Open access peer-reviewed chapter - ONLINE FIRST

Progress in Technology of the Chromatographic Columns in HPLC

Written By

Serban C. Moldoveanu and Victor David

Submitted: January 27th, 2022 Reviewed: March 2nd, 2022 Published: May 13th, 2022

DOI: 10.5772/intechopen.104123

Analytical Liquid Chromatography - New Perspectives Edited by Serban Moldoveanu

From the Edited Volume

Analytical Liquid Chromatography - New Perspectives [Working Title]

Dr. Serban Moldoveanu and Prof. Victor David

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Chromatographic column is an essential part of a any HPLC separation, and significant progress has been made in developing columns with better performance to provide better separation, a shorter separation time, resilience to a wider pH range of the mobile phase, longer lifetime, use of lower volumes of mobile phase, etc. All these characteristics were achieved by the introduction of novel technologies and improvements of the older ones. These include smaller particle used to fill the column, more homogeneous spherical particles, core-shell particles, monolithic columns, more pure silica as a stationary phase support, use of ethylene bridge silica, a wider variety of active phases, use of mixed mode stationary phases, use of polymers as stationary phase, use of various endcapping techniques, etc. Miniaturization and progress in the instrumentation played an important role for the chromatographic column development. All these aspects are summarized in the present chapter.


  • chromatographic column
  • silica
  • support derivatization
  • reversed phase
  • ion exchange
  • chiral columns

1. Introduction

The chromatographic separation is based on the differences in the retention of the components of a sample dissolved in a mobile phase when passing through a stationary phase typically contained in a chromatographic column. In HPLC, the mobile phase is a liquid and the characteristics of high performance (of the separation) and high pressure (used for the mobile phase) lead to the acronym HPLC. Although cartridges and micro-fluidic chips can be used to contain the stationary phase, a column is much more frequently utilized for this purpose [1, 2]. The external body of the column is a tube made from stainless steel or a strong polymer (e.g., polyether ether ketone or PEEK). This tube is filled with the stationary phase. Stationary phase can be in the form of particles or as monoliths. Both particles and the monoliths usually have a rigid porous support that may also act as the active phase, but more frequently the support has on the surface a chemically bonded or physically coated active phase used for the separation. The progress in the making of chromatographic columns is very important for the development of HPLC. A large body of information describes the progress in column construction including peer reviewed papers, books, and information on the Internet [3, 4, 5, 6, 7, 8, 9, 10, 11]. Present chapter describes some of the more recent progress in column construction and indicates potential for new developments. This progress takes place into two main directions: 1) the improvement of mainstream-type columns that are widely used in everyday work for practical analyses and 2) the development of exploratory new materials for the stationary phase and miniaturization.


2. Short theoretical background

In a chromatographic separation, the components of a mixture are eluting from the column, then are detected, and the detection electric signal is converted into a graphic output as peaksin a chromatogram. The peaks have ideally a Gaussian shape. Each peak has a specific retention time tR. For a compound X, tR(X)is the time (usually measured in min) from the injection of the sample into the chromatographic system to the time of elution of the compound. A time slightly longer than the retention time of the last peak in a chromatogram is indicated as run time.The retention time for an unretained compound is known as dead time t0. The dead time t0 is defined by the ratio L/uwhere uis the linear flow rateof the mobile phase and Lis the column length. In HPLC instrumentation, the controlled parameter by the user is the volumetric flow rate Uand not u. The two parameters, Uand u, are related by the expression:


In formula (1), dis the inner diameter of the column, and ε* is a constant depending on column packing porosity (an average value for ε* is 0.7 although this may vary considerably depending on the stationary phase particle size and structure).

The separation in a chromatographic process between two compounds Xand Y(Xeluting first in the separation) is overall characterized by a parameter termed resolution R. The expression for Ris given by the formula:


In formula (2) parameter k’termed retention factoris defined by the formula:


Parameter k’depends on chemical nature of the separated compound X, on the nature of the mobile phase, on the chemical composition and physical characteristics of the stationary phase as well as on a parameter termed phase ratio Ψ. The value of Ψis given by the ratio Vst/V0 where Vst is the volume of the active part of the stationary phase involved in separation process, and V0 is the dead volume of the column (V0 = t0 U). The retention factor k’is proportional with Ψ. Parameter αis the selectivity, which is defined as the ratio k’(Y)/k’(X), and Nis a parameter termed theoretical plate number, which describes the peak broadening in a separation and estimate the column efficiency. The value of Ndepends on column length and a related parameter to Nindependent on Lis the height equivalent to a theoretical plate H(HETP) defined as L/N.

For achieving a good separation, the value of Rshould be higher than 1.0 and Ris larger when α, Nand k’are larger. These parameters can be increased by improving the columns properties. Since a larger k’ indicates a longer retention time tR, which is not usually desired, the increasing of Ris achieved mainly by increasing αand N. The increase of Nfor a given Lis achieved by decreasing H. The value of Hdepends on the linear flow rate uby the following expression known as van Deemter equation:


In formula (4), Dis the diffusion coefficient of the mobile phase, dpis the diameter of the particles in the column, and A’, B′, and C′are coefficients that depend on the nature of stationary and mobile phase.

