Perspective Chapter: Mixed-Mode Chromatography

Abstract

In this chapter, we present mixed-mode stationary phases and their applications in the determination of nonpolar, polar, and charged compounds, as well as larger molecules such as peptides or proteins using a single column. Mixed-mode chromatography (MMC) has been growing rapidly in recent years, owing to the new generation of mixed-mode stationary phases and a better understanding of multimode interactions. Mixed-mode chromatography provides a wide range of selectivities and adequate retention of a variety of compounds, especially polar and charged molecules. In summary, this technique is particularly useful in the pharmaceutical analysis of drugs, impurities, biopharmaceuticals, and polar compounds in natural products.

1. Introduction

The development of liquid chromatography is one of the most active areas of research in separation science, with applications in various fields, such as drug analysis, medicinal chemistry, agriculture, food chemistry, and bioanalysis. This study aims to determine the optimal working conditions for the effective and selective separation of chemical compounds. In the research process, the choice of optimal chromatography conditions is of prime importance, including the determination of a suitable liquid chromatography mode and the investigation of mobile phase characteristics (pH, type of organic modifier, mobile phase additive, etc.) [1].

Chromatographic retention processes can be divided into many types, such as normal-phase, reversed-phase, ion-exchange, hydrophobic interaction, hydrophilic interaction, and metal coordination chromatography. These chromatographic methods are known as single-mode chromatography because the retention of solutes in these chromatograms is dependent on a single-retention mechanism. For instance, in reversed-phase chromatography, problems may be encountered during the analysis of highly polar (or charged) compounds. Hydrophilic interaction chromatography (HILIC) is designed for the analysis of polar compounds; however, it is still affected by a range of challenges, such as low solubility in highly organic media, the amount of organic solvents used, the sample matrix that affects retention, and the retention extent of hydrophobic analytes that can be controlled. Ion-exchange chromatography can be used for charged molecules, but not for neutral analytes. Therefore, mixed-mode chromatography (MMC) can be utilized to resolve some of the problems associated with each of the other mechanisms [2].

Mixed-mode chromatography (MMC) separates solutes by using a stationary phase that involves in the separation two or more types of interactions. Compared to single-mode chromatography, mixed-mode chromatography can simultaneously act on different functional groups of the solute, such as hydrophobic and ionic groups [3]. Mixed-mode chromatography is not a new technique. Many chromatographic matrices are based on rigid supports, such as cellulose, agarose, polyacrylamide, or silica gel, which are modified to produce specific functionalities on their surfaces. If the solute is a substance with numerous functional groups, such as amino acids, nucleic acids, peptides, and proteins, which are commonly found in biological samples, mixed-mode chromatography will exhibit a distinct behavior as opposed to that of single-mode chromatography [4].

Recently, MMC has been receiving increasing attention as an alternative or complementary tool to traditional chromatography (reversed phase, ion exchange, and normal phase) in pharmaceutical and biopharmaceutical applications because of its efficient selectivity and adequate retention of a variety of compounds—particularly polar and charged molecules. To achieve better solute retention characteristics, selectivity, and separation capabilities, mixed-mode stationary phases must be designed and synthesized based on the specific structural characteristics of different compounds. Additionally, the diversity of the mixed-mode stationary phase depends on the diversity of the analyte structure and its properties. It is expected that the applications of mixed-mode chromatography will increase in the future and serve as a power resolution for the separation and purification of biological substances.

2. Mechanisms of mixed-mode chromatography

2.1 Mechanisms related to stationary phases

2.1.1 Classification of stationary phases by chemistry design

In MMCs, stationary phases have been prepared using several types of stationary phases involving different mechanisms. According to the study design, mixed-mode stationary phases can be divided into four categories [2, 3].

Type 1: A mixed-mode stationary phase is created by combining two types of stationary phase particles (each with a single chemistry) and packing them into a single column (Figure 1). However, the major drawbacks of this approach are the non-homogeneity of the stationary phases and low batch-to-batch reproducibility.

Figure 1.

Types of mixed-mode stationary phases classified by chemistry designs [5].

Type 2: The surface of the stationary phase is modified with a mixture of ligands of different chemistries. This is a second-generation approach, but its disadvantages are similar to those of Type 1. Thus, Types 1 and 2 are not commonly used because of their performance limitations.

