Ethylene-bridged hybrid inorganic–organic (BEH) particle technology has allowed for the improvement in separation performance for a range of separation modes, including reversed-phase, hydrophilic interaction and size-exclusion chromatography. In this report, the process of preparing BEH particles, the characterization of these materials and their use for unique chromatographic applications are explored.

The development of column packing materials is a competitive field that is the focus of many industrial research and development (R&D) groups worldwide. The result of these efforts is the commercialization each year of new families of separation columns — all claiming to have improvements over the prior technologies. End users may wonder why we need so many different column brands and phases. 

The question asked in an industrial R&D setting is: How can a new family of column packings be differentiated from others in the marketplace? To achieve a successful product, R&D efforts need to be focused on user needs. As shown in Figure 1, there are a number of user considerations for new separation columns. A new column family may try to differentiate on one or more of these user needs.  

Figure 1: Understanding how to optimize research parameters to meet user needs is a critical factor in the development of new chromatographic materials.			

Because user needs change over time, it is critical for an R&D team to be able to feel the pulse of these changing requirements. In the 1990s, users were struggling with column reproducibility and as a result, industrial R&D efforts focused on improving the reproducibility and purity of porous silica packing materials (1). At the turn of the century, a major user need was increased selectivity options and research groups addressed that need with the development of new stationary phases (2).  

Our exploration of hybrid inorganic–organic materials (denoted

) started in the mid-1990s as a way to address the needs of scientists using reversed-phase chromatography for improved selectivity, efficiency, resolution, peak shape, stability and reproducibility (3). We found hybrid particles to be high performance materials that are well suited to meet these needs. This technology has expanded into the preparation of new materials to address user requirements in hydrophilic interaction (HILIC) and size-exclusion chromatography (SEC). In the following sections, we will detail how hybrid particles are prepared and how their unique qualities can be used to improve separations.  

Hybrid materials have a mixed composition of silica and organosiloxanes. Our initial research into hybrid particles focused on the use of methylsiloxanes and these products were commercialized in 1999 as the XTerra family of reversed-phase columns (4,5). These columns demonstrated improved chemical stability and reduced silanol activity over silica-based reversed-phase columns.  

As hybrid columns started to be adopted, our research focused on preparing novel hybrid formulations and exploring new chromatographic applications for these materials. Early in this research, we determined that improved performance can be achieved with a new hybrid formulation based on an ethylene-bridged hybrid group. This became the foundation of our BEH particle technology and the basis of a number of commercial products since the initial introduction in 2004 (6,7).  

Figure 2: Chemical structure and scanning electron micrograph (SEM) image of a BEH particle.			

The BEH particle (Figure 2) is formed in a patented, multistep manufacturing process, as shown in Figure 3 (8,9). In the first step of this process, two high-purity monomers, tetraethoxysilane and 1,2-bis(triethoxysilyl)ethane, are co-condensed in a 4:1 molar ratio to form a water-immiscible polyethoxyoligosilane polymer (denoted 

). In the next step, the PEOS is emulsified in water (o/w emulsion) to form highly spherical oil droplets. Surfactants and high shear mixers are employed for control of droplet size. Upon addition of a catalyst, the condensation reaction completes to yield highly spherical, porous hybrid particles. The pore volume of BEH materials is controlled in this step through the use of chemical additives called 

. Modification of porogen concentration can be used in this process step to produce new materials that differ in pore volume over a wide range (0.3–1.6 cm

/g). BEH particles used in reversed-phase and HILIC separations have a 0.7-cm

/g pore volume. In contrast, BEH particles used in SEC have a 1.3-cm

Figure 3: The manufacturing of BEH particles is a fully optimized multistep process that allows for the control of critical design parameters at different steps in the process.			

Next, we modify the surface area and pore diameter of these materials with a pore ripening step (8). This step exposes the particles to elevated temperatures and pH to allow for increased pore diameters and decreased surface areas. By changing the conditions for this process step we have been able to produce hybrid particles with average pore diameters that range between 40 and 760 Å. The pore diameter of the BEH particles is tailored for specific separations. The BEH particles used in small molecule reversed-phase and HILIC separations have a 130-Å pore diameter; materials used for peptide and protein reversed-phase separations have a pore diameter of 300 Å; and materials used for size-exclusion chromatography have 125-Å or 200-Å pore diameters. 

Additional steps shown in Figure 3 include particle sizing, bonding and column packing. These parameters are fully optimized for individual applications. Following this process, hybrid particles have been produced with particle sizes of 1.7–10 μm. 

While the process of producing hybrid particles is multistep, this approach is well understood and has been fully scaled to the multikilogram level. During the initial development program, we determined how every step of the process impacted the critical design parameters. We found that using separate reaction steps to control critical design parameters ensures a high level of batch-to-batch reproducibility. This process is also useful for tailoring new materials for specific applications (10). For example, high resolution, faster SEC separations can be achieved using 1.7-μm BEH particles that have increased pore volume. Alternatively, wide-pore BEH particles that are used for reversed-phase separations of proteins and peptides are prepared using the same BEH precursors as those used for analytical small molecule separations, but with a different pore ripening step. Table 1 details some physical parameters of these tailored BEH particles.  

Table 1: Selected physical parameters of BEH technology columns.			

The BEH particles have many similarities to high-purity porous silica. For example, these materials have surface silanols that can be derivatized with silanes to yield a variety of different reversed-phase materials (such as, C18, C8, phenyl-hexyl and fluoro-phenyl). These similarities allow most separations using BEH columns to use a similar method development as traditional silica columns.  

The BEH materials differ from silica because ethylene-bridged hybrid groups are homogeneously distributed throughout the particle. These internal and surface hybrid groups modify the chemical properties of these materials in a number of ways that are advantageous for use in separations. This includes improved chemical stability and reduced silanol activity over silica columns. 

Acid Stability of Bonded Phases on Hybrid Particles

When developing a new chromatographic stationary phase, it is important to assess chemical stability. The use of accelerated stability tests allows one to determine how extreme operating conditions impact the efficiency or retentivity of a column. Loss in efficiency can be an indicator of poor mechanical stability or the dissolution of the packing material. Changes in retentivity are often indicative of bonded-phase hydrolysis.  

When reversed-phase columns are evaluated using an accelerated acid stability test, the stability concern is bonded-phase hydrolysis and not particle dissolution. In these tests, we monitor the loss in retention for neutral analytes (11). Stability under acidic conditions is bonded-phase dependent, and not a function of the base particle chemical formulation. As a result, similarly bonded hybrid and silica reversed-phase columns have comparable stability results. Improving acidic stability requires improvements in bonded phase technologies. The use of trifunctional bondings, for example those based on octadecyltrichlorosilane, allows for improved acid stability over monofunctional bondings, such as those based on octadecyldimethylchlorosilane. However, trifunctional bonded phases have historically suffered from poor reproducibility. Fully optimizing trifunctional bondings was initially a challenge. Our prelaunch prototypes consisted of more than 130 different experiments over a two-year timeframe. Each material had to be bonded, optimally packed, and fully evaluated using a series of tests (for example, isocratic and gradient; pH 3, 7 or 10; UV or mass spectrometry [MS] detection; and van Deemter analysis). In order to process this large dataset, we used similar design of experiments (DoE) and quality by design (QbD) methodologies that many of our customers use. The end result of this was a new approach to trifunctional bonding and endcapping that yielded a high level of reproducibility and improved low pH stability of our packing materials. This advanced bonding technology is an important part of BEH particle technology.  