In addition to R, many other parameters are used for the characterization of an HPLC separation such as peak asymmetry As, which shows the deviation from the ideal Gaussian shape of the chromatographic peak, column backpressure Δp, which is the difference between the pressure at the column inlet and that at the outlet of the column, etc. Column backpressure is described by the following formula (known as Darcy equation):


In formula (5), ηis the mobile phase viscosity, and ϕris a column flow resistance factor. Also, the columns are characterized by several other parameters and properties such as the construction of particles (porous, core-shell, etc.), uniformity of particles dimensions, porosity of the stationary phase, percent coverage of the solid support with the active phase, resilience of the stationary phase to a specific pH range of the mobile phase, resilience to dewetting, etc. A more detailed description of many parameters and properties describing a separation and characterizing the chromatographic column can be found in various books about HPLC (e.g., [12]). Significant progress in HPLC is being made such that to obtain better separation (higher R), shorter run times, lower values for Asand Δp, reproducible separations, etc. A main source of progress is the improvement in the making of the chromatographic column by modifying parameters such as L, d, dp, ε*, k’, H, As, ϕr, etc., in a manner that will lead to better chromatography. Other properties of the modern columns that are not captured with these parameters are also being improved and will be further discussed.


3. Trends in the column physical dimensions

The column physical dimensions are its length and internal diameter (i.d.). The common column lengths are between 30 mm and 250 mm with typical lengths of 50, 100, 150, 250 mm. The i.d. of the column is used to classify the columns as standard (3.0–4.6 mm i.d.), minibore (2.0–3.0 mm i.d.), microbore (0.5–2.0 mm i.d.), capillary (0.2–0.5 mm i.d.), and nanoscale (0.05–0.2 mm i.d.). The tendency of modern columns is to have them shorter and narrower leading to shorter run times and the use of less solvent. However, for a given H, the decrease in column length Lleads to a lower value for N. The improvements in the stationary phase such that the columns have lower Hallow the use of shorter columns maintaining a desirable R.

The use of narrower columns leads to higher linear flow rate ufor a given volumetric flow rate Uresulting in shorter retention times. This can be seen based on Eq. (3) and from the dependence of t0 on uthat give tR=k+1L/u. Although sorter retention times are desirable, the increase in uis limited by the decrease in the value of H(as indicated by van Deemter Eq. (4)) and by the increase in column backpressure (as indicated by formula (5)). For this reason, the most commonly utilized columns in current practice are those with standard and minibore i.d. The use of microbore, capillary, and nanoscale column encounters problems with a decrease efficiency (decreased α, increased H) [13, 14]. In addition to that, the microbore and narrower columns have a low loading capacity (maximum amount of sample that can be loaded on the column) leading to requirements for the increased sensitivity of the detector. For narrower columns, a compromise in setting Umust be made such that a faster chromatography is obtained but the associated increase in the value of Hdoes not preclude a good separation. A study for the evaluation of the possibilities to use narrower columns indicated that an optimum i.d. is around d = 1.5 mm, which achieves short retention times and low solvent use with good column performance [5].

The tendency to use shorter and narrower column in order to achieve shorter run times and use of less volume of mobile phase will continue in the future [15]. The production of columns with smaller H, higher α, and the progress in the instrumentation allowing the use of higher backpressure for the chromatographic columns as achieved currently with ultrahigh-pressure chromatographs (or ultra-performance LC, UPLC) that can generate up to 1300 bar, will continue to allow the decrease in column length and diameter. In parallel with the developments of commonly used columns for routine analytical laboratories, significant effort is made in developing novel experimental columns using miniaturization, very high column backpressure, as well as special active phases, etc. The progress in the sensitivity of the detectors used in HPLC/UPLC will allow the use of smaller and more diluted samples to overcome the lower loading capacity of smaller columns.


4. Trends in the structure and composition of solid support of stationary phase

The most common type of stationary phase in HPLC and UPLC is made from small particles (typically 1.7–10.0 μm in diameter), which are packed in the body of the column. Monolithic columns are also utilized and are made from a single rod of a solid porous material. Because hydrated porous silica can have a very large surface and can be derivatized to bind an active phase, it is the most common material used as solid support to make the particles and also some monoliths for HPLC. This silica usually has a bonded, grafted, or coated layer of organic material. This organic layer is the active part of stationary phase involved in the separation process, but the silanol groups from the uncovered surface of silica also participate in the separation. In case of hydrophilic interaction liquid chromatography (HILIC) and in normal phase chromatography (NPC), bare silica can be used as active phase without additional coverage due to its polar character.

Not only silica can be used as support for the active stationary phase. Materials such as hybrid organic–inorganic still based on a hydrated silica but containing organic groups such as -CH2-CH2- in its structure can be used as support. Also hydrated zirconia, titania, ceramic hydroxyapatite, or organic polymers can be used as solid support. The progress regarding the solid support is made in two directions, one being the physical characteristics of the support and the other its chemical properties. These characteristics are further discussed separately.

4.1 Physical characteristics of stationary phase support

For particles used as solid support, one first characteristic is the physical type, which can be fully porous, core-shell, or pellicular. Porous particles (1.7–10 μm in diameter) have a porous structure for the entire particle, core-shell have a solid nonporous core 1.5–3 μm in diameter surrounded by a porous outer shell 0.3–0.5 μm in depth. Pellicular particles are solid nonporous spheres covered with a thin layer of stationary phase. Fully porous and core-shell particles are widely utilized in common HPLC practice, while pellicular particles are less common because of their reduced loading capacity. Core-shell particles offer better peak shape (lower Hvalues) compared with fully porous particles and are likely to continue to be used even more frequently in the future. Generally, they are characterized by higher values for phase volume ratio Ψthan monolithic columns, but lower Ψthan fully porous particles [16].

