●; Inertsil SIL, ◇; PCHILIC, ▲; Inertsil Amide, ; ZIC-HILIC, ◆. Column temperature: 35 °C. Mobile phase: acetonitrile–10 mmol l−1 ammonium formate buffer (pH 3.0) = 75/25 (v/v). Linear velocity: 58 mm min−1. Fluorescence detection: ex; 375 nm, em; 510 nm.
  90. 90. Effect of (A) acetonitrile content, (B) pH of ammonium formate buffer and (C) concentration ofammonium formate buffer on retention time of SBD–thiols. Symbols: SBD– MPG, ●; SBD–NAC,◇; SBD– CA, ▲; SBD–Hcy, ; SBD–Cys, ◆; SBD–CysGly, ○; SBD–GSH, ■; SBD–γ-GluCys, △. Column: ZIC-HILIC (150 mm × 2.1 mm i.d., 5 μm, Merck). Column temperature: 35 °C. Mobile phase: (A) acetonitrile–10 mmol l−1 ammonium formate buffer (pH 3.0), (B) acetonitrile–10 mmol l−1 ammonium formate buffer = 75/25 (v/v), and (C) acetonitrile– ammonium formate buffer (pH 3.0) = 75/25. Flow rate: 0.2 ml min−1. Fluorescence detection: ex 375 nm, em 510 nm. Injection sample: 5 μl, containing 90%acetonitrile.
  91. 91. Comparison of five HILIC columns: amide, hybrid silica, diol, bare silica and zwitterionic phase, for the separation of a test mixture of acidic compounds: (AT) coumachlor, (BJ) hydrochlorothiazide, (BP) ketoprofen, (AY) diclofenac, (AD) acetylsalicylic acid, (BH) furosemide. Column dimensions: 2.1 mm × 50 mm, 1.7 μm, except for RRHD 1.8 μm. Flow rate of 0.5 mL/min, λ = 230 nm, injected volume = 1 μL. Condition: ammonium formate (50 mmol/L, pH 4), gradient profile: 95%ACN for 1 min, then 95–65%ACN in 3 min with T = 30 °C.
  92. 92. Comparison of five HILIC columns: amide, hybrid silica, diol, bare silica and zwitterionic phase, for the separation of a mixture of basic compounds: (CJ) noscapine, (AF) alprazolam, (BI) heroin, (CV) sotalol, (AS) codeine, (BK) hydromorphone, (AZ) dihydrocodeine. Column dimensions: 2.1 mm × 50 mm, 1.7 μm, except for RRHD 1.8 μm. Flow rate of 0.5 mL/min, λ = 249 nm, injected volume = 1 μL. Condition: ammonium formate (50 mmol/L, pH 4), gradient profile: 95% ACN for 1 min, then 95–65% ACN in 3 min with T = 30 °C.
  93. 93. Effect of pH on selectivity. Column: bare silica (2.1 mm × 50 mm, 1.8 μm), flow rate of 0.5 mL/min., λ = 249 nm, injected volume = 1 μL. Condition: pH 3 ammonium formate 50 mmol/L, pH 4 ammonium formate 50 mmol/L, pH 5 ammonium acetate 50 mmol/L, pH 6 ammonium acetate 50 mmol/L, gradient profile: 95% ACN for 1 min, then 95–65% ACN in 3 min withT = 30 °C. Peak label: (CL) oxazepam, (AY) diclofenac, (BQ) lidocaine, (CP) pindolol, (CZ) thebaine, (BK) hydromorphone, (AZ) dihydrocodeine
  94. 94. Effect of organic modifier on selectivity: ACN, ACN/IPA 80:20 v/v, and ACN/MeOH 80:20 v/v. Column: silica bare, flow rate of 0.5 mL/min, λ = 249 nm, injected volume = 1 μL. Condition: ammonium formate (50 mmol/L, pH 4), gradient profile: 5% buffer for 1 min, then 5–35% buffer in 3 min with T = 30 °C. Peak label: (CL) oxazepam, (AY) diclofenac, (BQ) lidocaine, (CP) pindolol, (CZ) thebaine, (BK) hydromorphone, (AZ) dihydrocodeine.