Base Stability of Bonded Phases on Hybrid Particles 

Chromatographers using reversed-phase chromatography sometimes prefer to work under high-pH conditions for reasons of increased retention of basic compounds, increased compound stability, improved selectivity or the ability to use volatile MS-compatible buffers like ammonia. One of the most notable attributes of BEH columns is stability when used under basic conditions (7). The BEH particles have increased chemical stability at elevated pH because of the hydrophobicity and internal cross-linking of the ethylene-bridged hybrid group. A standard test for base stability challenges reversed-phase columns at elevated pH (10) and temperatures (50 °C). The traditional failure mode of silica-based reversed-phase columns under these conditions is catastrophic loss of column efficiency because of particle dissolution (12,13). Visual inspection of silica-based reversed-phase columns after testing often reveals the formation of voids. The BEH columns are stable under these conditions. To demonstrate a clear change in performance for BEH columns, this base stability test needed to be modified to further increase the pH to 12.3 using 0.02 N sodium hydroxide. Such demanding conditions were only used to compare hybrid columns, as silica columns fail with a small number of injections. For hybrid columns, failure generally occurs because of a loss of retention resulting from bonded phase hydrolysis, rather than a loss in efficiency because of particle dissolution. It is important to have strong bonded-phase attachment to the particle to match the outstanding base stability of hybrid particles. The use of exhaustively endcapped, trifunctionally bonded hybrid columns provides improved retention stability under these demanding high-pH conditions. 

Hybrid Particles Used in Reversed-Phase Chromatography 

This extreme pH stability can be used to optimize reversed-phase selectivity because changes in pH impact the ionization of both analytes and the particle surface. As shown in Figure 4 for an Acquity UPLC BEH C18 column, a diverse set of analytes can be separated under reversed-phase conditions using pH 3 or pH 10 gradients. For the pH 3 separations, acidic analytes (shown in red) are normally unionized and basic analytes (shown in blue) are ionized. Ionized analytes have lower retention times than their unionized forms. At pH 10, the basic analytes are generally in an unionized state and are more retained, while acidic analytes are ionized and less retained. As expected, neutral analytes are not greatly affected by changes in mobile-phase pH.  

Figure 4: Effect of pH on the separation of a diverse set of acidic, basic or neutral compounds using an Acquity UPLC instrument with a PDA detector (254 nm). Column: 50 mm Ã 2.1 mm, 1.7-Î¼m Acquity UPLC BEH C18; column temperature: 30 Â°C; gradient: 5â90% B in 5 min using a linear gradient curve, with a 0.5-min hold at 90% B; mobile-phase A: 15.4 mM ammonium formate (pH 3) or 10 mM ammonium bicarbonate (pH 10); mobile-phase B: acetonitrile; flow rate: 500 Î¼L/min; injection volume: 5.0 Î¼L. The column was equilibrated in 5% B for 2 min; total run time was 7.5 min. Peaks: 1 = thiourea, 2 = 2-nitrobenzoic acid, 3 = caffeine, 4 = metoprolol, 5 = 2-chlorobenzoic acid, 6 = papaverine, 7 = 4-nitrophenol, 8 = propranolol, 9 = pyrenesulphonic acid, 10 = amitriptyline, 11 = diethylphthalate, 12 = fenoprofen, 13 = dipropylphthalate. The peaks for acidic analytes are in red, basic analytes are in blue and neutral analytes are in black.			

Modifying the mobile-phase pH in HILIC separations can also be used to change selectivity (14). However, because of the high organic mobile phases required in HILIC separations, other considerations are needed to fully understand pH impacts on retention times, for example the polarity of the ionized molecule and stationary phase ionization. One major advantage of using high pH in HILIC separations is MS response. By having analytes in an ionized form we often observe sharp increases in electrospray MS signal intensities. These can be compared using the ratio of peak areas from pH 9 and pH 3 separations, which have been shown to have a 0.7–35-fold improvement at pH 9 (15). 

An example of using a hybrid column under HILIC conditions at elevated pH would be carbohydrate separations using an Acquity UPLC BEH Amide column. Carbohydrate analysis can be challenging and is often impacted by slow interconversion of reducing sugars anomers, impacting the speed, resolution and unreliable quantitation. The interconversion of α/β anomers can be accelerated at high temperatures or when exposed to elevated pH. Although traditional amino bonded silica-phases allow for rapid anomerization because of the localized surface alkalinity, these phases suffer from poor chemical stability because of autocatalyzed bonded phase hydrolysis. The reaction of reducing sugars with the amino group of the stationary phase may also lead to decreases in peak areas (16–19). 

BEH Amide columns have greatly improved particle and bonded phase chemical stability compared to amino bonded silica-columns. Accelerated base stability tests performed on BEH Amide columns (0.05% triethylamine, 80% acetone, pH 10.35, 90 °C) showed minimal loss in retention over 2000 injections. This improved chemical stability at both elevated pH and temperature (Figure 5) allows for rapid interconversion of reducing sugar anomers (20).  

Figure 5: Isocratic separation of carbohydrates using an Acquity UPLC instrument with evaporative light scattering detection. Column: 150 mm Ã 2.1 mm, 1.7-Î¼m Acquity UPLC BEH Amide; mobile phase: 75% acetonitrile at 35 Â°C (top), 75% acetonitrile with 0.2% triethylamine at 35 Â°C (middle), or 80% acetone with 0.05% triethylamine at 90 Â°C (bottom); flow rate: 0.29 mL/min. Peaks (1 mg/mL): 1 = fructose, 2 = glucose, 3 = sucrose, 4 = maltose, 5 = lactose.			

Chromatographic Impact of Silanol Activity  

Underivatized (unbonded) silanol groups on the surface of packing materials can impact the ionic properties as well as the retention of ionizable analytes for reversed-phase and HILIC separations because of ion-exchange or ion-exclusion interactions. The acidity of these silanol groups is impacted by the composition of the material. Impurities can often result in increased silanol acidity, which can negatively impact chromatographic performance. The need for improved reproducibility and peak shape for basic analytes was a major user need that drove the research into higher purity silica in the 1990s. Although the advent of higher purity silica led to improved peak shapes for basic analytes, there was still a need for further improvement.  

Silanol acidity of silica and hybrid packing materials has been examined for underivatized and C18-bonded particles by measuring retention changes of an ionized analyte (bretylium tosylate or LiNO

) under different mobile-phase conditions (pH 2–11 and from water to 80% acetonitrile) (21,22). These studies showed a higher ionization pH for hybrid particles over silica (pH 10 and pH 7, respectively). Because hybrid materials are a mixture of silica and organosiloxane groups, the acidity of the surface silanol groups is not expected to be the same as that of silica.  

