Login Register Cart 0
Generic placeholder image

Current Analytical Chemistry

Editor-in-Chief

ISSN (Print): 1573-4110
ISSN (Online): 1875-6727

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Exploring the Effect of Buffer Strength on the Retention Time of Weak Acids, Neutral and Weak Bases in Hydrophilic Interaction Liquid Chromatography (HILIC) Mode

Author(s): Naser F. Al-Tannak*, Sami Bawazeer and David G. Watson

Volume 15, Issue 1, 2019

Page: [34 - 46] Pages: 13

DOI: 10.2174/1573411014666180806152818

Price: $65

Abstract

Background: Hydrophilic Interaction Liquid Chromatography (HILIC) orthogonal to conventional reversed phase High-Performance Liquid Chromatography (HPLC) mode allowing separation of polar compounds. HILIC has been reported to be an alternative to normal phase liquid chromatography, yet the separation mechanism reported in HILIC is much more complicated than that in normal phase liquid chromatography.

Objective: To investigate the effect of water layer thickness on silica gel and the amount of ammonium ions present within the buffer on retention mechanism in hydrophilic interaction chromatography.

Methodology: A test system was designed which used weak acids, neutrals and weak bases as probes with three different strengths (5, 10 and 20 mM) of ammonium acetate, ammonium formate and ammonium propionate as the counter-ions to compete with the test probes with ionised silanol groups and water present in the stationary phase. A Kromasil 60-5SIL column (150 mm×4.6 mm×4 μm, pore size 60Å) was used as the stationary phase to perform the study.

Results: Retention times were examined for the test probes at 90% acetonitrile (ACN) with 10% of 5, 10 and 20 mM of ammonium acetate, ammonium formate and ammonium propionate. As the buffer strength increases, the thickness of the water layer on the surface of the silica gel increases and also the repulsion between ionized silanol groups and acidic test probes will decrease. On the other hand, such increase in buffer strength will increase the competition between the ammonium ions and basic test probes. In addition, the hydration energy of buffer’s counter ions and hydrophilicity may be important in retention mechanism in HILIC mode.

Conclusion: At 20 mM buffer strength acidic probes with low log P values retain more due to reduced repulsion by silanol groups, while basic probes retention time will decrease due to increased competition from ammonium counter ions. However, in 5 mM buffer strength basic probes with low logP value will be retained longer, while acidic probes will be eluted earlier due to the repulsion between ionized acids and ionized silanol groups.

Keywords: Buffer strength, counter-ion, HILIC, kormasil column, retention mechanism, silica gel, water solubility.