  95. 95. Performance comparison of two HILIC columns: (A) an Acquity BEH HILIC (2.1 mm id × 150 mm, 1.7 μm) and (B) an Acquity HILIC amide (2.1 mm id × 150 mm, 1.7 μm) for the analysis of a mixture of hypoxanthine (1: 80 μg/mL), cytosine (2: 10 μg/mL), nicotinic acid (3: 30 μg/mL) and procainamide (4: 30 μg/mL) dissolved in pure ACN. Conditions: mobile phase: ammonium formate (50 mM, pH 3.14) modified with ACN, gradient profile: 95% ACN for 6 min, then 95– 75% ACN in 5 min for (A) and isocratic conditions: 94% ACN for (B), flow rate of 500 μL/min, λ = 214 nm, volume injected = 5 μL, T = 30 °C.
  96. 96. Comparison of the chromatographic performance obtained by (A) RPLC vs. (B) HILIC for the analysis of a mixture of 9 peptides: (1) lysine vasopressin (20 μg/mL), (2) arginine vasopressin (12 μg/mL), (3) peptide D (20 μg/mL), (4) triptorelin (5 μg/mL), (5) peptide A (20 μg/mL), (6) insulin (60 μg/mL), (7) peptide B (6 μg/mL), (8) peptide E (25 μg/mL), (9) peptide C (6 μg/mL) dissolved in water for (A) and in IPA for (B); the star (*) designates an impurity present in synthetic peptides. Conditions: (A) Column Acquity BEH C18 (2.1 mm id × 150 mm, 1.7 μm), flow rate of 400 μL/min, λ = 214 nm, volume injected = 5 μL, gradient profile: 10–90% ACN in 20 min with T = 30 °C, (B) Column Acquity HILIC amide (2.1 mm id × 150 mm, 1.7 μm), flow rate of 500 μL/min, λ = 214 nm, volume injected = 5 μL, gradient profile: 90% ACN for 3 min, then 90–62% ACN in 9 min with T = 30 °C.
  97. 97. size exclusion chromatography
  98. 98. Illustrative description of separation in SEC
  99. 99. HONG, Paula; KOZA, Stephan; BOUVIER, Edouard SP. A REVIEW SIZE-EXCLUSION CHROMATOGRAPHY FOR THE ANALYSIS OF PROTEIN BIOTHERAPEUTICS AND THEIR AGGREGATES. Journal of liquid chromatography & related technologies, v. 35, n. 20, p. 2923-2950, 2012. FEKETE, Szabolcs et al. Theory and practice of Size Exclusion Chromatography for the analysis of protein aggregates. Journal of Pharmaceutical and Biomedical Analysis, 2014.
  100. 100. The free energy change of a chromatographic process can be described by, where ΔG 0, ΔH 0, and ΔS 0 are the standard free energy, enthalpy, and entropy differences, respectively; R is the gas constant: T is absolute temperature, and k is the partition coefficient. For most chromatographic modes of separation, the enthalpy of adsorption is the dominant contributor to the overall change in free energy. SEC is unique in that partitioning is driven entirely by entropic processes as there ideally is no adsorption, ΔH = 0. Thus the previous equation becomes: where K D is the thermodynamic retention factor in SEC. Thus, in SEC separations, temperature should have no impact on retention. In practice, temperature can indirectly impact retention to a small degree by altering the conformation of the proteins, as well as by affecting mobile phase viscosity and analyte diffusivity.
  101. 101. The thermodynamic SEC retention factor is the fraction of intraparticle pore volume that is accessible to the analyte: where V R , V 0, and V i are the respective retention volumes of the analyte of interest, the interstitial volume, and the intra-particle volume. K D will range from a value of 0 where the analyte is fully excluded from the pores of the stationary phase, to a value of 1 where the analyte fully accesses the intraparticle pores.
  102. 102. Separation of (1) thyroglobulin, (2) IgG, (3) BSA, (4) Myoglobin, and (5) Uracil on a Waters ACQUITY UPLC BEH200 SEC, 1.7 μ, 4.6 × 150 mm. Mobile phase: 100 mM sodium phosphate, pH 6.8. Flow rate: 0.3 mL/min. Temperature: 30°C (black), 40°C (blue), 50°C (red). Reproduced with permission from Waters Corporation, Milford, MA
  103. 103. Typical SEC calibration curve
  104. 104. Theoretically expected impact of the particle size and mobile phase temperature on column performance. (For the calculations, a 50 kDa protein was assumed.).