This lower silanol acidity for hybrids compared to silica allows for improved peak shape for basic analytes under reversed-phase conditions. Aqueous SEC is another application where performance may be negatively affected by silanol activity (16,23,24). For optimal SEC separations one wants to have the highest possible particle porosity and no secondary interactions. To limit secondary interactions because of acidic silanols of silica-based SEC particles, hydrophilic diol bonded phases are often used. A thick coating of the diol phase, which can greatly decrease the porosity of these materials, is required to appropriately shield the electrostatic charge of the acidic silanols from the analytes. Even with optimal diol bondings the effects of surface silanols are still observed, requiring the use of mobile phases with high ionic strength to minimize ionic retention.  

One method to test for acidic silanols on SEC columns is to monitor the chromatographic behavior of the basic protein lysozyme (pI ~11) at pH 7 using mobile phases containing different phosphate buffer concentrations. When comparing silica-based SEC columns with the BEH SEC column, as shown in Figure 6, the results are clear. With low ionic strength mobile phases (10–25 mM) the BEH column exhibits less adsorption of the basic protein lysozyme, when compared to a commercially available silica SEC column.  

Figure 6: Effect of sodium phosphate concentration on lysozyme peak shape and retention time. For each column, the chromatograms obtained using three different mobile phases (differing in the concentration of sodium phosphate) are overlaid. Secondary interactions are minimized with BEH packing material when compared to silica-based packing material. Instrument: Acquity UPLC system with UV detection (280 nm); flow rate: 0.5 mL/min; mobile phase: 10, 25 or 100 mM sodium phosphate (pH 6); temperature: 30 Â°C. 			

As noted above, user needs continue to change. Today, there are still difficult problems facing scientists that require new column solutions, including additional selectivity options and improved LC–MS separation solutions. Many LC–MS users experience problems with commercially available reversed-phase columns that are attributable to poor performance with MS-compatible mobile phases (for example, formic acid, acetic acid or ammonia). Specific problems include slow equilibration and poor peak shape for basic analytes at low pH (25–27). These issues have been linked to a variation in the surface charge of the packing materials, which is amplified by the use of low-ionic-strength mobile phases.   

We undertook a two-year research programme to better understand these issues (28). Slight changes in surface charge because of mobile phase contaminants or impurities left from the particle synthesis or bonding process can impact chromatographic performance with low ionic strength mobile phases. Such issues are masked with higher-ionic-strength mobile phases or ion-pairing reagents (22,29,30). These effects impact both hybrid and silica materials and are evident on most modern high-purity stationary phases.   

Rather than continue research controlling impurity levels on the surface of these particles, we explored a different path in controlling surface charge through surface modification using ionizable silanes. The end result of this research was the development of a new family of columns based on BEH particle technology, called 

(CSH) (28). In this approach, BEH particles were surface-modified with a low concentration of a weakly basic ionizable silane, followed by bonding of the primary phase (for example, C18) and endcapping. The optimal surface concentration of the ionizable silane groups is more than an order of magnitude lower than the primary bonded phase. The weakly basic silane group is protonated at a low pH and is neutral above pH 7. 

CSH columns are available in a 1.7-μm particle size as Acquity UPLC CSH columns as well as 2.5–5 μm particle sizes as part of the XSelect CSH column family. These columns are available in C18, phenyl-hexyl and fluoro-phenyl bonded phases, allowing for a large breadth of selectivity options.  

CSH columns have notable improvements in peak shape for basic analytes with low-ionic-strength mobile phases compared to other C18 columns. The CSH C18 column maintains nearly linear-isotherm behaviour for amitriptyline at mass loads that approach those used in purification applications. This improvement in peak shape for basic analytes is also realized in gradient separations, and allows for notable improvements in peak capacity for basic analytes. As shown in Figure 7, a 1.7-μm BEH C18 column has poor peak shapes for the bases metoprolol and amitriptyline when used with a 0.1% formic acid mobile phase because of mass overloading (31). In contrast, the 1.7-μm CSH C18 column does not suffer this limitation, so that the peak capacities for these bases are similar to those of the neutral analytes prednisone and caffeine. The CSH C18 column can take full advantage of the efficiency gains from using sub-2-μm packing materials when using formic acid mobile phases for basic analytes (32,33).  

Figure 7: Comparison of peak capacities for gradient separations on an Acquity UPLC instrument with TUVe detection. Columns (50 mm Ã 2.1 mm): 1.7-Î¼m Acquity UPLC CSH C18 (in red) and BEH C18 (in blue); mobile-phase A: 0.1% formic acid in Milli-Q water; mobile-phase B: acetonitrile; gradient: 15â95% B in 7.4 min with a hold at 95% B for 1.4 min; injection volume: 2 Î¼L; flow rate: 400 Î¼L/min; column temperature: 30 Â°C; detection wavelength: 260 nm. The sample diluent was 17% methanol in Milli-Q water. Peak capacities were calculated for each analyte using 4Ï peak widths and the gradient time of 7.4 min. Peaks: 1 = caffeine (10 Î¼g/mL), 2 = prednisone (10 Î¼g/mL), 3  = metoprolol tartrate (200 Î¼g/mL), 4 = amitriptyline (50 Î¼g/mL). 			

Articles

How ethylene-bridged hybrid inorganic-organic (BEH) particles are prepared, and how their special quallities can be used to improve separations.

Synthesis and applications of BEH particles in liquid chromatography

 in late 2011 showed that since 2008, hydrophilic interaction liquid chromatography (HILIC) has more than doubled its application. HILIC is finding more and more applications for polar compounds that are either unretained or weakly retained on reversed-phase chromatographic columns. The technique has also been used for the separation of biomolecules. This article reviews the newer stationary phases intended for HILIC and studies performed to categorize columns for optimized separations based on their selectivities.

Hydrophilic interaction liquid chromatography (HILIC) has become an established chromatographic technique in widespread use in most application areas. This great interest has led to an accompanying increase in the number of new stationary phases explored, some of which have become commercially available. For the user, this multiplication of phases is beneficial, but it also presents some challenges. Where to start? What column to choose? How can one get optimal selectivity for a specific application? Therefore, approaches to classify different HILIC stationary phases into categories have emerged. This article will summarize the progress in this direction since a previous article was published on this topic in the 2008 North American supplement (1). 

reader survey (2) that the use of HILIC mode had doubled since the year 2007. This means that roughly 25% of chromatographers are using HILIC today. I had the pleasure to be responsible for the market introduction of the column brand ZIC-HILIC in January 2002, which was immediately well received by researchers at AstraZeneca. At that time, there were about 70 papers on HILIC in the scientific literature published since the first paper by Alpert in 1990 (3). Back then few chromatographers had a complete picture of the retention mechanism of HILIC or which column to use, but there was a multitude of unsolved application problems that were delaying research in several branches of science. Most important, the orthogonal selectivity of HILIC to reversed-phase chromatography was driving the users interest.  

