Liquid Chromatography Separation Mechanism: Comparison
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Separation is a critical process to isolate a particular compound, whether it is a natural product or a synthetic product. Studies of a compound’s characteristics and elucidation structure provides reliable results for pure compounds because there is no interference from other compounds. The primary source of difficulty in a separation process is the high similarity between two or more compounds, such as racemic and homologous mixtures. Liquid chromatography has proven to be an effective solution to those problems. The key to liquid chromatography separation is a sustainable retention and elution process. Stationary phases are essential for separating compounds in liquid chromatography. Various liquid chromatography columns of both preparative and quantitative types have been used and continue to develop. This research will discuss the separation mechanism in liquid chromatography.

  • liquid chromatography
  • stationary phase
  • separation mechanism

1. Introduction

Separation is a critical process to isolate a particular compound, whether it is a natural product or a synthetic product. Studies of a compound’s characteristics and elucidation structure provides reliable results for pure compounds because there is no interference from other compounds. The primary source of difficulty in a separation process is the high similarity between two or more compounds, such as racemic and homologous mixtures. Liquid chromatography has proven to be an effective solution to those problems. The key to liquid chromatography separation is a sustainable retention and elution process. Stationary phases essential for separating compounds in liquid chromatography. Various liquid chromatography columns of both preparative and quantitative types have been used and continue to develop. For this reason, multiple studies and publications related to liquid chromatography can be found and accessed easily.