Types 3 and 4: Embedded (Type 3) and tipped ligands (Type 4) are the third-generation mixed-mode phases that improve the reproducibility and homogeneity of the stationary phase. The functional groups (polar or ionic groups) of the embedded ligands are close to the pore surface, and the hydrophobic parts of the ligands extend to the mobile phase. In contrast, the tipped ligands have functional groups at the ends of the hydrophobic chains.

2.1.2 Combinations of separation modes in MMC

2.1.2.1 Reversed-phase ion-exchange stationary phases (RP-IEX)

Polar compounds, such as biologically active molecules, natural products, and drug metabolites containing several functional groups, tend to be weakly retained in the reversed phase, resulting in poor separation. With the combination of hydrophobic and ion-exchange mechanisms in the mixed-mode stationary phases, the selectivity and retention of both the hydrophobic and polar compounds are improved [4]. In addition, it is the most popular ligand in MMC and is mainly used for the separation of peptides, nucleotides, basic drugs, and their metabolites. The ligands consist of a hydrophobic part (alkyl chains or aromatic hydrocarbons) and an ionic part embedded in the end, middle, or vicinity of the hydrophobic part. Depending on the structure of the ionic part, four ion-exchange modes can be classified: quaternary amines are used as strong cation-exchange groups (SCX); primary, secondary, or tertiary amines are used as weak cation-exchange groups (WCX); sulfonic acids are used as strong anion-exchange groups (SAX); and carboxyl groups are used as weak anion-exchange groups (WAX) (Figure 2) [6]. The retention mechanism of this mixed-mode phase was based on the formation of a divalent complex involving hydrophobic and oppositely charged analytes. Moreover, repulsive ionic interactions with identically charged functional groups also affect analyte retention in mixed-mode stationary phases. Thus, separation can be optimized by adjusting the mobile phase parameters, such as pH, ionic strength (including the concentration of buffers and modifiers), and solvent strength [4]. For example, the C18/SAX column shows strong retention of acidic compounds due to electrostatic attraction under basic conditions. In addition, the retention values increase with increasing alkyl chain length of the analytes. Elution can be achieved using acidic conditions with high percentages of organic solvents, and/or high ionic strength to make neutral the acidic compounds and weaken the hydrophobic interactions. In contrast, the RP/SCX and RP/WCX columns can effectively retain base compounds, such as peptides and alkaloids, under acidic conditions. If a mobile phase with a neutral pH and a low ionic strength is used, the retention of these compounds is strongly influenced by hydrophobic interactions [7].

Figure 2.

Structures of some RP-IEX mixed-mode stationary phases: (a) RP-WCX, (b) RP-SCX, (c) RP-SAX, and (d) RP-WAX [6].

In the field of mixed-mode reversed-phase/ion-exchange stationary phase, mixed-mode RP/AX based on ethylene-bridged hybrid (BEH) organic/inorganic particles was recently developed, named Atlantis BEH C18 AX. The intermediate C18 surface concentration (1.6 μmol/m²) together with tertiary alkylamine groups) makes BEH C18 AX compatible with highly aqueous mobile phases. The BEH particles used for the BEH C18 AX stationary phase have an average pore diameter of 95 Å that increases retention, stemming from the 46% higher surface area. Furthermore, hydrophilic anion-exchange group of them create positive surface charge, which show stronger retention of negatively charged compounds in a wider pH range while using with buffers of pH 3.0–6.9 in the survey. The extended upper pH limit of BEH C18 AX allows it to be used with a wider range of mobile phase pH values. For samples containing ionizable analytes, mobile phase pH has been demonstrated to be a key variable to use in optimizing RP separations [8, 9].

2.1.2.2 Reversed-phase hydrophilic stationary phases (RP-HILIC)

RP-HILIC mixed-mode stationary phases have shown advantages in the separation of both hydrophobic and hydrophilic compounds, especially proteins. This combination is equivalent to combining the HILIC properties with the reversed-phase properties to analyze complicated compounds and matrices with a wide range of polarities in a single run. The ligands are composed of hydrophobic and polar groups. The hydrophobic parts can be alkyl or aromatic groups, and the hydrophilic parts can be charged or neutral functional groups, such as diol, amide, cyano, and ionic groups (Figure 3) [10]. For compounds with hydrophilic and polar parts, ligands containing nonpolar and polar groups can interact separately with their corresponding nonpolar and polar groups. Therefore, it is possible to improve analyte retention and separation selectivity through multivalent effects, including hydrophobic and hydrophilic interactions. In recent years, many mixed-mode stationary phases have been synthesized and applied to the analysis of surfactants, peptides, nucleotides, and proteins (Table 1).