Later on, in April 2008 when the previous review in this series was written by McCalley (1), he had to consider in total 350 HILIC papers. Today, this number has risen to almost 1300 papers, with more than 300 new contributions only last year (see Figure 1). Now, scientific literature has become a valuable source of separation applications using HILIC. The question of whether separation is possible can often be answered based on the published work already done. More focus can be given to how it is done and to other aspects of analysis such as sample preparation, throughput and detection. Readers are referred to special journal issues on HILIC (4–6), to a textbook with various chapters addressing successful applications (7), and to reviews on different application areas such as pharmaceutical analysis (8), proteomics (9), metabonomic and metabolomic analysis (10), glycomics (11) and bioanalysis (12).  

Figure 1: The number of HILIC papers per year (43).			

It is clear that the improved understanding of HILIC and growing interest for selectivity, in general, also has led to better sample handling by complementary pretreatment protocols and, in many methods, to an overall faster throughput.  

Even by the Hemström and Irgum review in 2006 (13) it became clear that a wide range of polar stationary phases had been explored and used in HILIC mode applications. However, recently new stationary phases have been introduced specifically for HILIC separations. The rapidly increasing interest and demand for HILIC applications pushed column manufacturers to provide HILIC branded products. It was my experience that among users this created the impression that so-called "HILIC columns" were interchangeable just as reversed-phase columns (a somewhat faulty impression) are supposed to be. This belief persisted despite the often very obvious difference in chemical structure of the functional group on the stationary phase of different HILIC columns.  

A few years ago, during the so-called "Chinese tainted milk scandal" and the urgent need for reliable assays of melamine and cyanuric acid (14), my personal experience was rather shattering. It was generally accepted that a HILIC column was required for the analysis, but it turned out that just a few were suitable and those few required quite different experimental conditions. Thus, column providers contributed to the confusion experienced in food laboratories, of whom from just one day to the next had to be introduced to a totally new separation mode — HILIC. 

For this reason, I believe that HILIC columns require further classification. From a user perspective, during method development, it makes sense to try several different categories of columns and to avoid repeating experiments on similar types. In an early attempt to classify HILIC columns, a range of probes targeting various interactions were selected and run on different polar stationary phases. Principal component analysis (PCA) then clearly revealed that the columns could be divided into different classes and that selection of columns from the different categories actually made sense in method development (15). Another aspect linked to the column classification is the possibility to predict the analyte retention or at least to get a rough idea if HILIC is a suitable separation mode and to find starting conditions. Based on retention data for 40 different probes evaluated by multivariate analysis, we developed a small "predictor program" (16) that made it possible to get an initial idea on the suitability of the ZIC-HILIC column for a certain analyte. At least it confirmed that it was possible to predict retention based on the chemical nature of a solute. 

Using the criteria that any polar column used under HILIC conditions can be denoted as a "HILIC column," a recent literature search revealed that a wide range of columns were still being used (17,18). In his HILIC tutorial at HPLC 2011 in Budapest, McCalley (19) suggested five main classes of HILIC columns as follows: 

			1. Silica gel (Si-OH or charged form Si-O

			2. Neutral bonded ligands (for example, amide and diol) 

			3. Charged ligands (for example, positively charge amino phase; negative charge poly-2-sulphoethylaspartamide) 

			4. Zwitterionic phases (for example, sulphobetaine and phosphorylcholine) 

			5. Mixed reversed phase/HILIC phase (for example, alkyl chain diol). 

In application work, three of them were identified as the most used: bare silica, neutral bonded ligand (amide), and zwitterionic bonded ligand (sulphobetaine), according to the literature search (17,18). Notably, the three most popular phases also represented three different categories of stationary-phase functional group chemistries. The popularity of these was also reported in a recent review on food analysis (20). Now, I will explore these three phase types in greater detail. 

Silica gel has been used as a normal-phase HPLC packing and in reversed-phase chromatography as the base for bonded phases for a long time. Bare (nonbonded, plain) silica possesses silanol groups that can ionize at mid-values of pH give rise to a negatively charged surface and exhibit cation-exchanger behaviour. Apparently, the surface silanols on silica are sufficient to create a surface water layer and the material can provide "HILIC retention." It is well known that the silica sorbent quality has a significant impact on its suitability as a HILIC stationary phase (21), so indeed nonbonded phases like bare silica also can be divided into subcategories.  

The often high surface area of bare silica greatly contributes to its ability to establish HILIC retention. McCalley (22) showed that bare silica columns better preserved high efficiency at high flow rates compared to bonded sulphobetaine and amide phases, which were elsewhere found to be even more suitable for high speed analysis than reversed-phase columns (23). It should be noted that the performance also differs between various types of bare silica columns. It is well known that the metal content of the silica influences its acidity, but more recently the selectivity differences between unbonded ethylene bridged hybrid silica (BEH) and the Atlantis HILIC silica were demonstrated. Moreover, bonding amide and diol functionalities to the BEH silica also created distinct selectivity differences (24). Actually, a method with these bonded BEH HILIC columns just recently, in time for the doping controls at the Olympic Games in London, proved to be superior to reversed-phase chromatography (BEH-based columns) for the analysis of ephedrines (25). 

Among the bare silica materials recently introduced, the fused-core superficially porous shell particles have generated the most interest, marketed under the brand names Halo HILIC (AMT), Poroshell (Agilent Technologies), Ascentis Express Fused Core (Supelco) and Kinetex HILIC (Phenomenex). They seem to have a somewhat limited capacity, but bonded phases such as Nucleoshell HILIC (Macherey-Nagel) and Halo Penta HILIC (AMT) have also become available. The Halo Penta HILIC is a neutral hydroxy phase, which according to the report by the manufacturer show less silanolic interactions. However, at least the Nucleoshell particle has a significantly lower carbon load (1.3%) than the corresponding Nucleodur HILIC particle (7%) and it seems that the bonding density has been compromised on the core–shell particles. Thus, it is quite likely that these bonded phases also show some bare silica–related interactions. 

The hypothetical benefit of a nonbonded bare silica column is that in absence of a functional group the risk for bleeding and reduced mass spectrometry (MS) detection sensitivity is eliminated. However, to my knowledge, there is no paper confirming this assumption. According to my experience, stationary phase bleeding generally is a smaller problem with HILIC columns, at least compared to mixed-mode columns used in reversed-phase chromatography. The lack of stationary-phase bleed may result from the relatively mild mobile-phase conditions typically used in HILIC separations — that is, dilute buffers and a low ratio of water, often at neutral pH. Some HILIC amino columns have been reported to bleed, but no paper was found where bleeding from different columns was compared. Sometimes a suspected bleeding "problem" can be explained by the release of buffer salts, matrix components or other solutes that in an earlier stage have been adsorbed or precipitated on the stationary phase (by the user). A proper washing of the column often solves this type of "bleeding problem." 