  105. 105. Effect of linear velocity on plate height for (a) ribonuclease A (red) and (b) a monoclonal antibody (blue) on two columns varying in particle size. The 4.6 × 150 mm columns were packed with either 1.7 micron (solid line) or 2.6 micron particles (dashed line). Pore size of stationary phase sorbent: 200 Å. Mobile phase consisted of 100 mM sodium phosphate, pH 6.8. Reproduced with permission from Waters Corporation, Milford, MA.
  106. 106. Comparison of Columns: Effect of Particle Size on Efficiency and Resolution for a Reduced Antibody Theoretical Plates [-17pt] Columns Dimension s (m i.d. × mm length) Particle size (μm) Pore sizes (Å) HC LC Resolution TSKgel G3000SW 7.5 × 300 10 250 1980 3845 3 TSKgel G3000SWxl 7.8 × 300 5 250 5060 10674 4 Shodex KW-804 8.0 × 300 7 250 4952 8859 2 Protein-Pak 300SW 7.5 × 300 10 250 2078 4271 3 BioSuite 250 7.8 × 300 5 250 5149 9403 3
  107. 107. The drug aprotinin (Trasylol, previously Bayer and now Nordic Group pharmaceuticals), is the small protein bovine pancreatic trypsin inhibitor, or BPTI, which inhibits trypsin and related proteolytic enzymes. Under the trade name Trasylol, aprotinin was used as a medication administered by injection to reduce bleeding during complex surgery, such as heart and liver surgery. Its main effect is the slowing down of fibrinolysis, the process that leads to the breakdown of blood clots. The aim in its use was to decrease the need for blood transfusions during surgery, as well as end-organ damage due to hypotension (low blood pressure) as a result of marked blood loss. The drug was temporarily withdrawn worldwide in 2007 after studies suggested that its use increased the risk of complications or death
  108. 108. Schematic representation of some of the key steps in non-native aggregation
  109. 109. Plate heights (HETP) vs. linear velocity (u0) plots of Panitumumab (A), chicken ovalbumin (B) and β-lactoglobulin (C). Columns: Acquity UPLC BEH200 SEC 1.7 μm, 150 mm × 4.6 mm (operated at 30, and 60 °C), YMC-Pack-Diol- 200 5 μm, 300 mm × 6 mm and Phenomenex Yarra SEC- 3000 3 μm, 300 mm × 4.6 mm (operated at 30 and 50 °C). Mobile phase: 20 mM disodium hydrogen-phosphate buffer of pH = 6.8. Journal of Pharmaceutical and Biomedical Analysis 78– 79 (2013) 141– 149
  110. 110. Effect of pressure (A) and temperature (B) on the observed aggregates. Column: Acquity UPLC BEH200 SEC 1.7 μm, 150 mm × 4.6 mm. Mobile phase: 20 mM disodium hydrogen-phosphate buffer of pH = 6.8. Flow rate: 200 μl/min, detection: FL (Ex: 280 nm, Em: 360 nm). The column pressure (head pressure) was varied by adding restrictor capillaries to the column outlet (131, 271, 406 and 465 bar were generated, including the column pressure) on (A). Sample: heat stressed panitumumab.
  111. 111. Representative chromatograms on the effect of column temperature (A) and pressure (B) on the observed amount of antibody aggregates
  112. 112. Representative chromatograms on BEH 1.7 μm column (A) and on YMC diol 5 μm column (B) obtained by injecting the same sample (native panitumumab). Mobile phase: 20 mM disodium hydrogen-phosphate buffer of pH = 6.8. Flow rate: 500 μl/min, detection: FL (Ex: 280 nm, Em: 360 nm), mobile phase temperature: 30 °C. The generated pressure was 274 bar (A) and 73 bar (B).