Figure 3.

Structures of some RP-HILIC mixed-mode stationary phases with (a) diol, (b) amide, and (c) amine polar groups [6].

Hydrophobic part	Hydrophilic part	Application	References
Alkyl chain	Glycol terminus groups	Nonionic ethoxylated surfactants: of alkylphenol ethoxylates and fatty alcohol ethoxylates	[11]
Alkyl chain	Amide groups	Nucleosides and phenolic compounds	[12]
Alkyl chain of L-lysine	Terminal amine group of L-lysine	Aniline compounds (aniline, 2-nitroaniline, 4-aminophenol, 1-amino-2-methylbenzene, and 2,4-dinitroaniline)	[13]
Alkyl chain of small peptide	Amine and amide groups of small peptide Boc-Phe-Aib-Phe	Polycyclic aromatic hydrocarbons, steroids (hydrophobic compounds), nucleosides (hydrophilic compounds)	[14]
β-Hydroxyl fatty acid	Surfactin, a peptide loop including seven amino acids	Chiral separation	[15]

Table 1.

Applications of reversed-phase hydrophilic stationary phases.

2.1.2.3 Hydrophilic ion-exchange stationary phases (HILIC-IEX)

The combination of hydrophilic and ion-exchange groups presented strong advantages for analyzing charged polar compounds. The multivalent effects of these mixed-mode phases provide unique selectivity, higher retention efficiency, and a wider range of application than any single-mode phase for peptide analysis [16]. The main application of this combination is the separation of proteins and peptides. In this mode, the retention mechanism of polar compounds depends on the percentage of an organic solvent (such as acetonitrile (ACN)) in the mobile phase. If a mobile phase has a low percentage of the organic solvent, the analyte retention is dominated by ion-exchange mechanisms. An increase in the percentage of acetonitrile promoted more hydrophilic interactions than ionic ones. At a high concentration of acetonitrile, the electrostatic interactions decreased significantly, whereas the hydrophilic interactions dominated the analyte retention. Bo et al. prepared a HILIC-IEX phase with adjustable selectivity to separate nucleosides and β-agonists, which were synthesized by controlling the mixture ratio of the two functional monomers [17]. In addition, the use of ionic liquids to develop HILIC-IEX stationary phases can provide an environment for multiple interactions, such as electrostatic, dipole-dipole, and π-π interactions, and hydrogen bonding. Quiao et al. developed a new HILIC-SAX phase by using glucaminium-based ionic liquids to separate nucleosides [18]. According to studies reported by Mant et al. [16], the hydrophilic cation-exchange column (HILIC-CEX) has a higher separation efficiency than the RP-LC for peptide analysis, and the highly charged peptides are best resolved by this column [16]. Hartmann et al. [19] separated amphipathic α-helical peptides using a HILIC-CEX column and an RP-LC column. Both columns presented an adequate efficiency but displayed different selectivities. With the HILIC-CEX column, the temperature had a stronger influence on the separation of peptide columns than that with the RP-LC column. The results showed that both the resolution and retention of peptides in the HILIC-CEX phase significantly improved with increasing temperature [19].

2.1.3 Other combinations

Inclusion hydrophobic mixed-mode: The ligands are composed of hydrophobic parts and cavities, cages, or cryptates, which form an inclusion complex with the analytes. Thus, the multivalent effects of this mode include both the inclusion complexation and the hydrophobic interactions. A representative example of this combination is crown ether immobilized on a solid matrix. A tripartite hydrogen bond can form between the six oxygen atoms of the crown ether and three hydrogen atoms of the protonated primary amine. Therefore, these ligands can be used to retain and separate the primary amines or other protonated molecules. Additionally, hydrophobic interactions can form between the methylene on the crown ether and the alkyl chain of the analytes, resulting in improvement of analyte retention [4].

Inclusion hydrophilic mixed-mode: In this mode, the ligands are composed of a cavity, cage, or cryptate, and a polar group. An example of this chromatography process is the binding of crown ethers with primary amines. The hydrogen bonding between the primary amines and crown ether can be enhanced with a polar organic solvent mobile phase, leading to enhanced inclusion effects.