The particles are also characterized by (average) diameter dp, the shape of the particles, which can be irregular or spherical, the uniformity of the particles dimension, the surface area, the pore size and volume, the tortuosity and the uniformity of the channels in the particle, the structural rigidity. The dimension of particles dpwith diameters of 5 μm, 3 μm, 2.1 μm, 1.8 μm, 1.7 μm is commonly used, and smaller particles lead to lower Hvalues as indicated by formula (4). An empirical formula shows how Ndepends on dpas follows:


In formula (6), Ctcan be 2, 2.5, or even 3, depending on other particle characteristics. The use of core-shell particles and dimensions of 1.7–1.8 μm leads to columns having the values for Nper m (L = 1 m) as high as 200,000–300,000 [17].

At the same time with increasing N, lower dpleads to higher Δpas indicated by formula (5). This increase imposes limitations on how small the particles can be made. However, the tendency to use smaller particles to obtain columns with higher Nis likely to continue in the future in spite of the increase in the backpressure, as the capability of the HPLC/UPLC pumps to deliver higher pressures increases.

Regarding particle shape, spherical particles show lower Hvalues compared with particles of irregular form, and more uniform values of particle size show lower Hvalues compared with particles of various sizes (particle size distribution is described by a parameter d90/d10 and values lower than 1.2–1.3 indicate good homogeneity). Particles in modern columns have spherical shape and low d90/d10 values, and these characteristics will continue to be maintained. Similar to the external aspect of particles, the internal structure regarding the channels in the porous material can be more or less homogeneous. The uniformity of particles interior is also contributing to a lower Hvalue for the column.

Another physical characteristic of the stationary phase is its surface area [18]. For silica, common values for surface area are between 100 m2/g and 300 m2/g. The trend for modern stationary phases is to have particles with larger surface area since they can be coated with more active phase (increased Ψvalue). However, the strength of stationary phase tends to decrease when stationary phase surface area increases. High silica strength (HSS) support is available now, allowing its use without restrictions in UPLC-type conditions where the back pressure of the column can be up to 900–1000 bar.

The pore size of the porous solid support is commonly characterized as small pores (with diameter below 60 Å), medium (in the range 60–150 Å), and large (of about 300 Å or larger). Common silica pore size is around 100 Å. However, the selection of the pose size depends on the type of molecules to be separated on the stationary phase. For small molecules (with Mwlower than about 3000 Da), the pore size of 100 Å is adequate, but for larger molecules, special phases with large pore size (about 250 Å) should be used. The adequacy of the pore size for the type of molecules to be separated, and in particular for the separation of proteins, is a field where significant development takes place [19].

Monoliths have a porous structure characterized by mesopores (pores between 2 and 50 nm in diameter) and macropores (about 4000–20,000 nm in diameter). For silica monoliths, the silica skeleton is 1–2 μm thick and has a void volume of almost 80% of the entire column volume. Polymeric monoliths have similar void volume. Since monolithic columns produce a lower pressure drop as compared with columns containing particles with similar characteristics, the monoliths are a promising material to be used as support for chromatographic columns. Monoliths are also successfully utilized in the construction of capillary and nano-LC columns [20].

4.2 Chemical characteristics of stationary phase support

The two main aspects of the chemical characteristics of solid support, which are of interest, include: 1) its internal chemical composition and 2) the chemical functionalities allowing the binding of the active phase (in cases when the solid support does not act itself as the active phase). Regarding the internal chemical composition, the solid support can be made from silica, ethylene or propylene bridged silica, hydrated zirconia, hydrated alumina, aluminosilicates, porous graphitic carbon, zeolites, or various organic polymers such as polystyrene cross-linked with divinylbenzene (PS-DVB) [21], methacrylates, etc. More recently, metal–organic frameworks (MOFs) were experimentally evaluated as support for HPLC stationary phases [22].

The most common support material is hydrated silica, which is obtained in principle from a chemical reaction that generates silicic acids followed by condensation reaction of the type:

The resulting material contains numerous silanol groups that are further used for bonding the active phase. The purity of resulting hydrated silica is very important since the presence of metal ions in its structure leads to undesired effects such as peak tailing in chromatography. Very high purity silica (indicated as Type B) is now common as support in chromatographic columns. The ethylene bridge silica (indicated as BEH technology by Waters or TWIN technology or EVO by Phenomenex) [23] is also a common support offering excellent resilience to the strong acidic or basic character of the mobile phase (pH range of stability 1 to 12). This is a significant advantage compared with the range of pH stability of common silica, which is between 2.5 and 7.5. Ethylene bridge silica can be prepared from hydrolytic condensation of bis(triethoxysilyl)ethane and tetraethoxysilane using a small amount of water in a reaction schematically written as follows:

Ethylene bridge silica is an excellent material to be used as solid support for the stationary phase in HPLC, and its use will continue probably becoming even more common.

Regarding the other materials, columns based on hydrated zirconia are commercially available, but in general, they have lower chromatographic performance compared with those based on silica mainly due to numerous Lewis acid sites present on the stationary phase. Commercially available are also porous graphitic carbon columns. In order to achieve a large surface area, graphitic stationary phases are made using silica as template on which a layer of an organic material is applied followed by pyrolysis in an inert atmosphere to generate graphite. This is followed by the dissolution of silica template [24]. This type of column has a strong hydrophobic character, but some problems with surface homogeneity remain to be solved.

