This document discusses the use of reversed-phase ultrahigh pressure liquid chromatography to analyze macro molecules. It examines the effect of pressure and hydrophobic mobile phase additives on the retention of small molecules and proteins. The document presents data on the physicochemical characteristics and retention factors of various compounds under different pressure and mobile phase conditions. It finds that increasing pressure and adding liophilic agents like NaClO4 and KPF6 increases the retention of proteins and other compounds. This allows for improved separation selectivity and resolution of mixtures like lysozyme and cytochrome C.

1. 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.

2. Physicochemical Characteristics of Compounds Used in the Study
Compound Name
Molecular
Weight,
Dalton pKa
b
Molar Volume,
cm3/mol
Urea b 60 14.43 49.52
Pyridine b 79 5.23 82.66
Uracil b 112 8.95 & 15.6 84.81
Naphthalene b 128 NA 123.55
Propylparaben b 180 8.23 158.77
RDX b 222 −15.06 117.01
Nortriptyline b 263 10.00 242.85
Amitriptyline b 277 9.18 257.79
HMX b 296 −16.32 151.76
Labetolol b 328 9.30 273.56
CL-20 b 438 −18.95 167.37
Bradykinin a 1,060 – 707.00
Insulin a 5,808 – 3872.00
Aprotinin a 6,512 – 4341.00
Cytochrome C a 12,400 – 8267.00
Rybonuclease a 13,700 – 9133.00
Lysozyme 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.).

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

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

5. Retention Factor Shift Upon Pressure Increase from 6000 psi to
9000 psi with Different Liophilic Additives
k' H3PO4 H3PO4 NaClO4 NaClO4 KPF6 KPF6
Psi 6000 9000 6000 9000 6000 9000
Nortriptyline 0.42 0.59 1.85 2.18 3.06 3.53
Amitriptyline 0.48 0.66 2.06 2.44 3.48 4.03
Pyridine NA NA 0.03 0.04 0.13 0.13
Naphthalene 6.71 7.45 6.54 7.16 6.40 6.88
Insulin NA NA 0.12 0.16 1.10 1.41
Cytochrome C 0.11 0.12 0.11 0.13 12.92 19.75
Aprotinin NA NA 0.09 0.10 1.12 1.30
Myoglobin 2.41 3.63 4.14 5.29 6.39 7.89
Lysozyme NA NA 0.18 0.29 18.86 28.88
Bradykinin NA NA 0.11 0.13 0.46 0.51
Rybonuclease NA NA NA NA 3.04 3.87
Propylparaben 1.72 1.91 1.69 1.86 1.65 1.77
Labetolol 0.11 0.11 0.45 0.51 0.75 0.82
CL-20 4.06 4.96 3.88 4.34 3.73 4.07
HMX 0.82 0.93 0.79 0.86 0.77 0.82
RDX 0.93 1.04 0.90 0.97 0.88 0.92

6. 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, R
5000 17.0 1.16 1281 1.3
9000 28.9 1.23 1242 1.7

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.

8. 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.