2. The Principle of Separation of Compounds in Liquid Chromatography

Separation using liquid chromatography is possible because of the different interactions between the compounds present in the sample with the stationary and the mobile phases in the liquid chromatography system (Figure 1). Stationary phases can be developed to enable compound separation based on several modes, such as (A) differences in the affinity to the compounds, (B) differences in the strength of electrostatic forces with the target compounds, and (C) size differences of target compounds. One or more of these modes of interaction will result in compound separation using liquid chromatography. Choosing the appropriate columns, whether commercial or under development, could be somewhat confusing for a user. Understanding the modes of interaction will be helpful to assist users in selecting the appropriate columns for a particular type of analyte.
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Figure 1. Scheme of physical interactions between column and target molecules, based on (A) affinity, (B) electrostatic forces, and (C) size difference in liquid chromatography systems.
In affinity-based separation (Figure 1A), stationary particles (black) with specific functional groups (red) interact with the appropriate compounds (green) through specific binding via a network of interactions, resulting in retention in the column. The stationary phase does not suspend noninteracting compounds (purple), and the compounds flow without retention. The common interactions in this mode of separation are hydrogen bonding, dipole–dipoles interaction, London forces, and complex formation. The combination of hydrogen and dipole-dipole interaction facilitates a hydrophilic interaction, while the London force is hydrophobic interaction.
Complex formation commonly occurs in chromatography where the analytes are proteins [1][2][3], DNA [4], and RNA [5][6]. The stationary phase is modified so that it has a metal-complex binding site that can accept ligands from macromolecules. Stronger interaction between compounds and the stationary phase increases retention time, while a weaker interaction decreases retention time. It is almost impossible to find compounds that have no affinity interactions at all. Even though stationary particles and compounds have very different polarities and no functional groups can interact, London forces always exist. Each molecule interacts differently due to its different functional groups, atomic numbers, and 3D structures. This difference allows for the separation of analog [7] and homologous compounds [8][9].
Affinity chromatography is commonly employed in the separation of biomacromolecules. Further, reverse-phase chromatography (RPC) and hydrophilic interaction liquid chromatography (HILIC) are common for the separation of smaller organic compounds, in which affinity-based separation occurs as a result of interactions based on hydrophobicity and hydrophilicity.
The mode of separation that is based solely on electrostatic separation requires coulombic forces to occur between cationic species and anionic species. Stationary particles can function either as cations or anions, depending on the type of functional groups and the experimental conditions. As exemplified in Figure 1B, the blue-colored anion, which binds to the stationary cation (black) via electrostatic forces, is replaced by the analytes (red) due to a stronger interaction. This process is an ion-exchange mechanism. Green-colored compound, which is neither charged nor negatively charged, is eluted from the column without being retained, as no coulombic interaction occurs between the green-colored compound and the stationary cation. The same principle applies to the separation of cationic compounds using anionic stationary phases. A greater charge density of molecules will produce a stronger electrostatic force, resulting in a longer retention time.
Such electrostatic forces are widely used in ion-exchange chromatography (IEC) or ion chromatography (IC) systems. The charge of the stationary phase and analyte can be changed by adjusting the pH or composition of the mobile phase system so that separation can be carried out. The pH setting affects molecules that are weak acid or weak base. The new temporary molecules or the changes of 3D conformation can result from the changes of the mobile phase. The variables that can change in a mobile phase system are salinity, buffer type, and complexing ligand. The types of samples are small ions [10][11] and macromolecules [12].
Separation based on size difference is better known as size exclusion chromatography (SEC) or gel permeation chromatography (GPC). In this type of chromatography, compounds are separated based on the possibility of the analyte being trapped in stationary particle pores. Commonly, the smaller-sized analyte is retained for a longer time in the stationary pores, so the retention time is increased compared to bigger compounds, as shown in Figure 1C. Generally, this system is used to separate compounds with large molecular masses between 2000 and 20,000,000 amu, such as proteins [13][14], carbohydrates [15], surfactants [16], and polymers [17].
Combining two or three of the above modes of separation could result in more effective compound separation compared to applying only one mode of separation. For instance, chiral chromatography combines the principles of size exclusion and affinity-based separation. Electrochromatography (EC) [18][19][20], widely used to separate proteins and DNA, combines size and electrostatic separation principles. Separation in EC is based on the ratio of the charge density of the compounds. Larger charge density compounds elute faster than the low ones. Mixed-mode chromatography (MMC) [8][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] is a system that can be used for at least two types of chromatography depending on the measurement conditions, especially in the mobile phase. Multi-column chromatography (MCC) is used to separate compounds from complex matrices utilizing a combination of the three separation principles above.
