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Efeito do contra ion e de sua 
concentração na retenção de β- 
bloqueadores.
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.
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.
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.
The effect of different anionic additives on retention, peak symmetry and efficiency of 
narcotine.
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.
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.
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.
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.
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.
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.
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
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).
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).
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.
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.
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.
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.
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.
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.
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.
Chromatograms of mixtures of β-blockers obtained by the use of different mobile phases. The 
peaks order: atenolol, pindolol, nadolol, metoprolol, acebutolol.
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.
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).
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.

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Ion par chromatography

  • 1. Efeito do contra ion e de sua concentração na retenção de β- bloqueadores.
  • 2. 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.
  • 3. 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.
  • 4. 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.
  • 5. The effect of different anionic additives on retention, peak symmetry and efficiency of narcotine.
  • 6. 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.
  • 7. 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.
  • 8. 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.
  • 9. 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.
  • 10. 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.
  • 11. 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.
  • 12. 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
  • 13. 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).
  • 14. 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).
  • 15. 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.
  • 16. 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.
  • 17. 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.
  • 18. 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.
  • 19. 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.
  • 20. 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.
  • 21. 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.
  • 22. Chromatograms of mixtures of β-blockers obtained by the use of different mobile phases. The peaks order: atenolol, pindolol, nadolol, metoprolol, acebutolol.
  • 23. 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.
  • 24. 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).
  • 25. 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.