  113. 113. Fast separation of the aggregate and native form of β-lactoglobulin (A) and of chicken egg ovalbumin (B). Column: Acquity UPLC BEH200 SEC 1.7 μm, 150 mm × 4.6 mm. Mobile phase: 20 mM disodium hydrogen-phosphate buffer of pH = 6.8. For β-lactoglobulin: flow rate: 700 μl/min, mobile phase temperature: 45 °C and for egg ovalbumin: flow rate: 850 μl/min, mobile phase temperature: 60 °C. Detection: FL (Ex: 280 nm, Em: 360 nm) for both cases. Peaks: 1, 2 and 3: high molecular weight species.
  114. 114. C. Wong et al. / J. Chromatogr. A 1270 (2012) 153– 161 SE-HPLC chromatogram profile at 280 nm showing fronting shoulder on monomer of a drug product at time zero (red) and 40 °C 2 months stability sample (black). Column TSKgel BioAsisst G3SWXL
  115. 115. Representative chromatograms of formulated bulk separation using 0.25 M NaCl (black), 0.5 M NaCl (blue), 0.75 M NaCl (green), 1.0 M NaCl (cyan), 1.25 M NaCl (magenta), 1.5 M NaCl (purple), and 1.7 M NaCl (red) sodium chloride in 20 mM sodium phosphate, pH 7.0 mobile phase, and Waters Acquity BHE200 4.6 mm × 300 mm column shown at 280 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
  116. 116. Representative chromatogram of (A) formulated bulk material (blue) and purified monomer fraction (black), and (B) 40 °C, 9 months stability sample (blue) and purified pre-peak fraction (black) using the final mixed mode UPLC method at 280 nm. Mobile phase containing 20 mM sodium phosphate at pH 7.0 and a flow rate of 0.15 mL/min showed the best separation for the mixed mode UPLC method. The pre-peak and the monomer peak were fractionated using the final mixed mode UPLC method for characterization
  117. 117. J. Sep. Sci. 2013, 36, 2718–2727 Chromatograms of PS standard (Mp ∼ 11 600 g/mol, -D- ∼ 1.03) obtained on XBridge(TM) C18 and ACQUITY R C18 columns (4.6 × 150 mm) packed with different size particles: 10, 5, 3.5, and 1.7 m. Mobile phase, THF; flow rate, 1 mL/min; detection, UV at 254 nm.
  118. 118. Stacked chromatograms of SEC separation of two proprietary polymers on BEH 45 unbonded and BEH 45 TMS columns, 4.6 × 150mmin THF at 1 mL/min using UV detection at 254 nm. (A) Polymer A on BEH 45 unbonded; (B) polymer B on BEH 45 unbonded; (C) polymer A on BEH 45 TMS; (D) polymer B on BEH 45 TMS
  119. 119. Chromatograms of PS standards on (A) BEH 200 diol, 4.6 × 150 mm, detection, UV at 254 nm; (B) same as (A), but with ELS detection. Five replicate injections are shown, demonstrating repeatability; (C) 5 m PLgel MiniMix D (4.6 × 250 mm); detection, UV at 254 nm. In all cases, mobile phase was THF and flow rate was 1 mL/min.
  120. 120. Chromatograms of (A) fourteencomponent mixture of PS standards obtained on two 4.6 × 150 mm columns connected in series. First column, 1.7 m BEH diol 200 A° ; second column, 1.7 m BEH diol 450 A° . Mobile phase, THF; flow rate, 1 mL/min; detection, ELS, (B) twelve-component PS standards obtained on three PL gel SEC columns (7.5 × 300 mm each) packed with 5 m Psdivinylbenzene particles with pore sizes labeled as 10E2, 10E3, and 10E4 A° . Mobile phase, THF; flow rate, 1 mL/min; detection, RI.
  121. 121. Chromatograms of PMMA standards and corresponding calibration curve (fifth-order fit). Mobile phase, THF; flow rate, 0.4 mL/min; column, 4.6 × 150 mm, packed with 1.7 mdiol-bonded particles with amean pore size of 200 A° ; detection, ELS.
  122. 122. Efeito do contra ion e de sua concentração na retenção de β- bloqueadores.