For the organic polymers, various procedures are used to obtain porous materials [21]. In some cases, these porous polymers may also act as the active phase, and in other cases they contain reactive groups on which an active phase is further bound. Among the problems with organic polymers as support material are the limitation of their structural rigidity and propensity to swelling in certain mobile phase compositions. Although for some types of HPLC (e.g., reversed phase or HILIC), the use of silica-based support is by far more common, organic polymers are frequently used in ion exchange chromatography and in size exclusion chromatography. Also, organic polymers are frequently used for making monolith-type columns. [25].

The second important property of the stationary phase support is its capacity to react with a derivatization reagent with the goal of attaching a desired type of functionality such as aliphatic chains of 8 or 18 carbon atoms (C8 or C18) typically used in reversed phase (RP) type of HPLC. For silica support, the reacting capacity is assured by the presence of numerous silanol groups on the silica surface. The number of OH groups per unit mass of silica is characterized by silanol density αOHexpressed by the formula:


In formula (9), Ssurfis the surface in m2/g, and δOHis the amount of silanol groups (in mmol/g). The value of αOHtypically varies between 4.1 and 5.6 OH groups per nm2.

A different type of phase support still based on silica but with different active groups is hydride-based silica (known as type C silica [26]). This material is obtained using a reaction of the type indicated below:

This type of silica can be used as normal phase without further derivatization or can react to attaching further organic groups that will operate as active phase.

Reactive groups used for further derivatization can also be present in various organic polymers. For example, many acrylate-type polymers are synthesized to contain glycidyl groups. These act as reactive sites on the porous polymer surface on which the desired functionalities can be bound. The use of organic polymers as solid support is an attractive alternative in particular related to the efforts toward miniaturization of HPLC columns, where 3D printing technology can be applied to make capillary and nano columns [27].

As described in this section, the most common stationary phase support is based on silica. Although numerous other types of support are continuously evaluated, significant progress is also being made in generating silica with better properties. One of the most promising directions is the preparation of ethylene-bridge silica, which offers an excellent stationary phase support, with good reactivity for binding the desired functional groups and with high resilience to the mobile-phase extreme pH values or composition. The use of ethylene-bridge silica in combination with core-shell type phase will continue to expand, and further progress is likely to continue for this type of phases.


5. Trends in the making of the active part of stationary phase

In many types of chromatographic columns, the active phase intended to be involved in the separation is bound or coated on a porous solid support with a large surface area. In some types of HPLC/UPLC, the solid support acts as the active phase without being further chemically modified, and some details about this type of phases will also be further presented, but this section is dedicated to bonded active phases. The bonded phase on a solid support is a key part of the type of chromatography for which the phase is made. For example, for RP-HPLC, which is the most common type of chromatography, the bonded phase is made to have a hydrophobic character. For this purpose, hydrocarbon moieties with different number of carbon atoms are attached to the porous support. The most common such groups contain 18 aliphatic linear carbon chains (C18) or eight carbons chains (C8), but other hydrophobic groups can be bound. For HILIC-type columns, organic fragments containing diol groups, amide, amino, sulfonylethyl, etc., can be bound. For ion-exchange-type chromatography, the bonded groups can be -COO, -SO3, or -NH3+,- N(CH3)3+, etc.

5.1 Progress in chemical reactions used for generating the active phase

Various chemical reactions are utilized for derivatizing the solid porous support of a stationary phase. The active phase can be directly attached to the silica surface, but variants of this procedure including the use of a pre-derivatization followed by a second one are also used. A typical derivatization reaction can be written as follows:

The reactive substituent X can be Cl, but also OCH3, OC2H5, etc. The substituent Rwill determine the active phase (C8, C18, amino, cyano, and many others). Numerous variants of reaction (11) were applied for the derivatization of the silica solid support. In some of these variants, the CH3 groups are replaced with other reactive substituents such as OC2H5, and the resulting material has the capability to further react. The procedure of using di- or tri-functional reagents (containing two or three reacting groups) leads to surfaces with different degree of coverage [28]. One important type of variant in derivatization is the formation of a single layer of attached active groups (indicated as horizontal polymerization) [29], or the formation of a multiple layer polymer on the silica surface (vertical polymerization). In vertical polymerization, a small amount of water is usually added during the derivatization process such that some of the bounded groups containing reactive fragments such as OC2H5 will be hydrolyzed generation active -OH functionalities that can be further reacting with the derivatization reagent. In this manner, the derivatization can be repeated a number of times [30]. For the preparation of hydrophobic phases with the use of vertical polymerization, various levels of carbon load (C%) can be placed on silica surface, C% varying depending on the procedure between 5% and 30%.

As the sensitivity of detection in HPLC/UPLC is becoming higher and higher in particular with the development of mass spectrometric (MS) and MS/MS detectors, one important quality of the chromatographic columns is to have a very low background that may be caused by small molecule “leaking” into the mobile phase left from the manufacturing process of the stationary phase. The use of trifunctional reagents and new procedures to achieve the derivatization of the solid support (usually of silica) led to chromatographic columns with very low bleed, higher resilience to a wide range of composition for the mobile phase and good reproducibility of the separation.