« Previous Next »
Graphical Abstract

[1]
Alpert, A. Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. J. Chromatogr. A, 1990, 499, 177-196.
[2]
Naidong, W. Bioanalytical Liquid Chromatography Tandem Mass Spectrometry methods on underivatized silica columns with aqueous/organic mobile phases. J. Chromatogr. B ., 2003, 796, 209-224.
[3]
Hemström, P.; Irgum, K. Hydrophilic interaction chromatography. J. Sep. Sci., 2006, 29, 1784-1821.
[4]
Buszewski, B.; Noga, S. Hydrophilic interaction liquid chromatography (HILIC)--a powerful separation technique. Anal. Bioanal. Chem., 2012, 402, 31-47.
[5]
Santali, E. Edwards. D.; Sutcliffe, O.; Bailes, S.; Euerby, M.; Watson, D. A comparison of silica C and silica gel in HILIC mode: The effect of stationary phase surface area. Chromatographia, 2014, 77, 873-881.
[6]
Zhang, T.; Watson, D. High Performance Liquid Chromatographic Approaches to Mass Spectrometry based metabolomics. Curr. Metabol., 2013, 1, 58-83.
[7]
Tang, D.; Zou, L.; Yin, X.; Ong, C. HILIC-MS for metabolomics: An attractive and complementary approach to RPLC-MS. Mass Spectrom. Rev., 2016, 35(5), 574-600.
[8]
Alpert, A.; Shukla, M.; Shukla, A.; Zieske, L.; Yuen, S.; Ferguson, M.; Mehlert, A.; Pauly, M.; Orlando, R. Hydrophilicinteraction chromatography of complex carbohydrates. J. Chromatogr. A, 1994, 676, 191-202.
[9]
Churms, S. Recent progress in carbohydrate separation by high-performance liquid chromatography based on size exclusion. J. Chromatogr. A, 1996, 720, 151-166.
[10]
Oyler, A.; Armstrong, B.L.; Cha, J.; Zhou, M.; Yang, Q.; Robinson, R.; Dunphy, R.; Burinsky, D. Hydrophilic interaction chromatography on amino-silica phases complements reversed-phase high-performance liquid chromatography and capillary electrophoresis for peptide analysis. J. Chromatogr. A, 1996, 724, 378-383.
[11]
Yoshida, T. Peptide separation by Hydrophilic-Interaction Chromatography: A review. J. Biochem. Biophys. Methods, 2004, 60, 265-280.
[12]
Hao, Z.; Lu, C.; Xiao, B.; Wenig, N.; Parker, B.; Knapp, M.; Ho, C. Separation of amino acids, peptides and corresponding Amadori compounds on a silica column at elevated temperature. J. Chromatogr. A, 2007, 1147, 165-171.
[13]
Li, R.; Huang, J. Chromatographic behavior of epirubicin and its analogues on high-purity silica in hydrophilic interaction chromatography. J. Chromatogr. A, 2004, 1041, 163-169.
[14]
Strege, M.; Stevenson, S.; Lawrence, S. Mixed-Mode Anion-Cation Exchange/Hydrophilic Interaction Liquid Chromatography-Electrospray Mass Spectrometry as an alternative to reversed phase for small molecule drug discovery. Anal. Chem., 2000, 72, 4629-4633.
[15]
Greco, G.; Letzel, T. Main interactions and influences of the chromatographic parameters in HILIC separations. J. Chromatogr. Sci., 2013, 51(7), 684-693.
[16]
Guo, Y. Recent progress in the fundamental understanding of hydrophilic interaction chromatography (HILIC). Analyst, 2015, 140(19), 6452-6466.
[17]
Heydari, R. A new HPLC method for the simultaneous determination of acetaminophen, phenylephrine, dextromethorphan and chlorpheniramine in pharmaceutical formulations. Anal. Lett., 2008, 41, 965-976.
[18]
Nawrocki, J. The silanol group and its role in liquid chromatography. J. Chromatogr. A, 1997, 779, 29-71.
[19]
Al-Tannak, N.F.; Bawazeer, S.; Siddiqui, T.; Watson, D. The hydrophilic interaction like properties of some reversed phase high performance liquid chromatography columns in the analysis of basic compounds. J. Chromatogr. A, 2011, 1218, 486-491.
[20]
McCalley, D.; Neu, U. Estimation of the extent of the water-rich layer associated with the silica surface in hydrophilic interaction chromatography. J. Chromatogr. A, 2008, 1192, 225-229.
[21]
Gritti, F.; dos Santos, P.A.; Sandra, P.; Guiochon, G. Comparison of the adsorption mechanisms of pyridine in hydrophilic interaction chromatography and in reversed-phase aqueous liquid chromatography. J. Chromatogr. A, 2009, 1216, 8496-8504.
[22]
Kazakevich, Y.; Lo Brutto, R. HPLC for pharmaceutical scientists; Wiley & Sons: New York, 2007.
[23]
Melnikov, S.; Höltzel, A.; Seidel-Morgenstern, A.; Tallarek, U. Adsorption of Water-Acetonitrile mixtures to model silica surfaces. J. Phys. Chem. C, 2013, 117, 6620-6631.
[24]
Smith, D. Ionic hydration enthalpies. J. Chem. Educ., 1977, 54(9), 540.
[25]
Kang, Y.; Nemethy, G.; Scheraga, H. Free energies of hydration of solute molecules. 3. Application of the hydration shell model to charged organic molecules. J. Phys. Chem., 1987, 91(15), 4118-4120.
[26]
Roses, M. Ionic equilibria in non-aqueous solvents: Part 1. General equations for calculation of pH, dissociation constants and reference potentials from potentiometric data. Anal. Chim. Acta, 1993, 276, 211-221.

Rights & Permissions Print Cite
Article Metrics
36
3
© 2024 Bentham Science Publishers | Privacy Policy