  123. 123. FLIEGER, J. The effect of chaotropic mobile phase additives on the separation of selected alkaloids in reversed-phase high-performance liquid chromatography. Journal of Chromatography A, v. 1113, n. 1, p. 37-44, 2006.
  124. 124. Chromatograms of a mixtures of alkaloids (A—caffenine, B— laudanozine, C— colchicine, D—boldine, E—strychnine, F— cinchonine, G—quinine) with different organic anions in the mobile phase.
  125. 125. Effect of anionic additive type on the retention of investigated alkaloids. (*) For emetine and berberine the strongest retention was observed when hexafluorophosphate salt was added to the mobile phase. Their retention factors were higher than 25.
  126. 126. The effect of different anionic additives on retention, peak symmetry and efficiency of narcotine.
  127. 127. Jones, Alan, Rosario LoBrutto, and Yuri Kazakevich. "Effect of the counter-anion type and concentration on the liquid chromatography retention of β-blockers." Journal of Chromatography A 964.1 (2002): 179-187.
  128. 128. Dependence of the retention factors for labetolol, acebutolol, and nadolol versus the concentration of perchlorate counter-anion in the mobile phase. Chromatographic conditions: column: Zorbax Eclipse XDB-C18 (150×4.6 mm), mobile phase: aqueous adjusted with perchloric acid and/or sodium perchlorate (pH 3.0)–acetonitrile (70:30), flow-rate: 1 ml/min, detection: UV at 225 nm.
  129. 129. Dependence of the retention factors for metoprolol, pindolol, and nadolol versus the concentration of perchlorate counter-anion in the mobile phase. Chromatographic conditions: column: Zorbax Eclipse XDB-C18 (150×4.6 mm), mobile phase: aqueous adjusted with perchloric acid and/or sodium perchlorate (pH 3.0)–acetonitrile (70:30), flow-rate: 1 ml/min, detection: UV at 225 nm.
  130. 130. Plot of the acebutolol retention factors versus counter-anion concentration in the mobile phase for different counter-anions used. Chromatographic conditions: column: Zorbax Eclipse XDB-C18 (150×4.6 mm), mobile phase: aqueous (pH 3.0)–acetonitrile (70:30), flow-rate: 1 ml/min, detection: UV at 225 nm.
  131. 131. Plot of the acebutolol retention factors versus counter-anion concentration in the mobile phase for different counter-anions used. Chromatographic conditions: column: Zorbax Eclipse XDB-C18 (150×4.6 mm), mobile phase: aqueous (pH 3.0)–acetonitrile (70:30), flow-rate: 1 ml/min, detection: UV at 225 nm.
  132. 132. Chromatograms of a mixture of β-blockers and o-chloroaniline analyzed at constant pH and increasing perchlorate concentration.. Chromatographic conditions: column: Zorbax Eclipse XDB-C18 (150×4.6 mm), mobile phase: aqueous (pH 3.0)–acetonitrile (70:30), flow-rate: 1 ml/min, detection: UV at 225 nm.
  133. 133. FLIEGER, J. Effect of mobile phase composition on the retention of selected alkaloids in reversed-phase liquid chromatography with chaotropic salts. Journal of chromatography A, v. 1175, n. 2, p. 207- 216, 2007
  134. 134. Experimental retention factors obtained for investigated alkaloids vs. trifluoroacetate and hexafluorophosphate concentration in mobile phase: 30% ACN/10 mM phosphate buffer (dashed lines) and 30% ACN/30 mM phosphate buffer pH = 2.7 (continuous lines).
  135. 135. Graphic comparison of the desolvation parameters obtained using hexafluorophosphate as the counter-anion for two eluent systems: 25% THF/30 mM phosphate buffer (THF) and 30% ACN/30 mM phosphate buffer (ACN).
  136. 136. Chromatograms of mixtures of alkaloids obtained by the use of different mobile phases: (A) 35% ACN/10 mM phosphate buffer (pH 2.7) + 30 mM NaPF6, (B) 40% MeOH/10 mM phosphate buffer (pH 2.7) + 30 mM NaPF6, (C) 25% THF/10 mM phosphate buffer (pH 2.7) + 30 mM NaPF6.