π-π hydrophilic mixed-mode: The ligands are designed by combining two groups: a π-electron donor or π-electron acceptor group, and a polar group. In π-π interactions, the electron-rich π system (π-electron donor) can interact with the electron-deficient π system or other π-electron acceptor groups, through electrostatic interactions. In this chromatography process, the π-interacting groups of ligands can interact with the π-interacting groups of analytes through π-π interactions, and the polar parts of the ligands can interact with the polar parts of the analytes through hydrogen bonding and/or dipole-dipole interactions. The main application of this combination is the separation of chiral compounds. An example of this mode is Pirkle-type chromatography ligands [20].

Π-π ion-exchange mixed-mode: In this mode, the stationary phase can interact with analytes through π-π interactions, dipole-dipole interactions, van der Waals forces, and electrostatic interactions. A representative example of this combination is the cinchona alkaloid derivative phase developed by Lämmerhofer M and Lindner W. Depending on the structure of the derivatives, this phase can exhibit many types of separation, such as anion exchange, cation exchange, and amphoteric ion exchange for chiral chromatography. Therefore, the main application of this phase is the separation of chiral acids, ionic chiral compounds with a wide range of polarities, and amphoteric compounds such as amino acids and small peptides. In particular, the amphoteric ion exchange can also be considered as an example of a multifunctional stationary phase because this ligand can present three ion-exchange modes for chiral separation under the conditions of a polar organic mobile phase. Thus, the anion-exchange mode is utilized for chiral acid separation, the cation-exchange mode for chiral amine separation, and the zwitterion mode for amphoteric compound separation [21].

Polymeric mixed-mode: Several novel polymeric MMC sorbents have been designed specifically for the separation of proteins, mainly serum albumins and immunoglobulins (IgGs). Heterocyclic compounds are unique as MMC ligands with specific aromaticity/hydrophobicity and dissociation properties compared with common aliphatic and aromatic compounds with capability to relatively selectively interact with some proteins, albumins, also antibodies and monoclonal antibodies [22].

Capillary-channeled polymer (C-CP) fiber stationary phases: The unique shape of C-CP gives them high surface area and when packed into columns, the fibers self-align, providing a monolith-like structure with parallel channels of 1–5 μm size. In relation to the size of proteins, the C-CP fibers surface is nonporous, which significantly reduce mass transfer resistance. Thus, separation can be run at high linear velocities and at low pressures without detrimental effect on the separation efficiency. All the studied C-CP stationary phases were able to separate a BSA/hemoglobin/lysozyme mixture at high mobile phase velocity and with acceptable elution characteristics [22].

2.2 Composition of mobile phases and their effects on mixed-mode chromatography

2.2.1 Polar organic solvents

The mobile phase used in mixed-mode chromatography usually involves a polar organic solvent, water, or a buffer. The following four properties of the solvent have significant effects on the retention and separation of analytes: solvent viscosity, dielectric constant, dipole moment, and surface tension. Solvent viscosity affects the chromatography process in various ways, especially when gradient conditions are used. Firstly, an increase in the viscosity of the mobile phase is the prime reason for an increase in the backpressure. Moreover, the column efficiency is influenced by the viscosity of the mobile phase. For example, a mixed solution of methanol and water has a higher viscosity than pure methanol. As a result, it reduces the diffusion coefficient of the solutes and exhibits a slow mass transfer, leading to a reduction in the column efficiency [23]. The dielectric constant (ε) and dipole moment (μ) characterize the polar nature of the solvent. A solvent with a higher ε value is usually considered a weaker eluent in reversed-phase chromatography, whereas the dipole moment is related to solvent polarity and has important effects on the interactions between the analytes and the ligands in hydrophilic chromatography. Finally, the surface tension of a solvent can affect analyte separation. A mobile phase with higher surface tension can lead to stronger analyte retention. In addition, the UV wavelength cutoff of the solvent must also be considered when a UV-Vis detector is used to measure the concentration of the analytes [24].