After derivatization, silica surface (and also the surface of hydrated zirconia or alumina) still remains with a considerable number of underivatized OH groups (silanols in case of silica). These silanol groups interact with the analytes from the mobile phase such that not only the Rgroups forming the active phase influence the separation but also the silanols. This effect is undesirable in some cases, and the process of endcapping is used for diminishing (or removing) the silanol interference. The endcapping consists of additional derivatization that places on silanols small organic groups such as Si(CH3)3. Steric hindrance that precludes the dense covering of the silica surface with larger groups such as C8 or C18 is avoided by using derivatization with small groups such as trimethylsilyl (TMS). Repeated derivatization with the endcapping reagent such as chlorotrimethylsilane is usually performed when most silanol groups are intended to be covered.

The process of silica surface derivatization offers numerous possibilities to generate stationary phases with different properties [31]. A large variety of columns is commercially available, and they are tailored for specific utilization. Derivatization and endcapping are used to obtain stationary phases with higher resistance to extreme pH of the mobile phase (e.g., controlled surface charge or CSH type columns), with extra dense bonding (XDB) of the active phase, with different degrees of hydrophobicity, with polar endcapping groups (e.g., CH2OH), or with embedded polar groups in the hydrophobic chain of the active phase [32, 33]. Besides the procedures summarily indicated above to derivatize the solid support, various other derivatization procedures are reported in the literature [34]. Also, alternative procedures to obtain the active phase such as direct synthesis of silica materials with an active bonded phase surface [35] can be used. Progress in the synthesis of monoliths, as well as of stationary phases based on organic polymers, is also being made [36]. One such example is the production of latex-agglomerated ion exchangers.

A variety of other procedures are available for producing the active phase for HPLC and UPLC columns (e.g., [37]). Some of these procedures are kept undisclosed by the column manufacturers and some are reported in the literature. Also, a variety of novel procedures for attaching the active phase on the solid support are developed, such as grafting of pre-synthesized polymers [38, 39], or direct synthesis of the stationary phase containing the desired functionalities [40, 41].

Stationary phases with better performance including better resolution R, lower asymmetry As, resilience to a wider pH range of the mobile phase, capability to work in 100% aqueous mobile phase (resilience to de-wetting), and production of phases with more complex structure than a single functionality are achieved using a diversity of procedures to derivatize the porous solid support. The use of trifunctional derivatization reagents and special endcapping techniques was among the important procedures to achieve this goal, and the use of these procedures is likely to continue to be improved in the future.

5.2 Improved properties of active stationary phase

The modern columns have various benefits from the improvements in the synthesis of the active phase. For example, from the derivatization with trifunctional reagents, the active phase is more homogeneous and stable, with reduced access of the analytes to the free silanols and more reproducible chromatography. The horizontal polymerization (derivatization) has the advantage of higher homogeneity and reduced presence of free silanols, while vertical polymerization leads to phases with a higher mass of active phase (larger Ψ). The new active phases allow the separation to be based on wider types of interaction, and preparation of phases with mixed mode functionalities is more and more common. The progress made in the endcapping process, the capability to use polar endcapping, and the introduction of controlled surface charge (CSH) contribute to the extension of pH range of column stability. Also, the stability of columns in time (to be used for a larger number of injections), the low bleed allowing the use of the columns with very sensitive detectors without generating a high background signal are important factors in the increase of column quality.

Besides the making of columns with improved characteristics, the increased variety of available columns is another direction in which considerable progress is being made. This variety of columns allows a better selection for a specific task, and also, as bidimensional HPLC is sometimes needed for the separation of complex samples, the column variety offers choices for orthogonal separations [42].


6. Diversity of HPLC columns

Under the acronyms HPLC or UPLC are included a number of similar techniques that have significant differences regarding the mechanism involved in the separation. According to the separation mechanism, a specific type of chromatographic column is used. Some of HPLC techniques are common, and some are more special having lower utilization. One main type of common HPLC/UPLC is RP-HPLC, which is used for the separation of molecules having in their structure hydrophobic moieties but frequently additional polar groups. Other common HPLC types are HILIC used for the separation of strongly polar molecules, ion exchange HPLC used for the separation of molecules capable to ionize, chiral HPLC used for the separation of enantiomeric molecules, size exclusion HPLC used for the separation of molecules based on their molecular size (more precisely hydrodynamic volume), and affinity/immunoaffinity HPLC. Various other techniques less frequently utilized are derived from the main types, and examples of such techniques are ion pair chromatography, hydrophobic interaction, normal phase, ion moderated, etc. The active stationary phase for each of those techniques has specific structures. Regardless of the column type, all modern columns benefit from the progress in the solid support in particular by using high-purity silica and ethylene bridge silica, from the use of core-shell particle construction and the advances in the making of monoliths. Some specific aspects for different types of HPLC/UPLC are further discussed.