  137. 137. PAN, Li et al. Influence of inorganic mobile phase additives on the retention, efficiency and peak symmetry of protonated basic compounds in reversed-phase liquid chromatography. Journal of Chromatography A, v. 1049, n. 1, p. 63-73, 2004.
  138. 138. Effect of analyte load on: (A) N(h/2) and (B) tailing factor. Chromatographic conditions: 0.1% (v/v) H3PO4:acetonitrile eluent; benzylamine (5% acetonitrile), toluene (50% acetonitrile), Labetalol and 4- nitrophenol (25% acetonitrile), flow rate: 1.0 mL/min; temperature: 25 °C; analyte load: 0.5– 50 μg.
  139. 139. Chromatographic overlays of Labetalol analyzed at different analyte concentrations using increasing mobile phase concentration of perchlorate anion. Chromatographic conditions: analyte load: 3.3, 6.5, 31.2 μg, (a) 75%:0.1% (v/v) H3PO4:25% acetonitrile; (b) 75%:0.05% (v/v) HClO4:25% acetonitrile; (c) 75%:0.3% (v/v) HClO4:25% acetonitrile; (d) 75%:0.4% (v/v) HClO4:25% acetonitrile; (e) 75%:0.5% (v/v) HClO4:25% acetonitrile.
  140. 140. Chromatographic overlays of Dorzolamide HCl analyzed at different analyte concentrations using increasing mobile phase concentration of perchlorate anion. Chromatographic conditions: Analyte load: 1.4, 5.2, 9.2, 48 μg, (a) 90%:0.1% (v/v) H3PO4:10% acetonitrile; (b) 90%:0.05% (v/v) HClO4:10% acetonitrile; (c) 90%:0.3% (v/v) HClO4:10% acetonitrile; (d) 90%:0.4% (v/v) HClO4:10% acetonitrile; (e) 90%:0.5% (v/v) HClO4:10% acetonitrile.
  141. 141. Effect of counteranion type and concentration on analyte retention, peak efficiency, N(h/2), and tailing factor. Chromatographic conditions: Mobile phase: 75% aqueous:25% acetonitrile. Effective counteranion concentration for each mobile phase indicated in figure legend, flow rate: 1.0 mL/min; temperature: 25 °C; analyte load: 0.5 μg; wavelength: 225 nm.
  142. 142. FLIEGER, J. Application of perfluorinated acids as ion-pairing reagents for reversed-phase chromatography and retention-hydrophobicity relationships studies of selected β-blockers. Journal of Chromatography A, v. 1217, n. 4, p. 540-549, 2010. Effect of ion-pairing reagent concentration in methanol/water mobile phase (acetic acid, AA; trifluoroacetic acid, TFAA; pentafluoropropionic acid, PFPA; heptafluorobutyric acid, HFBA) on retention coefficient of investigated β-blockers.
  143. 143. Chromatograms of mixtures of β-blockers obtained by the use of different mobile phases. The peaks order: atenolol, pindolol, nadolol, metoprolol, acebutolol.
  144. 144. XIE, Wenchun; TERAOKA, Iwao; GROSS, Richard A. Reversed phase ion-pairing chromatography of an oligolysine mixture in different mobile phases: effort of searching critical chromatography conditions. Journal of Chromatography A, v. 1304, p. 127-132, 2013. SIR mass chromatograms of a mixture of oligolysine (dp = 2–8) at different percentages of ACN in the mobile phase when heptafluorobutyric acid [HFBA] is 9.2 mM. Column Waters XBridge Shield RP18 column (50 mm × 4.6 mm i.d.; pore size 135 Å, particle size 3.5 μm) thermostated at 35 °C. The number on the top of each peak represents dp. Peaks corresponding to dp = 7 and 8 are shown as an inset for 23% ACN.
  145. 145. XIE, Wenchun et al. Cooperative effect in ion pairing of oligolysine with heptafluorobutyric acid in reversed-phase chromatography. Journal of Chromatography A, v. 1218, n. 43, p. 7765-7770, 2011. Effect of the HFBA concentration on the retention of oligolyisne. The number of lysine residues is indicated adjacent to each curve. (a) Results for all concentrations of HFBA. (b) Results at low concentrations. The y axis is in a log scale in (a) and in a linear scale in (b).