In RP-IEX mixed-mode chromatography, polar organic solvents (such as methanol, acetonitrile, ethanol, and tetrahydrofuran) were used as strong eluotropic components. Furthermore, organic solvents can control the retention and elution of analytes in the chromatography process, thereby providing scope to increase the solubility of analytes in the mobile phase. An increase in the organic solvent concentration causes the polarity of the mobile phase to decrease, and the hydrophobic interactions between the analytes and the ligands decrease, resulting in a decrease in retention. According to the eluotropic strength, the order of solvents is water < methanol < acetonitrile < propanol < isopropanol < tetrahydrofuran [23]. The most commonly used polar organic solvents are methanol and acetonitrile (ACN). To analyze peptides and proteins, acetonitrile is preferred over methanol because the mixed solution of acetonitrile and water has a low viscosity, leading to excellent mass transfer.

A binary mixture of organic solvents and buffers is commonly used in HILIC-IEX chromatography. An increase in the organic solvent concentration can reduce the polarity of the mobile phase, leading to a strengthening of the hydrophilic interaction between the analytes and the ligands. In contrast, decreasing the organic solvent concentration can weaken the hydrophilic interactions, facilitate ionic interactions, and lead to compound elution. Thus, the organic solvent acts as a polarity modifier. One of the most commonly used solvents is acetonitrile [4]. As acetonitrile is an aprotic solvent that does not possess a hydrogen bond donor capacity, it cannot compete with the analytes for the ligands. If the mobile phase has a high level of acetonitrile, analytes can be adsorbed to the stationary phases through polar interactions, and they can be resorbed by reducing the acetonitrile content. Therefore, in the HILIC-IEX mixed-mode, acetonitrile has an important effect on the retention and separation of analytes. At high acetonitrile levels (up to 90%, v/v), the hydrophilic interactions may dominate the electrostatic interactions, and this may become the main factor affecting analyte retention.

Furthermore, to elute proteins that are strongly bound to the mixed-mode stationary phase and are significantly affected by hydrophobic interactions, reducing the polarity of the mobile phase by increasing the organic solvent content can be used as a severe elution method instead of reducing the salt concentration.

2.2.2 Buffers and pH

In mixed-mode chromatography, buffers are usually added to the mobile phase to either maintain the pH at an almost constant value or to adjust the pH value. Buffer systems can be selected depending on the required pH range. Buffer systems are classified into two categories according to their components.

Type 1: A buffer system is composed of a weak acid and its conjugate base, or a weak base and its conjugate acid, such as acid acetic/sodium acetate or ammonium chloride. For example, when acetate buffers are used in anion-exchange mixed-mode chromatography, CH₃COO⁻ can participate in the ion-exchange process by binding to the positively charged ligands. Therefore, a buffer system with buffer ions having the same charge as the ligands is ideal for mixed-mode chromatography involving ion-exchange mechanisms. Positively charged buffer ions are preferred when using an anion-exchange mechanism (having positively charged ligands), and negatively charged buffer ions are recommended for the cation-exchange mechanism.

Type 2: A buffer system contains an organic amine or an amphoteric compound that can be used in both the anion-exchange and the cation-exchange chromatography. The examples of such a buffer system are N-2-hydroxyethylpiperazine-N-2′-2-ethanesulfonic acid (HEPES) and N, N-dihydroxyethylglycine (BICINE).

The mobile phase pH can influence the charged properties of the analytes and the nature of the ligands; therefore, it can be used to promote the adsorption and elution of the target compounds [24]. Certain rules are applicable for selecting a suitable pH value for the mobile phase. Firstly, the ideal pH should be selected according to the pKa of the analytes and the ionic groups of the ligands. For example, a target compound with amine groups will be positively charged when the pH value is lower than its pKa, thus resulting in adsorption by cation-exchange ligands. Generally, for the adsorption process, the pH should be selected to charge the analytes and facilitate the electrostatic interactions between the analytes and the oppositely charged ligands. Therefore, the pH should be lower than the pKa of the analytes by approximately 1–2 pH units when the adsorption is carried out on a cation-exchange ligand, while the pH should be higher than the pKa of analytes by approximately 1–2 pH units on anion-exchange ligands. In contrast, for the elution process, the pH should be adjusted to weaken or disrupt the interaction between the target compounds and ligands by charge repulsion. Secondly, the pH of the mobile phase should be within the stability range of the stationary phase. Finally, for protein analysis, it is necessary to select a pH value at which the proteins are stable and retain their biological activity [4].