A bonded phase implies that there is an opportunity for specific solute functional group interactions, as well as solute spacer interactions while achieving a desirable degree of solute to sorbent interactions. Moreover, the polar bonded phases used in HILIC may be divided into two groups depending on the surface grafting chemistry rendering: a monomeric or a polymeric surface layer. On the other hand a nonbonded phase, such as bare silica, actually relies on the functionality and interactions that the bonded category might be missing (or at least express less). So, this classification is indicative of the capacity and chemical interactions that can be expected to contribute to the selectivity. 

The properties of the polar bonded stationary phases are strongly related to the hydrophilicity of the functional group and consequently its ability to retain and establish a water layer on the stationary phase, which is assumed to promote a partitioning retention mechanism.  

A popular type of neutral bonded HILIC column has an amide functionality bonded onto silica gel as a support. In an interesting experiment, the effects of bonding a neutral hydrophilic amide to silica under comparable conditions was explored. The Daisogel silica (5 μm, 120 Å, 300 m

/g) was modified to a different extent (0–3.67 μmol/m

) with a urea-type ligand, thereby enabling a direct comparison of bare silica and bonded columns (26), see Figure 2. To link the results to commercially available amide-type columns, the TSKgel Amide-80 (Tosoh Bioscience), Unisol Amide (Agela Technologies) and XBridge Amide (Waters) columns were evaluated in parallel. The bonded experimental urea phase with the highest loading performed the most similar to the Unisol Amide. It was seen that all amide phases showed significant differences in performance. It was noted that the commercial amide phases studied varied considerably in specific surface area (185–450 m

/g), and the authors pointed out the importance of column selection during HILIC method development. The cationic behaviour of free silanols on bare silica leads to strong retention of bases and weak retention for acids, in particular at low mobile phase buffer concentrations. As expected, because of the influence on silanolic interactions, the retention of bases decreased and acids increased when the bonding ligand density increased. Also, the retention of neutral solutes increased and the bonded phase was more selective for this group, and the difference in solute charge state gave higher selectivity on bare silica because of a higher contribution by ionic interactions on that phase. For neutral solutes, it was seen that retention was not only promoted by increasing eluent ion strength, but also by higher ligand density.  

Figure 2: Stationary phase structural formula of an amide phase used for comparison of chromatographic performance of bare silica and the corresponding bonded phase at different surface coverage (0â3.67 Î¼mol/m2). Adapted from reference 26.			

The retention and selectivity of various stationary phases commonly used was recently reviewed by Guo and Gaiki (27). They chose the charge of the polar functional group as a criterion to divide columns into three categories: charged, neutral or zwitterionic phases. This distinction between phases might not be as clear as it sounds because silica is most often the base material that is functionalized. As mentioned earlier, at mobile-phase pH values > 4–5 free silanolic groups that are still present on many silica bonded phase materials may give even neutral materials a net negative charge and cation-exchanger behaviour. Apparently this is less evident if the functional groups are attached by a process creating a shielding stationary-phase layer (26). However, spacers or polymeric grafting may, on the other hand, introduce more hydrophobic properties to the phase that may affect the retentivity and selectivity of the column. McCalley (22) demonstrated that for strongly basic compounds all column categories showed ion-exchange contribution to the retention even at a 10 mM buffer concentration. It also was seen that for the more hydrophobic bases a larger extent of retention was attributed to ion exchange and less to other interaction processes. The neutral bonded and cross-linked diol column (Luna HILIC, Phenomenex) was an exception as it showed less electrostatic interactions, and presumably free silanols were more shielded. However, it was also the most hydrophobic bonded stationary phase providing the least retention (Figure 3). Because different column brands are likely to be based on different silica base materials these, therefore, will have different acidity and would provide different proportions of free silanolic groups available for ion-exchange interactions. Hypothetically, at its extreme, a less acidic bare silica could contain a similar number of ionized silanols as a bonded phase exposing free silanols from a more acidic silica. Actually, McCalley speculated that column manufacturers deliberately chose to use more acidic silica base material to also obtain this potential electrostatic interaction selectivity with the column. Still, metal impurities would need to be kept low to avoid tailing peaks (21).  

Figure 3: Average retention of neutral solutes on different HILIC columns. Mobile phase: 80:20 (v/v) acetonitrileâ25 mM aqueous ammonium acetate (pH 6.8); flow rate: 0.5 mL/min; temperature: 22 Â°C. Toluene was used as unretained marker to determine t0. On the LiChrospher CN, 5-Î¼m, 100-Ã column several analytes had less retention than toluene (data not shown). Data adapted from reference 33.			

Zwitterionic bonded phases are a relatively new type of stationary phases in chromatography and have been popular since the early days of HILIC. Ideally, the zwitterionic functionality should have a zero net charge and therefore be neutral. Because of the short but distinct charge separation within the functional group the phase expresses weak electrostatic interactions that contribute to the selectivity, but without being the dominating retention mechanism. The most popular sulphobetaine functionality bear a positively charged quaternary ammonium and a negatively charged sulphonic acid group and because both of these are strong ion-exchange groups, the stationary phase is "charged but neutral" regardless of the mobile-phase pH.  

The popularity of zwitterionic columns is also reflected among the new bonded phases. In a recent paper on the development of a weak zwitterionic stationary phase, a trimethylchlorosilane "end-capped" variant was investigated (28). The 3-P,P-diphenylphosphoniumpropylsulphonate functional group is presented in Figure 4. The most significant effect of "end-capping," using relatively low overall buffer concentration at pH 4.1, seemed to be less retention in general for various categories of analytes (strong bases not tested). This indicates that free silanols may play a role on the sterically hindered nonendcapped phase and contribute to the overall hydrophilicity and retentivity of that material. Thus, functional groups and functionalization chemistries introducing hydrophobic elements at the silica surface tend to give less retentive stationary phases. To some extent, this is because it may influence the electrostatic interactions, but probably more so because it counteracts a "water layer" on the stationary phase and retention by a hydrophilic partitioning "HILIC mechanism.  

Figure 4: A zwitterionic 3-P,P-diphenylphosphoniumpropylsulfonate stationary phase endcapped by trimethylchlorosilane functionalization. Adapted from reference 28.			

Another weak zwitterionic phase was developed by the functionalization of a silica monolith with a lysine moiety (29), and it proved to be more suitable for HILIC than a less hydrophilic diol type monolith. A range of zwitterionic stationary phases have also been studied in the development of polymeric monolithic columns (30), and recently a zwitterionic sulphobetaine silica monolith capillary column was introduced by Merck (CapRod ZIC-HILIC) (31). 

While selecting a column stationary phase based on "charge state" classification it is necessary to keep in mind that it depends on the mobile-phase pH. Functional groups like silica or amino columns that show weak ion exchanger behaviour are defined as charged (27). Also, depending on the surface coverage and type of silica base material, neutral phases on silica may show weak ion exchanger properties because of acidic silanols. Even zwitterionic functional groups may be affected by pH if they are "weak" rather than "strong" zwitterions.  