6.1 Columns for RP-HPLC

Frequently used for the analysis of a large range of compounds, from small molecules to proteins and from highly hydrophobic to rather polar ones, RP-HPLC is the most commonly applied HPLC technique. To this extensive use is associated a significant number of RP type columns many of them commercially available. For RP-HPLC the active stationary phase contains hydrophobic groups, the most common being C18 and C8 phases. The hydrophobic character of the stationary phase in RP-HPLC can be modified by using the active phase with specific groups. Besides C18 and C8 that are very common, aliphatic C2, C4, C12, C14, C20, C22, C27, C30, cyclohexyl, phenyl, diphenyl, C6-linked phenyl, pentafluorophenyl, cyanopropyl, etc., can be used to create a hydrophobic surface. The hydrophobic character of these phases represents one criterion to differentiate them. However, even for columns containing the same type of phase, such as C18, many variations in the active phase structure are possible. The variations may include the type of bonding (mono, di, or trifunctional), the type of polymerization (horizontal or vertical), the carbon load, the density and uniformity of the coverage of solid support (e.g. of silica), and the variations in endcapping. Some hydrophobic stationary phase may contain polar imbedded groups [43]. Various imbedded groups in aliphatic chains were reported in the literature [32], and some are present in commercially available columns. Some of these groups include ether, amide, urea, carbamate, sulfone, thiocarbamate, etc. These groups are used to modulate separation of many types of organic compounds that have in their molecule polar groups. In addition, the imbedded polar groups “shield” the silica residual silanols for interacting with the analytes (in particular with highly basic ones) leading to a reduced silanol activity of the stationary phase (as in Symmetry Shield type columns) and also to better resilience to extreme pH values of the mobile phase.

Evolution of stationary phases in RP-HPLC (and RP-UPLC) took place in two directions: 1) perfecting common columns such as C18 or C8 columns and 2) exploring the binding of various less common groups on the solid support. Perfection of common columns is being done by working with either fully porous or core-shell particles, using special substrates usually high-purity silica or ethylene bridged silica, controlling the derivatization to be very homogeneous, and using special endcapping. By endcapping with TMS groups, the polarity of the silanols is reduced, but the extent of this process can vary from column type to column type, and some C18 columns are intentionally left with some silanol activity for interacting with polar molecules. The use of endcapping with small polar groups also brings distinctive properties to the RP-type columns. Adding special procedures such as CSH or XDB technologies, the variety of RP columns becomes even larger. CSH technology takes advantage that the silica surface is usually slightly negatively charged due to the dissociation of silanols. This charge can be neutralized by adding specific reagents such that the surface reactivity is decreased. The technology is applied to ethylene bridge particles by incorporating a low level of surface charges on stationary phase particles. Also, the construction of phases with C18 or C8 active phase but based on silica with specifically larger pores (e.g., 250 Å) is a promising path for the separation of large molecules such as proteins.

Regarding the binding of various less common groups on the solid support, special phases with bonded cholesterol or fullerene moieties were made, as well as columns with aliphatic chains having an unusual number of carbons (e.g., C3 or C4 for lower hydrophobicity or C30 for intended higher hydrophobicity) [44]. However, these types of experimental bonded phases did not generate columns with much different hydrophobic properties. The intimate mechanism of hydrophobic interactions caused by the “rejection” of the molecules containing hydrophobic moieties from a polar solvent and their “acceptance” in a hydrophobic stationary phase leads to a non-unique process of separation, as long as the accepting phase is less polar than the mobile phase (e.g., [12]). As a result, the choice of mobile phase composition in RP-HPLC plays an important role in the separation, and the differences in the properties of columns used in RP-HPLC are basically obtained by modulating the ratio of hydrophobicity and residual polar interactions and less by changing the phase hydrophobicity.

The use of hydrated zirconia as solid support, the use of coating of a silica base and not binding it, the use of organic polymers to make phases for RP-HPLC, or the use of porous graphitic carbon as stationary phase, although leading to a variety of columns to be used in RP-HPLC remained with a relatively limited utilization. Both trends of improving columns with common stationary phase such as C18, C8, phenyl, cyanopropyl, and experimenting with new active phases are likely to continue in the future. However, a considerably more impact for the progress is still expected from the improvements of common stationary phases.

6.2 Columns for HILIC and NPC

Important progress has been made in the construction of columns dedicated to HILIC separation. The active phase for these columns must be polar, and it is used with a mobile phase less polar than the stationary phase and containing water plus an organic solvent. Similar phases are used for NPC, but in this case the mobile phase is non-aqueous. Bare silica can be used as stationary phase in HILIC, and the improvements in the silica purity and homogeneity of silanol coverage made these columns rather common. Bonded phases with groups such as diol, ether embedded+diol, amide terminal, polyamide, cyano (also used in RP HPLC) are common. Propylamine, diethylamine, or triazole groups are used to generate weak anionic active phases, sulfonylethyl groups are used to generate weak cationic active phase, and amino+sulfonic, amino+carboxylic groups are used to generate zwitterionic phases. Various other types of phases for HILIC applications were synthesized [45]. These phases have various polarities, but the spacer (handle) molecular fragment connecting the polar group with the silica base plays an important role in the separation. The same features as for RP-HPLC columns, including the coverage of support with the bonded phase, the pH resilience, the preparation procedure using mono-, bi-, or tri-functional reagents, the phase ratio are important for the column quality. Since in the HILIC separations not only the polar interactions are important in the separation, but also the hydrophobic interactions play a role, the carbon load (caused by the spacer) also influences the separation characteristics. Some HILIC columns are also endcapped, and this process changes the stationary phase characteristics in a similar manner as for the RP-HPLC. Besides common phases used in HILIC separations, special stationary phases were also known. Such phases were made with bonded cyclodextrin, bonded perhydroxyl-cucurbit[6]uril, polyhydroxyethyl-aspatamide, polysuccinimide [46], etc. One example of a structure of a zwitterionic stationary phase containing sulfonylalkylbetaine groups used in HILIC separations is indicated below:

Because of the proximity of the positive and negative charged groups in the structure, the phase is not used as a zwitterionic ion exchanger.