  146. 146. LONG, Zhen et al. Strong cation exchange column allow for symmetrical peak shape and increased sample loading in the separation of basic compounds. Journal of Chromatography A, v. 1256, p. 67- 71, 2012. Chromatograms of basic compounds separated on the Sunfire C18 column (A), XBridge C18 column (B) and the XCharge SCX column (C); Loading amounts on columns were 0.09035 mg, 0.9035 mg, and 3.614 mg from (a) to (c). Peaks: 1 = propranolol, 2 = berberine, 3 = amitriptyline. The mobile phases used for the separation of basic compounds on XCharge SCX column were A: acetonitrile, B: 100 mmol/L NaH2PO4 (pH = 2.83) and C: water. The flow rate was 1.0 mL/min and peaks were recorded at 260 nm. Mobile phase composition on the XCharge SCX column was 50% A, 30% B. The optimized mobile phases on Sunfire C18 column were A: 0.1% FA in ACN (v/v) and B: 0.1% FA in water (v/v). Mobile phase composition on Sunfire C18 column started at 10% A and shifted to 35% A over 30 min. Mobile phases for the analysis of basic compounds on XBridge C18 column were A: acetonitrile, B: 100 mmol/L NH4HCO3 (pH adjusted to 10.12 with ammonia solution) and C: water. Mobile phase composition on XBridge C18 column started at 20% A, 10% B, shifted to 30% A, 10% B from 0 to 10 min, and finally shifted to 60% A, 10% B from 10 to 40 min.
  147. 147. Universidade Federal do Rio Grande do Norte Centro de Ciências Exatas e da Terra Instituto de Química Matéria de ensino: Química Analítica Detectores
  148. 148. DETECTORES Classificação UNIVERSAIS: Geram sinal para qualquer substância eluida. SELETIVOS: Detectam apenas substâncias com determinada propriedade físico-química. ESPECÍFICOS: Detectam substâncias que possuam determinado elemento ou grupo funcional em suas estruturas
  149. 149. DETECTORES Parâmetros Básicos de Desempenho SENSIBILIDADE Relação entre o incremento de área do pico e o incremento de massa do analito MASSA ÁREA Fator de Resposta, S: inclinação da reta Área do pico x Massa do analito o mesmo incremento de massa causa um maior incremento de área S Sensibilidade Na ausência de erros determinados: A = área do pico cromatográfico m = massa do analito
  150. 150. FAIXA LINEAR DINÂMICA Intervalo de massas dentro do qual a resposta do detector é linear MASSA ÁREA A partir de certo ponto o sinal não aumenta mais linearmente O fim da zona de linearidade pode ser detectado quando a razão (Área / Massa) diverge em mais de 5 % da inclinação da reta na região linear: MASSA ÁREA / MASSA 1,05 S 0,95 S
  151. 151. Detector de Ultra violeta (ultraviolet detector, UV) ou espectroquímico
  152. 152. Extrato de folhas da M. aquifolium depois de um processo de hidrolise, detecção a 270 nm Chromatography Research Inte national Volume 2012 (2012), Article ID 691509, 7 pages http://dx.doi.org/10.1155/2012/691509
  153. 153. Uma solução 0,1 mol/L de uma substância de coloração intensa é analisada por LC-UV, mas nenhum pico é observado. Por que?
  154. 154. Detector de índice de refração (refractive index detector, RI) (A) Indice de refração quando não há analito e (B) quando há analito.