In mixed-mode chromatography involving ion-exchange mechanisms, the charged properties of weakly acidic and basic ligands can be significantly affected by the pH value. For example, if the mixed-mode stationary phase contains weakly basic groups when the pH of the mobile phase is higher than the pKa of the ligand, then the ligand is neutrally charged and its hydrophobicity increases. Contrastingly, the ligand is positively charged and has a high hydrophilicity when the pH is lower than the pKa of the ligand. In the elution stage, pH gradient changes can be utilized to obtain a higher selectivity of the separation when the change in solvent polarity and the change in ionic strength produce no improvement in the separation efficiency. By changing the pH, the analytes and the ligands can have the same charge; therefore, the analytes can be eluted by charge repulsion. For example, in the experiment of Hostein et al. [25], α-Lactalbumin, β-lactoglobulin A, and trypsin inhibitor with pIs (protein’s isoelectric point) of 4.5, 5.1, and 4.5, respectively, were separated by using a linear pH gradient from pH 3.8 to 8.0 (0.05 pH units/min) on the multimodal cation exchanger Capto MMC. When the pH value of the mobile phase was higher than that of the pIs, these proteins were negatively charged, as with the ligands, and eluted by electrostatic repulsion. The farther the pH value is from the pIs, the more negatively charged these proteins are, leading to their stronger hydrophilicity. It was observed that a shallower gradient (0.05 pH units/min to 0.01 pH units/min) reduces the sharpness of the peaks but improves the protein resolution.

2.2.3 Salts

In MMCs, salts are usually added to the mobile phase to adjust their ionic strength. Sodium chloride is usually used in the ion-exchange mode, whereas salts with higher solubility in organic solvents (such as sodium perchlorate and ammonium perchlorate) are preferred in the hydrophilic mode. In the hydrophobic mode, salts are classified into two categories: salting-out and salting-in. Salting-out salts, such as sodium sulfate, ammonium sulfate, and potassium sulfate, can be used to stabilize proteins and promote hydrophobic interactions between the proteins and the ligands. In contrast, salting-in salts, such as calcium chloride, magnesium chloride, and zinc nitrate, can increase the solubility of the proteins in water and promote protein denaturation and unfolding [26].

The ionic strength of the mobile phase has a significant effect on the retention and the elution of analytes in both the ion-exchange and the hydrophobic modes. In the mixed-mode chromatography involving ion-exchange mechanisms, because an increase in the ionic strength can suppress the electrostatic interaction between the analytes and the charged groups of the ligand, it may result in the weakening of analyte binding on the ligands, thereby leading to a decrease in the analyte retention or elution [24]. Moreover, in the hydrophobic mode, the increase in the ionic strength of the mobile phase can cause the analytes to strengthen their binding to the hydrophobic parts of the ligands, leading to an increase in the analyte retention. Hydrophobic mixed-mode stationary phases are typically used for protein separation. In this mode, the proteins are adsorbed at high salting-out salt concentrations and eluted at low salt concentrations. Therefore, reducing the salt concentration in the mobile phase can be used in the elution mode [4].

2.2.4 Other additives

For protein separation, proteins can be eluted with ease by changing the pH or by reducing the polarity or the ionic strength of the mobile phase. However, when the proteins are firmly bound to the ligand, the recovery and the biological activity of the protein can decrease during the elution step. Therefore, additives are usually added to the mobile phase to reduce its polarity, resulting in the weakening of protein binding to the hydrophobic parts of ligands, and leading to an enhanced protein recovery. Some of the commonly used additives and their functions are listed in Table 2.

Additive	Function	Mechanism	References
Magnesium chloride	Improving recovery and maintaining biological activity of proteins	Promoting protein dissolution	[27]
Ethylene glycol	Reducing hydrophobic interactions	Causing a slight increase in electrostatic interactions	[28]
Glycerol	Stabilizing proteins	Inhibiting protein unfolding; Interacting with hydrophobic surface regions of proteins	[29]
Urea	Affecting to hydrophobic and hydrogen bonding interactions; Causing a slight decrease in electrostatic interactions	Interacting with the polar side chain and backbone of proteins; Changing the solvation of proteins	[30]
Arginine	Reducing hydrophobic and electrostatic interactions and hydrogen bonding	Interaction with the polar side chain and aromatic moieties of proteins	[31]
Caprylic acid	Causing a large decline in the retention of proteins	Binding to the region which is also the binding site of proteins to mixed-mode ligands	[32]

Table 2.

Commonly used additives in mixed-mode chromatography process.