Classification and Phase Selection in HILIC

To select the optimal stationary phase for a separation problem, one has to know the retention, selectivity and efficiency of HILIC columns. Recently, two comprehensive HILIC column characterization studies were reported in which 14 columns and 13 test compounds (32), and 22 columns and 21 test compounds (33) were used to categorize columns and study the HILIC retention mechanisms. The aim was also to find probes that may be in common use for HILIC column testing. Both studies were designed to avoid probing pH effects either by the choice of test solutes or by using constant chromatographic conditions with respect to pH. Changing pH in the mobile phase is very important for controlling selectivity, but it is not a desired effect to probe in column comparisons as the ionization state may change for both solutes and for some stationary phases. The test compounds in these studies were selected to probe all kinds of interactions that are relevant for retention and selectivity and the results were evaluated by principal component analysis (PCA).  

Clearly, classification of columns into different categories is possible, as this was also found in early studies (15). Kawachi and colleagues (26) found three clusters in the PCA analysis and summarized the column properties in radar plots. As previously seen, even columns with (a supposed) equal functionality show different chromatographic behaviour. They therefore suggest that HILIC columns can be divided into two classes: 

			1.  Strongly retentive showing high selectivity (amides, sulphobetaine, sulphonates) 

			2.  Weakly retentive with limited selectivity (silica, diol, amino, hydroxy, triazol).  

Although peak shape was not evaluated in either of these studies, both papers underline the importance to avoid or control strong electrostatic interactions leading to a dominating ion-exchange mechanism, which otherwise ruin both peak shape and column efficiency. It was also concluded that increased ion strength of the mobile phase not only reduces ion-exchange contribution, but also reduces the selectivity differences between columns. Moreover, the differences between columns and the individual column selectivity are promoted by a high content of acetonitrile in the eluent. Thus, the conditions providing the most between-column and in-column selectivity are also generally ideal for the most used (17) MS detection sensitivity.  

The findings of these two studies were also coherent in comments on details in selectivity differences found and they manifest that there is no significant correlation between retention and selectivity. The average retention for 22 commercially available columns is presented in Figure 3. When these columns were investigated by injection of the test solutes at identical conditions the PCA evaluation revealed clustering into four major groups (see Figure 5) providing different main selectivity characteristics; 

			I:   Bare silica phases — cation exchange and adsorption 

			II:  Bonded neutral phases — low specificity 

			III: Bonded amino phases — anion exchange 

			IV: Bonded zwitterionic, sulphoethyl amino phases — partitioning and weak ion exchange  

Some of the newer hydrophilic interaction liquid chromatography (HILIC) columns are reviewed, and experiments performed to categorize columns for optimized performance.

Advances in the understanding of stationary phases for HILIC

Chiral separations remain a decided area of interest, as well as an all-too-often puzzle and on-going problem for many industries. High performance liquid chromatography (HPLC) remains the method of choice for most separations because of its robustness, transferability and instrument availability. Several chiral stationary phases continue to enjoy wide use in part because of their broad application. More recently, developed phases such as cyclofructans are finding increased use in HPLC and separation techniques. This brief review is an update on some of the latest chiral offerings.

Chiral separations in high performance liquid chromatography (HPLC) continue to be of great interest because of their prevalence in diverse areas such as the pharmaceutical industry, agrochemicals and food additives. Separation of enantiomers can be challenging because of their identical physical and chemical properties in an achiral environment. The advent of chiral stationary phases (CSPs) has been a boon to the field of enantioseparations. While research on specialized separation techniques using novel CSPs continues for the resolution of specific individual enantiomers, chiral separations on commercially available CSPs are a mature and widely used technique. Polysaccharide and macrocyclic glycopeptide CSPs remain the most widely used commercial chiral phases, with cyclodextrin-, cyclofructan-, π-complex- and protein-based CSPs also finding use. For non-HPLC separations, such as gas chromatography (GC) and capillary electrophoresis (CE), cyclodextrins dominate. The HPLC enantioseparation of larger molecules and multiple chiral centres has increased as the influence of biotech grows, and more polar molecules are of interest in discovery laboratories. Recently, the most important and widely used CSPs for HPLC were reviewed with sections on enantiorecognition mechanisms (1). There are plenty of resources and information available for one needing to perform chiral separations. Reviews have recently been published on general chiral HPLC methods for pharmaceutical analysis (2–4) and for supercritical fluid chromatography (SFC) separations of pharmaceuticals (5,6), including preparative-scale separations.   

Chiral separation by HPLC continues to be the primary technique of choice, with many companies seeing an increase in the number of enantioseparations in their laboratories. The market continues to enjoy growth and maturation as older technologies are replaced by newer and improved technologies. A case in point is the expiration of the patent for the traditional coated Daicel columns (Daicel Corporation). Coated-type amylose and cellulose derivative CSPs are now offered by many companies and they remain one of the most important commercial CSPs. Daicel has started offering new bonded polysaccharide CSPs, which are advantageous for reversed-phase modes, mass spectrometry (MS) detection and preparative separations. A new class of commercial CSPs comprises the cyclofructan phases invented by Professor Daniel W. Armstrong (available through AZYP and Supelco/Sigma-Aldrich as the LARIHC and FRULIC columns). These phases are useful for the enantioseparation of primary amines.   

Two apparent trends in the market are the increased use of SFC and smaller particles. The advantages of each are the reduced environmental impact and operating costs, as well as increased throughput. However, particles with diameters less than 3 μm are not commercially available, limiting microscale techniques. SFC has found more use particularly in preparative scale chiral separations, where the waste reduction and decreased solvent use is attractive to industry. 

Chiral separations achieved by polysaccharide CSPs in HPLC account for approximately one-third of all HPLC chiral separations in the literature, and most of these separations are carried out on commercially available columns. The most commonly used phase on cellulose or amylose is 3,5-dimethylphenylcarbamate, which includes immobilized phases such as Chiralpak IA (Chiral Technologies), Chiralpak IB (Chiral Technologies), Lux Cellulose-1 (Phenomenex), and coated columns such as Chiralcel OD-H (Chiral Technologies), Kromasil-CelluCoat and AmyCoat (Eka Chemicals). Chloro-substituted polysaccharide CSPs have also found a lot of use, including tris(3,5-dichlorophenylcarbamate) such as the Chiralpak IC; tris(3-chloro-4-methylphenylcarbamate) like the Lux Cellulose-2; tris(5-chloro-2-methylphenylcarbamate) like the Chiralpak AY-H; and tris(3-chloro-4-methylphenyl carbamate) like the Chiralcel OZ-H. The selectivity of the Chiralpak IC column toward different enantiomeric compounds is demonstrated in Figure 1.  

Figure 1: Normal-phase chiral separation of three compounds using a 250 mm Ã 4.6 mm, 5-Î¼m Chiralpak IC column. Mobile phase: 70:30:0.1 (v/v/v) n-heaneâethyl acetateâdiethylamine. Adapted from http://immobilizedchiralcolumns.com/csp-robustness (courtesy of Chiral Technologies).			