  155. 155. Detector Fluorescência. G. Iriarteet al.,J. Sep. Sci.,29, 2265 (2006)
  156. 156. Fontes de ionização para o LC-MS • Ionização por eletronebulização – ESI (Electrospray) – Modo ESI – Modo Ionspray (ISI) • Ionização Química a Pressão Atmosférica – APCI • Fotoionização a Pressão Atmosférica – APPI
  157. 157. Espectrometria de massas (mass spectrometry, MS Diagramas de blocos de um MS Modo de Ionização no Vácuo Interface / Fonte de Íons Interface / Fonte de Íons Analisador de Massas ALTO VÁCUO Detector ALTO VÁCUO Analisador de Massas Detector Modo de Ionização à Pressão Atmosférica (API) Introdução dos analitos Introdução dos analitos Exemplos: EI, CI, PI, MALDI, TSI, FAB, SIMS, FI/FD Exemplos: ESI, APCI, APPI, AP-MALDI, DESI, DART, EASI
  158. 158. Escolhendo o modo de ionização e a polaridade  ESI: solutos iônicos e polares (PM 100-150x103 dalton). Moléculas maiores adquirem mais que uma carga.  APCI: solutos de polaridade média e não-polares (PM 100-2000 dalton).  APPI: mesmos solutos que APCI mas resposta melhor para moléculas de alta apolaridade.
  159. 159. Electrospray – ESI Vazões: 100 nL/min a 5 μL/min
  160. 160. Como você acha que a percentagem de modificador orgânico na fase móvel? ESI pode ser usado com que modos da cromatografia líquida NP, RP, IEX, SE e HILIC? HILIC: A Critical Evaluation http://www.chromatographyonline.com/lcgc/article/articleDetail.jsp?id=835539&pageID=1
  161. 161. Ionização Química a Pressão Atmosférica (APCI)
  162. 162. Fotoionização a Pressão Atmosférica (APPI) Saída HPLC Gás de nebulização Nebulizador (sprayer) Vaporizador (aquecedor) Gás de secagem Capilar Lâmpada UV • Pode ser usado um dopante Ex. Tolueno
  163. 163. http://www.shimadzu.com/an/lcms/lcmsittof/ittof-8.html
  164. 164. Baixa abundância do íon [M+H]+ em m/z 253. Íon [M+H]+ mais abundante Presença de M+ em m/z 252 Isótopo C13 em m/z 254 Abundância 5 x maior que APCI e 20 x maior que ESI. Análise de benzo[a]pireno;100 picomoles; modo positivo; injeção em fluxo. Agilent Technologies, www.agilent.com/chem, 2001.
  165. 165.  Ésteres de testosterona: baixo PM e polaridade média Que modo de ionização vc escolheria? ESI ou APCI?
  166. 166.  Ésteres de testosterona: baixo PM e polaridade média 1 propionato de testosterona 2 fenilpropionato 3 caproato 4 decanoato (b) Pico 2 em APCI (c) Pico 2 em ESI Sandra, P. et al., LC-GC Europe, 2001.
  167. 167. Modos de ionização para MS(/MS) Massa Molecular (GC) EI (GC) CI Apolar Polaridade Muito Polar Fonte: Agilent Technologies, www.agilent.com/chem, 2001.
  168. 168. Analisadores para MS • Setor Magnético e Setor Eletrostático • Quadrupolo • Ion Trap • Tempo de Voo • FT-ICR • (FT) Orbitrap
  169. 169. Focalização Dupla • Vantagens – Alta resolução e exatidão – Excelente estabilidade = resultados quatitativos – Permite MS/MS • Desvantagens – Caro -- Difícil de usar – Velocidade de varredura limitada (histerese) – Detectabilidade é dependente da velocidade
  170. 170. Tempo de Voo - TOF
  171. 171. Quadrupolo
  172. 172. Modos de operação - Espectro dos produtos ou “íons filhos” MS1 Estático Massa do precursor (pai) Câmara de colisão Fragmentação CID Modo RF (passam todas as massas) MS2 Scan Espectro dos produtos (ions filhos)
  173. 173. QqQ – Aplicação MRM (frente) x SIM (trás) Mercaptobenzotiazol SIM: m/z 166 MRM: m/z 166 → 134 Mercaptobenzoxazol SIM: m/z 150 MRM: m/z 150 → 58 Tempo de Retenção, minutos Trends Anal. Chem., 2001, 20, 533-542.
  174. 174. Nigericina Salinomicina Narasina Lasalocida Monensina Cromatograma obtido a partir da análise de matriz fortificada com LAS (20 μg kg-1), MON (10 μg kg-1), SAL (100 μg kg-1) e NAR (15 μg kg-1) na concentração de seus respectivos LMRs e NIG (50 μg kg-1).

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