3. Pharmaceutical analysis application of mixed-mode stationary phases

3.1 Drugs and impurities in drugs

A mixed-mode column with a stationary phase of 50% hydrophobic C₁₈ phase and 50% strong cation exchanger allows for a simultaneous detection of the ionic and hydrophobic analytes [33]. Acetaminophen and its related impurities, which ionize based on the mobile phase pH, are often separated for drug examination using ion-pair chromatography, which is a technique for organic charged compounds. Despite its numerous advantages, the corrosive effect of a large number of counterions on the stationary phase of the column is a practical drawback of the ion-pair chromatography. As a result, the mixed-mode stationary phases can overcome the limitations of ion-pair chromatography, allowing for the simultaneous separation of ionic and neutral organic molecules without practical constraints [34]. Furthermore, because of the lack of UV chromophores in most drugs, refractive index (RI) and evaporative light-scattering detection (ELSD) detectors have been utilized. However, these approaches are insensitive or have compatibility issues with gradient elution. Recently, charged aerosol detection (CAD) has been developed as a new type of detector for high-performance liquid chromatography (HPLC) applications. CAD is a universal detection technique for nonvolatile and semi-volatile substances with higher sensitivity and reproducibility than other types of detectors. It is highly convenient in usage as it eliminates the necessity for parameter optimization [35]. Table 3 shows the combinations of MMC and CAD detectors, as well as the applications of various types of MMCs for drugs and impurities. Therefore, it is also a viable analytical tool for concurrently determining a wide range of drugs, pharmaceuticals, and their related compounds in a particular procedure [33].

Drugs
Compounds	Mechanisms	References
Atovaquone, proguanil, and its two main metabolites	50% C18 phase and 50% strong cation exchanger	[33]
Naproxen sodium and adenine hydrochloride; An undisclosed drug in hemifumarate salt form	Acclaim Trinity P1 stationary phase with CAD detector	[35]
Imidazole, pyrazole, pyridine, pyridazine, piperidine	Reversed-phase and ion-exchange characteristics	[36]
Etidronate disodium [(1-hydroxyethylidene) bisphosphonate]	Primesep SB column (anion-exchange reverse phase column) with CAD detector	[37]
Flurbiprofen, flufenamic acid, mefenamic acid, ibuprofen, loxoprofen, ketoprofen, carprofen, indoprofen; sulindac	C18-DTT stationary phase (dithiothreitol silica (SiO2); A reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography	[38]
metoprolol, salicylic acid, acetylsalicylic acid, propranolol, betamethasone, imipramine, clotrimazole, thioridazine, indomethacin flurbiprofen	Two UHPLC mixed-mode hybrid CSH (charged surface hybrid) stationary phases modified by C18 or Phenyl group	[39]

Impurity
Compounds	Impurity analysis	Mechanisms	References
Acetaminophen (paracetamol)	4-Nitrophenol, 4′-chloroacetanilide; 4-Aminophenol; P-Benzoquinone Hydroquinone	Octadecylsilane/strong cation exchanger (C18/SCX)	[34]
L-Methionine	N-Acetyl-dl-methionine; N-Acetyl-l-methionyl-l-methionine; N-Acetyl-l-methionyl-d-methionine and its enantiomers; L-Methionine-sulfoxide	Reversed phase/cation exchange	[40]
Bispecific IgG	Homodimer Impurities	Mixed-mode size exclusion chromatography (mmSEC)	[41]

Table 3.

Application of mixed-mode stationary phases in drugs and impurity.

3.2 Metabolomics applications

A common target of pharmacokinetic studies is the development of a biological analysis method for simultaneous observation of a wide range of drugs in a biological matrix. The tandem usage of reversed-phase and ion-exchange chromatography in MMCs has shown favorable results on the retention of polar and nonpolar small molecules in a single run [42]. In addition, efficient retention and separation of the above compounds were obtained under common and MS-friendly RP conditions, reaching a high point of selectivity and sensitivity. Therefore, MMC tandem mass spectrometry has been commonly applied in metabolic analysis. For instance, the study by Roverso et al. [43] demonstrated the effective retention of selected highly polar metabolites, which was performed by using a mixed cationic-RP column, and simultaneously obtained an efficient separation in the analysis without ion pair and derivatization of 2,4-diaminobutyric acid (DAB) and isobaric beta-methylamino-L-alanine (BMAA) [43]. The metabolomics applications are summarized in Table 4.