A reversed-phase study using tris chloro methylphenylcarbamate derivatives of cellulose and amylose concluded that using these CSPs in screening protocols have higher success rates in achieving baseline separations with shorter screening times (7). An updated generic separation strategy in normal-phase HPLC was reported using the commercial CSPs: Lux Cellulose-1, Lux Cellulose-2, Lux Amylose-2, Lux Cellulose-4, Chiralpak AD-H, Chiralcel OD-H and Chiralcel OJ-H (8). Reversed phase screening strategies for HPLC with Chiralpak IA, Chiralpak IB and Chiralpak IC with applications compatible with liquid chromatography–mass spectrometry (LC–MS) were also reported (9).  

Although no new cyclodextrin-based CSPs have been brought to market since the last review in 

 (10), the use of cyclodextrins still account for more than a fourth of all publications in chiral HPLC in recent years. The bonded cyclodextrin-based CSPs are used because of their robustness, wide selectivity and ability to enantioseparate in the reversed and polar organic phases. The mechanical stability of the cyclodextrin CSPs lends itself to wide use for preparative scale as well.  			

The macrocyclic glycoprotein CSPs continue to fill a wide and useful niche in chiral HPLC because of their unique versatility and broad selectivity. These CSPs are unsurpassed in the enantioseparation of chiral amino acids. Supelco/Sigma-Aldrich offers a Chirobiotic method development kit containing V2, T, R and TAG columns for screening in polar ionic, polar organic, reversed-phase and normal-phase modes. A review of macrocyclic glycopeptide-based CSPs in HPLC methods for amino acid enantiomers and related analogs was published in 2010 (11). 

The recently released cyclofructan-based LARIHC CSPs from AZYP, also available from Supelco/Sigma-Aldrich, show great promise in chiral HPLC separations. These columns include LARIHC CF6-P, which is considered the "king column" for separation of racemic primary amines; LARIHC CF6-RN, which separates nonprimary amines; and LARIHC CF7-DMP, which is the only commercialized cyclofructan 7 column and shows complementary enantioselectivity compared to the CF6-RN CSP.   

Figure 2: Examples of separations of pharmaceutical enantiomers on CF6 and CF7 CSPs. Top: LARIHC CF6 column; bottom: LARIHC CF7 column. Adapted from http://www.sigmaaldrich.com/etc/medialib/docs/Supelco/Posters/1/daw-chiral-010711.Par.0001.File.tmp/daw-chiral-010711.pdf (courtesy of Supelco/Sigma-Aldrich). 			

LARIHC CF-6P is the only column that separates primary amines in a nonaqueous solvent using the polar organic mode. All LARIHC CSPs reportedly work well in SFC because of their use in both the normal-phase and polar organic phase modes. In a bit of marketing wordplay, LARIHC is "chiral" spelled backwards. 

Figure 3: Chromatograms showing chiral separations in (a) normal-phase, (b) polar organic phase and (c) reversed-phase modes on LARIHC CF6-RN CSP. Adapted from http://www.sigmaaldrich.com/etc/medialib/docs/Supelco/Posters/1/daw-chiral-010711.Par.0001.File.tmp/daw-chiral-010711.pdf (courtesy of Supelco/Sigma-Aldrich).			

Although the chiral market is maturing and stable, perhaps the biggest need in the field is an increased understanding of overall chiral methodologies and more practical training. As information is largely supplied by vendors to newcomers to chiral HPLC, novices can sometimes stumble around for a while wading through the vendor's literature and advice. In addition to an understandable sales objective, vendors occasionally misunderstand the customers' end-to-end chiral operation, so promised increases are sometimes not realized at the laboratory level. Furthermore, increased performance is also sometimes not realized because of low-tech reasons that may be related more to laboratory layout and operation, rather than the lack of latest technology and equipment. Newcomers should make use of the short courses on chiral HPLC that give a wealth of unbiased information to the participants and are offered at major conferences such as Eastern Analytical Symposium (EAS), Pittcon, Chirality and occasionally at American Chemical Society meetings. 

Increased selectivity remains a challenge in chiral separations. With increased selectivity, loading can be increased in preparative chiral separations, which will provide the greatest cost savings. With significant cost savings in the separation process, companies can shift from using chiral selective synthesis for chiral purification to purification by chromatography. Although HPLC is used for preparative-scale separations, SFC continues to gain ground as the most cost-effective way to purify enough material for further studies.   

The market is maturing, but technology has not moved forward in any remarkable way. Chiral HPLC still lacks (and probably always will) a CSP that can operate in all modes for enantioseparation of molecules from low to high polarity. 

We gratefully thank Thomas Beesley of Chiral View LLC, Tracy Ascah of Sigma-Aldrich and Gary Yanik of PDR-Chemical LLC for their suggestions in the preparation of this manuscript. 

 is a professor of chemistry and the associate dean of sciences at Millsaps College in Jackson, Mississippi, USA. He received his BS degree from the University of Florida and his PhD from Texas Tech University. Dr Ward served as chair of the International Symposium on Chirality in 2007. His research interests include chiral separations, the development of analytical LC and CE methods, and their application to pharmaceutical and archaeological analysis. 

 is an instructor at Millsaps College in Jackson, Mississippi, USA. She received her BS from Texas A&M University and her MS from Texas Tech University. Ms Ward has worked previously in the pharmaceutical industry at the Analytical Environmental Research Division at Syntex Pharmaceuticals in Palo Alto, California. Direct correspondence to: Timothy J. Ward at: 

(1) A. Cavazzini, L. Pasti, A. Massi, N. Marchetti and F. Dondi, 

(2) Y. Zhang, S. Yao, H. Zeng and H. Song, 

(7) L. Peng, S. Jayapalan, B. Chankvetadze and T. Farkas, 

(8) A.A. Younes, D. Mangelings and Y. Vander Heyden, 

(11) I. Ilisz, Z. Pataj and A. Peter, in 

 D.W. Fitzpatrick and H.J. Ulrich, Eds. (Nova Publishers, 2010), pp. 129–157. 

An update on some of the latest chiral stationary phases and their use in various separation modes.

Recent progress in chiral stationary phase development and current chiral applications

This short review focuses on the physicochemical properties of core–shell particles and their resulting chromatographic efficiencies as they relate to the comparison of their mass transfer kinetics to those of columns packed with sub-2-μm fully porous particles. 

In recent years, column technologies have diversified for a multitude of separation applications. The improvement in separation efficiency attainable today on modern liquid chromatography (LC) columns results from the evolution of new packing materials and sorbents that offer analysts the choice to use fully porous particles, core–shell particles or silica–polymer monolithic columns, depending on the application (1). Among the new column packing materials, core–shell particles, also called 

of sub-3-μm diameter have gained considerable attention because of significant improvements in separation efficiency. Columns packed with core–shell particles can generate a minimum plate height below 

~ 3.5 (2,3) for small molecules, a similar efficiency to that of columns packed with fully porous sub-2-μm particles.  

