Analise de macro moléculas por RPLC.

MAKAROV, Alexey et al. INVESTIGATION OF THE EFFECT OF PRESSURE AND LIOPHILIC MOBILE PHASE ADDITIVES ON RETENTION OF SMALL MOLECULES AND PROTEINS USING REVERSED-PHASE ULTRAHIGH PRESSURE LIQUID CHROMATOGRAPHY. Journal of Liquid Chromatography & Related Technologies, v. 35, n. 3, p. 407-427, 2012.

Physicochemical Characteristics of Compounds Used in the Study

Compound Name

Molecular Weight, Dalton pKa b

Molar Volume, cm3/mol

Urea b 60 14.43 49.52Pyridine b 79 5.23 82.66Uracil b 112 8.95 & 15.6 84.81Naphthalene b 128 NA 123.55Propylparaben b 180 8.23 158.77RDX b 222 −15.06 117.01Nortriptyline b 263 10.00 242.85Amitriptyline b 277 9.18 257.79HMX b 296 −16.32 151.76Labetolol b 328 9.30 273.56CL-20 b 438 −18.95 167.37Bradykinin a 1,060 – 707.00Insulin a 5,808 – 3872.00Aprotinin a 6,512 – 4341.00Cytochrome C a 12,400 – 8267.00Rybonuclease a 13,700 – 9133.00Lysozyme 14,400 – 9769.0035

Myoglobin a 17,199 – 11466.00

a Estimated partial molar volume based on linear relationship between molecular weights and partial molar volumes from the paper by Imai et al.35.b Partial molar volumes and pKa's were calculated using ACD/Labs software Release 12.0 (Advanced Chemistry Development, Inc.).a Estimated partial molar volume based on linear relationship between molecular weights and partial molar volumes from the paper by Imai et al.35. b Partial molar volumes and pKa's were calculated using ACD/Labs software Release 12.0 (Advanced Chemistry Development, Inc.).

Retention factor versus flow rate at constant temperature in premixed mobile phase (55%aqueous 0.1v=v% H3PO4 with 35 mM KPF6=45% Acetonitrile).

Effect of pressure (at constant flow rate) on retention factor with and without liophilicadditives in premixed mobile phase : (A) No liophilic agents added; (B) 35 mM NaClO4 added; and (C) 35 mM KPF6 added.

Retention Factor Shift Upon Pressure Increase from 6000 psi to 9000 psi with Different Liophilic Additivesk' H3PO4 H3PO4 NaClO4 NaClO4 KPF6 KPF6

Psi 6000 9000 6000 9000 6000 9000Nortriptyline 0.42 0.59 1.85 2.18 3.06 3.53Amitriptyline 0.48 0.66 2.06 2.44 3.48 4.03Pyridine NA NA 0.03 0.04 0.13 0.13Naphthalene 6.71 7.45 6.54 7.16 6.40 6.88Insulin NA NA 0.12 0.16 1.10 1.41Cytochrome C 0.11 0.12 0.11 0.13 12.92 19.75Aprotinin NA NA 0.09 0.10 1.12 1.30Myoglobin 2.41 3.63 4.14 5.29 6.39 7.89Lysozyme NA NA 0.18 0.29 18.86 28.88Bradykinin NA NA 0.11 0.13 0.46 0.51Rybonuclease NA NA NA NA 3.04 3.87Propylparaben 1.72 1.91 1.69 1.86 1.65 1.77Labetolol 0.11 0.11 0.45 0.51 0.75 0.82CL-20 4.06 4.96 3.88 4.34 3.73 4.07HMX 0.82 0.93 0.79 0.86 0.77 0.82RDX 0.93 1.04 0.90 0.97 0.88 0.92

Chromatogram example of mixture separation by pressure increase. Conditions: Column: Acquity BEH C18 1.7 µm, 2.1*50 mm, Flow rate 0.5 mL/min, Temp. 45°C, Inj. 3 µL, Detection 250 nm, Mobile phase: 55%: 0.1% H3PO4 + 35 mM KPF6/45%: MeCN, Isocratic run 10 min. (Color figure available online.)

Chromatographic Merits for Mixture of Lysozyme and Cytochrome C at 5000 psi versus 9000 psi (Calculated for Lysozyme)

PSI k' Selectivity, α Plate Count, N Resolution, R5000 17.0 1.16 1281 1.3

9000 28.9 1.23 1242 1.7

MAKAROV, Alexey; LOBRUTTO, Rosario; KARPINSKI, Paul. Effect of pressure on secondary structure of proteins under ultra high pressure liquid chromatographic conditions. Journal of Chromatography A, v. 1318, p. 112-121, 2013.

Retention factor versus pressure at constant temperature and constant flow rate for various proteins and small basic molecules at different concentrations of acetonitrile in the mobile phase. Chromatographic conditions: column: Acquity BEH C-18 2.1 mm × 100 mm, 1.7-μm; flow rate 0.5 ml/min; column temperature 45 °C; mobile phase compositions: (65:35, v/v) 35 mM potassium hexafluorophosphate in 0.1 v/v% o-phosphoric acid: acetonitrile or (55:45, v/v) 35 mM potassium hexafluorophosphate in 0.1 v/v% o-phosphoric acid: acetonitrile or (50:50, v/v) 35 mM potassium hexafluorophosphate in 0.1 v/v% o-phosphoric acid: acetonitrile or (45:55, v/v) 35 mM potassium hexafluorophosphate in 0.1 v/v% o-phosphoric acid: acetonitrile.