Table 1 summarizes several examples of the types of chromatography that have been developed in the last 10 years. Most of the developed stationary phases are based on silica and organic polymers. The samples analyzed are also extensive, ranging from simple ions to uncharged macromolecules, from polar to nonpolar molecules, and from water-based to organic-based solvents. Various isomers (functional group, optical, and structural isomers) or molecule derivatives can be separated using an appropriate liquid chromatographic system.
Table 1. Types of liquid chromatography, separation principles, and new developments in the field of liquid chromatography.
Mode of Liquid Chromatography Separation Principle Stationary Phase Analyte Mobile Phase
Reverse-phase chromatography Affinity Silica modified with octadecyl acrylate and 2-vinyl-4,6-diamino-1,3,5-triazine [38] PAHs Methanol
Affinity Silica modified with octadecyl acrylate and N-methylmaleimide [39] PAHs and tocopherols Mixed of methanol and water
Affinity Silica modified with N-Boc-phenylalanine and cyclohexylamine [40] Phytohormones Mixed of phosphate buffer and acetonitrile
Affinity Zr6O4(OH)4 MOF modified with 2-amino-terephthalic acid or 4,4′-biphenyl-dicarboxylic acid [41] PAHs and aromatics compound Mixed of methanol and water
Hydrophilic interaction liquid chromatography Ionic Silica modified with (2-(methacryloyloxy)-ethyl)dimethyl-(3-sulfopropyl)ammonium hydroxide or 2-methacryloyloxyethyl phosphorylcholine [42] Mixed of toluene, formamide, dimethylformamide, and thiourea Mixed of water and acetonitrile
Affinity Amino silica modified with polyhedral oligomeric silsesquioxane and acrylamide derivatives [34] Nucleosides, organic acids, and β-agonists Mixed of acetonitrile and ammonium formate solution
Affinity Silica modified with EGDMA and maltose [32] Nucleobases and nucleotides Mixed of water and acetonitrile
Affinity Silica modified with vinyl silsesquioxane and dithiothreitol [43]    
Ionic and affinity Silica modified with pyrazinedicarboxylic anhydrate [44] Oligosaccharides, alkaloid, and organic acid groups Mixed of acetonitrile and ammonium formate solution
Mixed-mode chromatography Ionic and affinity Silica modified with 2-methacryloyloxyethyl phosphorylcholine [35] Protein and lysozyme Mixed of acetonitrile, ammonium formate solution, KH2PO4 solution, NaCl solution
Ionic and affinity Silica modified with octadecyl and diol groups [8] Aristolochic acid and derivatives Mixed of formic acid and acetonitrile
Ionic and affinity Silica modified with glutathione [26] Protein Mixed of water, formic acid, acetonitrile
Ionic and affinity poly(12-methacryloyl dodecylphosphatidic acid-co- ethylene glycol dimethacrylate) [45] Ketone aromatic, phenol and derivatives, small organic compounds Mixed of ammonium formate solution and acetonitrile
Affinity Amino silica modified with octadecyl and carbon dots [31] PAHs, nucleosides, and nucleobases Mixed of water and methanol, acetonitrile, and ammonium acetate solution
Affinity chromatography Ionic and affinity Agarose modified with 2-Mercapto-1-methylimidazole [46] Protein NaOH solution
Ionic and affinity Sepharose modified with ligand complex [3] Protein with histidine Mixed of Tris buffer, sodium chloride, and imidazole
Ionic and affinity Silica modified with N-methylimidazolium ionic liquid [28] Protein Mixed of acetonitrile, trifluoroacetic acid, NaClO4 solution, KH2PO4 solution, and NaCl solution
Ionic and affinity Amino silica modified with glutaraldehyde [47] Protein Phosphate buffer
Ionic chromatography Ionic Bentonite modified with chitosan and cetyltrimethylammonium bromide (CTAB) [11] Cr(III) and Cr(VI) solution Nitric acid solution for Cr(III) and ammonia solution for Cr(VI)
Ionic Polystyrene-methacrylate derivatives modified with poly(amidoamine) [10] Small anions like nitrate, sulfates, bromide, etc. NaOH solution
Chiral chromatography Size and affinity Polysaccharide modified with 3-chloro-4-methylphenylcarbamate [48] Paroxetine hydrochloride groups Mixed of supercritical CO2, methanol, and ammonium acetate solution
Affinity Isopropylcarbamate cyclofructan 6 groups [49] Methionine groups Mixed of methanol, acetonitrile, acetic acid, and triethylamine
Size Silica modified with 3,3′- phenyl-1,1′-binaphthyl-18-crown-6-ether [50] Amino acids and peptides Mixed of perchloric acid solution, acetonitrile, and methanol
Affinity Poly(styrene-divinylbenzene) coated with chitosan [9] Benzoin Mixed of water and acetonitrile
Electrochromatography Size and ionic Poly(POSS-co-META-co-DMMSA) [18] Benzoic acid, nucleosides, bases, glycopeptides Mixed of phosphate buffer, triethylamine, and acetonitrile
Affinity Silica modified with a metal-organic framework (MOF) [20] Benzenes and derivatives Mixed of phosphate buffer and acetonitrile
Size exclusion chromatography Size Poly(methacrylic acid-co-ethylene glycol dimethacrylate) [51] Protein Mixed of water and acetonitrile
The table also shows that most mobile phase types are water, acetonitrile, and methanol. Several modifications by adding organic or inorganic acids and bases were carried out to condition the pH and affinity in the chromatographic system. This selection depends on to the type of liquid chromatography that will be applied. Theoretically, the combination of mobile phases that can be used is unlimited. The compatibility between the stationary and mobile phases needs to be carefully considered. The mobile phase mixture must be completely miscible with each other. The mobile phase must not damage or dissolve the stationary phase.

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