Metabolites	Matrices	Mechanisms	References
Cytarabine (ara-C)	Mouse plasma	Reversed phase/ion exchange	[42]
Trimethylamine N-oxide (TMAO); Beta-methylamino-L-alanine (BMAA); 2,4-Diaminobutyric acid (DAB)	Plasma and urine	Reversed phase/cation exchange	[43]
S-Propargyl-cysteine (SPRC)	Rat plasma	Reversed phase/cation exchange	[44]
Phosphorylated carbohydrates	—	Reversed phase/weak anion exchange, combine with a charged aerosol detector	[45]

Table 4.

Metabolomics application of mixed-mode stationary phases.

In addition, combination of MMC with molecular imprinting technology is also improving recognition selectivity for protein BSA, which proved a potential combination of other chromatography modes and molecular imprinting technology [22].

3.3 Biopharmaceuticals and polar compounds in natural products

For biopharmaceutical analysis, Capto and HEA HyperCel MMC ligands with multimodal functionality have been commercialized. Capto includes a carboxyl group that exhibits the characteristics of a phenyl group involved in hydrophobic interactions, and a weak cation exchanger. HEA HyperCel contains a hexyl group that is involved in hydrophobic interactions, and a protonable amine localized in the spacer arm. The application of this type of MMC has been demonstrated in the research by Sophie Maria et al. for mAb determination [46]. Meanwhile, tri-mixed-mode chromatography and another dual combination of MMC are also useful tools for biopharmaceutical analysis. The applications are listed in Table 5.

Compounds	Mechanisms	References
Biopharmaceuticals
Recombinant monoclonal antibodies (mAbs)	Capto MMC resin	[46]
Model drug product was made by mixing an IgG1 mAb (MW 145 kDa, pI 8.4) with seven excipients from different property categories: sodium and potassium (cation), chloride and succinate (anion), histidine (zwitterions), trehalose (hydrophilic neutral sugar), and PS80 (hydrophobic nonionic surfactant).	Combination of four different separation mechanisms (size exclusion, anion exchange, cation exchange, and reverse phase)	[47]
Pembrolizumab; (Anti-PD1 IgG4 wild type and S228P mutant)	Weak hydrophobic interactions and size exclusion	[48]
Polar compounds in natural products
Theophylline, gastrodin, tetrahydropalmatine, lycorine, berberine, sinomenine, and tetrandrine	C18-DTT stationary phase [A reversed-phase liquid chromatography (RPLC)/hydrophilic interaction liquid chromatography (HILIC)]	[42]
H. diffusa and S. barbata aqueous extract	Strong anion exchange and reversed phase	[49]
Amino acids (L-pyroglutamic Acid, L-valine, L-tyrosine, L-proline); Carbohydrates (D-glucose, D-sucrose, α-D-glucopyranosyl-(1 → 2)-βD-fructofuranosyl-(1 → 1)-α-D-galactopyranose, and D-stachyose); Succinic acid; (Dan-Qi pair that make from Radix Salvia miltiorrhiza and Radix Panax notoginseng)	Directly coupled reversed-phase and hydrophilic interaction liquid chromatography–tandem mass spectrometry	[50]

Table 5.

Application of mixed-mode stationary phases in biopharmaceuticals and polar compounds in natural products.

Table 5 also illustrates the determination of polar compounds in natural products. Strong anion-exchange and reversed-phase mechanisms were analyzed in both the polar and more apolar ionic and nonionic compounds and have been used to determine Chinese herbal medicines that provide good retention for separation [49]. Thus, the combination of reversed phase and hydrophilic interactions is a common mechanism in this field because of its suitable characteristics for the detection of polar compounds, especially those of natural origin.

4. Conclusion

In this chapter, advanced applications of mixed-mode stationary phases are reviewed. By adjusting the ratio of organic matter and the mobile phase concentration, the reversed-phase, HILIC, and IEX modes can be successively used. In conclusion, RP-IEX, RP-HILIC, and HILIC-IEX are the most commonly preferred mixed-mode stationary phases.

Acknowledgments

We would like to express their hearty gratitude to Can Tho University of Medicine and Pharmacy. We also thank all of our colleagues for their excellent assistance.

We would like to thank Editage (www.editage.com) for English language editing.