Stationary phases based on organic polymers are also used for HILIC separations [47]. However, more common are still the silica-based columns.

6.3 Columns for ion exchange HPLC and related techniques

Ion exchange (IC) stationary phases are classified as cation exchange phases (weak, medium, and strong), anion exchange phases (also weak, medium, and strong), zwitterionic, and amphoteric. The phases contain groups attached through a handle on silica or on an organic polymeric support. Specific groups such as -COO, -PO3H, -SO3, etc., generate cationic phases, groups such as -NH3+, -NH2(CH3)]+, -N(CH3)3]+, −[N(CH3)2(CH2CH2OH)]+, −[N(C2H5)(CH3)2]+ generate anionic phases, and groups such as -N(CH3)2+-(CH2)n-SO3 or -CH(SO3)-(CH2)n-N(CH3)3+ generate zwitterionic phases. While for RP-HPLC and HILIC phases, the use of organic polymeric support is less common, for ion exchange phases the use of polymeric support is more common. A specific type of polymeric support is the latex agglomerated type. The latex agglomerated ion exchange particles contain an internal core that has ionic groups on its surface. On this surface is attached a monolayer of small diameter particles that carry functional groups having bonded ions with an opposite charge with those of the support. The groups of the outer particles have the double role of attaching the small particles to the support and also to act as an ion exchanger for the ions in the mobile phase. The advantages of this type of phase include its stability to a wide range of pH of mobile phase and resilience to higher column backpressure compared with common polymeric columns. This is possible because the cross-linking of the polymer from the core particles can be very high.

Because the loading capacity for the same amount of stationary phase is typically larger for IC columns compared with RP or HILIC columns, and because the separation mechanism is based on ionic interactions, which is different from that in RP-HPLC and HILIC, the capillary columns in IC are more successfully utilized. Such columns are used with a low flow rate (e.g., 0.01–0.02 mL/min) that increases the sensitivity of the conductivity detector used frequently in IC separations [48, 49].

Ion chromatography is extensively used in the separations of proteins and nucleic acids [50], and continuous progress is being made with new phases of IC type. Many such new phases are commercially available [10].

Special ion chromatographic columns are also applied in ion-moderated and ligand exchange chromatography. These types of columns are used for the separation of carbohydrates, sugar acids, as well as lipids. For example, difficult separation such as those between cis and trans lipids and fatty acids can be achieved using an ion-moderated columns containing Ag+ ions [51]. In spite of the need for ion-moderated chromatography for the separation of important types of analytes, some of the existent columns dedicated for ion-moderated chromatography require relatively long run times for the separation. For this reason, development of new ion-moderated type columns would be highly desirable.

The ion exchange stationary phases and columns are in continuous development, and in particular mixed mode phases containing ion exchange type moieties are demonstrated to be very useful in separations. A discussion dedicated to mixed mode phases is also included in this chapter.

6.4 Columns for chiral separations

The increased demand of analysis of a variety of pharmaceutical drugs, many of them with chiral character, required constant development of chiral columns. Other fields of chemical analysis also required chiral separation. For example, the increased use of vaping and the proliferation of companies producing synthetic nicotine required the development of sensitive methods for the analysis of nicotine enantiomers [52]. Active stationary phase for chiral separation can be of different types, which include: brush or “Pirkle” type, cellulose based, cyclodextrin or cyclofructan-based, amylose-based, crown-ether-based, macrocyclic antibiotic type, protein based, ligand exchange type, chiral synthetic polymer type [53, 54], etc. All these phases contain various types of chiral centers. In spite of the existence of such a variety of columns, the need for stationary phases offering better enantioresolution is still actual. Many chiral columns must be used in non-aqueous mobile phase (NPC type chromatography), and fewer phases allow the use of water in the mobile phase for RP, HILIC, or IC-type utilization. However, many chiral compounds are highly polar and some are even insoluble in non-aqueous media. In addition, the widespread electrospray type of MS detection (ESI-MS) generates weak or no response when a mobile phase with no water is used for the separation. For these reasons, continuous effort is made to develop chiral columns that work in RP, HILIC, or IC mode.

The improvements of stationary phases for chiral separations follow the same lines as the one utilized for other types of columns. The use of core-shell type particles (e.g., [55]), smaller particle size, monoliths, various types of phases containing chiral centers such as glicopeptides, and macrocyclic antibiotics, as well as more common ones such as derivatized polysaccharides [56, 57] is providing important tools for obtaining better, more efficient types of chiral chromatographic columns [58].

6.5 Columns for size exclusion HPLC

Size exclusion HPLC (SEC) is a technique used for the separation of analytes according to their molecular size (hydrodynamic volume), and it is applied for the separation of macromolecules of different sizes and of macromolecules from small molecules. Ideally, only the size of the molecule should contribute to the separation, but it is common that some energetic interactions (e.g., of polar type) also take place between the stationary phase and the analytes. These energetic effects can modify the intended purpose in which only the size affects the separation. As the molecular size is usually proportional with the molecular weight Mwof a molecule, size exclusion is also used for the evaluation of Mwfor macromolecules. Depending on the solubility of the polymers in an aqueous solvent or in an organic solvent, SEC is indicated as gel filtration chromatography (GFC) or as gel permeation chromatography (GPC), respectively. The stationary phases in SEC can be based on porous silica or on other inorganic materials, but very commonly on organic polymeric materials. Polymeric materials for making the stationary phase are more frequently used in SEC than in other HPLC procedures [59]. A common material for SEC stationary phase is polystyrene-divinylbenzene (PS-DVB) with different cross-linking degrees, but also gels based on dextran or agarose, hydroxylated poly(methyl methacrylate) (HPMMA), and polyvinylalcohol (PVA) copolymers are used. The separation phase should be made with large and controlled pore dimensions.

Among the requirements for a good stationary phase in SEC is to have homogeneous pores, to be as inert as possible and have minimal energetic interactions with the analytes, and to be resilient to high HPLC-type backpressure. These requirements are not very simple to achieve. The control of pose size such that they are as uniform as possible can pose difficulties during manufacturing. Silica-based SEC columns can be made using bare silica, but also bonded phases containing, for example, diol groups on silica are produced. The use of silica with large pores leads to lower resilience to the backpressure. In addition, the reduction of energetic interactions with the silanol groups on silica is not simple. For the polymeric phases, the problem of resilience to higher backpressure is even more stringent than it is for the silica-based phases. The use of special cross-linked polymers alleviates this problem. Also, SEC columns usually require long run times for separations, but new developments such as making core-shell type stationary phases shorten the separation time. Also, as the pressure resistance of the used materials is better, the reduction in the particle size of the phase contributes to improvements in SEC chromatography [60]. New stationary phases use all those procedures to improve the chromatographic columns for SEC.

6.6 Columns in affinity, immunoaffinity, and aptamer-type HPLC

In affinity/immunoaffinity chromatography (IAC), the stationary phase contains on its surface an immobilized biological complement of the analytes from the mobile phase [61]. Examples of pairs of biological complement and the analytes are antigens and their antibody, lectins and glycoproteins, metal ions and proteins containing amino acid residues that have affinity for the ion (e.g. histidine), biotin and avidin, etc. The solid support for the stationary phase can be silica, synthetic organic polymers, agarose (the neutral gelling fraction of the complex natural polysaccharide agar), cross-linked agarose, cross-linked dextrans (sepharose, sephacryl), cellulose, etc. It is typical for the solid support in affinity chromatography to have large pores, between 300 Å and 500 Å because the technique is used for the separation of large molecules (e.g., proteins and nucleic acids). The stationary phase particles can be porous] or nonporous [62] and also can be monolithic [63]. A variety of techniques are used to make stationary phases for IAC, using different procedures for the immobilization of biological complement ranging from covalent attachment to adsorption-based methods. For example, the immobilization of antibodies can be done through their amine groups by using a support that has been activated with reagents such as N,N′-carbonyldiimidazole, cyanogen bromide, N-hydroxysuccinimide, or tresyl chloride/tosyl chloride [64]. New phases are continuously reported for this technique, with a variety of active phases including different types of proteins, aptamers [65], and dye ligands [66]. Continuous progress is also made regarding stationary phases for biomimetic LC that mimic the interactions in natural biological systems [67, 68].

6.7 Mixed-mode HPLC columns

Preparation of stationary phases with mixed-mode active groups in which the separation is based on two or more types of main interactions is currently an important direction of development in HPLC [11]. Mixed-mode phases offer special separation capabilities and could be a simpler alternative to bidimensional separations that use orthogonal columns [69]. These phases may have reversed-phase and HILIC capabilities, reversed-phase and ion exchange capability, HILIC and ion exchange, or even more than two types of capability allowing for example reversed-phase/hydrophilic interaction/ion-exchange-type separations [70]. Some of the mixed mode phases also have chiral centers such that can be used for special chiral separations [71]. Porous or core-shell silica can be used for the preparation of mixed mode phases, and common functionalities such as C18, NH2, diol, SO3, etc., that are specific for one type of phase are used to obtain the mixed-mode phases. The main difference from single type of phase is that multiple functionalities are simultaneously present on the solid support. Synthesis of such phases frequently requires a sequence of derivatizations and strict control of the quality of the final product [72, 73]. The preparation of mixed-mode phases with organic polymers support, in the form of monoliths or using covalent organic frameworks, has also been described in the literature [74, 75]. Mixed-mode stationary phases can also be made as having the active functionality based on ionic liquids moieties [71, 76].


7. Conclusions

The chromatographic column is a key component of HPLC instrumentation, and the extensive use of HPLC promoted the effort for obtaining better columns. These columns provide better separations in a shorter time, generating reproducible chromatography, have minimal bleed avoiding background for the detectors, are resilient to a wide pH range of the mobile phase, can be used with the mobile phase having 100% water, and have a longer utilization life. Progress in making the chromatographic columns has been achieved by various procedures such as the optimizing the chromatographic column dimensions, the use of smaller particles for the stationary phase, the use of monoliths, the use of core-shell type particles. Significant progress was also made in chemistry of stationary phase, both regarding the solid support and the active phase bonded on it. Future progress is expected on the same lines of development for columns used in routine analyses. At the same time, experimental columns for HPLC miniaturization and enhanced efficiency are experimented and reported in the literature (e.g., [20, 77]). The parallel progress regarding the pumping system of HPLC instrumentation that can provide higher backpressure and well-controlled low flow rates, the precision of injecting systems (autosamplers), as well as the unprecedent increased sensitivity of detection in particular of MS and MS/MS type, were key for making possible some of the improvements in chromatographic column construction.


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Written By

Serban C. Moldoveanu and Victor David

Submitted: January 27th, 2022 Reviewed: March 2nd, 2022 Published: May 13th, 2022