The use of core–shell particles as column packing materials is not new; in fact, they have been in existence and have been used since the early days of high performance LC (HPLC) (4–6). The main purpose of pellicular particles in the early days of HPLC was to improve the mass transfer resistance of large molecules to facilitate faster separation (6). However, pellicular particles later met their demise because such particles were not as robust and refined as the fully porous particles that were introduced later. For detailed historical accounts of the development of various pellicular particles as packing materials for HPLC columns, readers should refer to reference 7. The new-generation core–shell particles display significant improvements in important physicochemical parameters, such as particle-size distribution; thickness of the porous shell, providing substantial increase in surface area for retention (8); surface roughness or smoothness; and the purity and robustness of the base core–shell silica. A number of current manufacturers and academic researchers are currently investing a great deal of time in the preparation of core–shell silica particles (9); a list appears in Table 2 in the Majors article in this issue (10). The new-generation core–shell particles have been solely designed to compete with the sub-2-μm fully porous particles by providing a substantial increase in column permeability; this results in faster separations while maintaining the same high column efficiencies. Recently, another set of core–shell particles (Phenomenex's Aeris core–shell silica particle) has been released and is claimed to be a rival to the sub-2-μm fully porous particles for separating large molecules (11). This short review will summarize the key physiochemical properties of the new generation of core–shell particles that results in superior performance of HPLC columns.  

Physical Properties of Core–Shell Particles

: In contrast to earlier core–shell packing materials, the volume fraction of the porous permeable layers around the solid impermeable core of the new generation of core–shell particles (which are generally 2.6, 2.7 or 1.7 μm in diameter) is 29–37%, as opposed to < 10% for the earlier core–shell particles (5,12). For the separation of small molecules, increasing the shell thickness is of paramount importance because it contributes to solute retentivity and minimizes the 

-term contribution to band broadening (13). For example, the 2.7- and 2.6-μm commercially available core–shell particles provide an excellent peak capacity under gradient elution with holdup time as low as 10 s, which requires an inlet pressure of 400 bar (14,15).  

Figure 1 shows electron micrographs of how porous shell thickness is controlled while keeping the overall core–shell particle diameter constant. Gritti and Guiochon (16) have examined in detail the improvement in column separation efficiency brought by the new core–shell particles as a result of their thicker porous shell. Modern-day core–shell particles now have larger surface areas (~100–150 m

/g) compared to their predecessors (< 1–13 m

/g), which results in significant gains in sample loading capacity and enhanced stability of the stationary phase.  

: Literature reports have shown that the surface roughness of core–shell particles plays a major role in enhanced film mass transfer resistance, an independent contribution to band broadening caused by the rugosity of the particle surface (17). A recent study by Gritti and Guiochon has shown that core–shell particles with large pore sizes can alleviate the film mass transfer resistance (18).  

Figure 2 shows the external surface structure of two sub-3-μm core–shell particles: prototype 2.6-μm core–shell particles synthesized in the ISSC Cork laboratory [Figure 2(a)]; Kinetex 2.6-μm core–shell particles (Phenomenex) [Figure 2(b)]; and 3.5-μm fully porous particles [Figure 2(c)]. The prototype material has a similar smooth surface in comparison to the fully porous particles. Conversely, the Kinetex 2.6-μm material is unique with its rough surface. The impact of the surface roughness on the packed bed porosity heterogeneity is still a matter of debate.  

Particle-Size Distribution and Column Packing

: The degree of the packed-bed radial heterogeneity of the external porosity across the column is directly proportional to the 

 term of the van Deemter equation. Traditionally, there is a general consensus that a narrow particle-size distribution is the major source of improvement of the 

 term of columns packed with core–shell particles (19,20). It was recently reported that subtle changes in the column packing methodology itself can greatly improve packed beds made of particles with a broader size distribution (21), provided fines of particles of the same fractional size of the external porosity are eliminated. Using a numerical packing algorithm, Tallarek and colleagues (22) studied the relationship of particle-size distribution in unconfined packing (without a wall) generated over a range of bed porosities (external porosities) to flow permeability and the 

 term is expected to be more sensitive to bed porosity heterogeneity than to particle-size distribution. An example of the relationship of particle-size distribution and efficiency was demonstrated at the ISSC Cork laboratory. As shown in Figure 3, two batches of prototype 2.6-μm core–shell particles were characterized and differ significantly in particle size distribution [Figure 3(a) and 3(b)]. These different packing materials were identically packed in 100 mm × 2.1 mm columns and their corresponding height equivalent to a theoretical plate (HETP) plot is given in Figure 3(c), which shows no discernible differences. This result implies that particle-size distribution has a minimal effect on overall column efficiency.  

Core–Shell Particle Chromatographic Efficiency

: Columns packed with 2.7- and 2.6-μm core–shell particles consistently generate reduced plates heights of 1.5 and 1.3, respectively, in 4.6 mm i.d. column formats, generally outperforming columns packed with fully porous particles (

 > 2.0) in the same column internal diameter under identical conditions. This improvement in core–shell column efficiency is attributed to the mechanism of the mass transfer kinetics, which ultimately plays a pivotal role in the contributions to the HETP. The HETP is a combination of eddy and longitudinal diffusion and resistance to solute mass transfer in the stationary phase present inside the pores and outside the particle surface (the latter is referred to as 

 are the coefficients of the eddy and longitudinal diffusion, respectively, 

 are the trans-particle and film mass transfer resistance coefficients, respectively. For a detailed description of the physicochemical aspects of the different mass transfer coefficients and their contributions to HETP, readers are referred to reference 23. 

: Conventional wisdom generally accepts that the 

 term of solute mass transfer is unaffected by flow velocity of the mobile phase and that the particle-size distribution is the determinant factor of the 

term. The evolution of modern core–shell particles appears to confirm a close relationship between particle-size distribution and the 

term. However, recent studies on packing reconstruction using computer simulation have elucidated the structure–transport relationship of packed beds and demonstrated that the current state of particle-size distribution commonly found in commercial packings of fully porous particles had only a minor effect on the 

 term (24,25). The relationship between the slurry packing methodology and the randomness of interparticle pore spaces present in the packed bed from the column centre to the wall is now becoming an avenue to explore (26), because this is the true parameter that influences eddy diffusion. Because the currently existing commercial sub-3-μm core–shell particles seem to generate bed porosity that mimics loose packing and can still generate very high efficiency, it can be said that the bed porosity is far less heterogeneous from the centre of the column to the wall in comparison to columns packed with fully porous particles. Gritti and Guiochon have demonstrated that there is a strong link between retention and the 

Figure 4 illustrates the relationship between retention factors of different solutes and the reduced 

 term for columns packed with fully porous particles and core–shell packing (16). The 

 term for nonretained solute (uracil) is far superior for the core–shell column than for the fully porous column at high flow rates. The improvement of the 

 term becomes more significant for well-retained solutes as a result of the impact of retention on the 

term, which ultimately is larger for fully porous particles.  

 term is related to the bulk molecular diffusivity, 

 term in the HETP equation is systematically reduced in core–shell particles compared to the fully porous packing. Given that solute diffusion in the stationary phase is bound to be slower than that in the bulk eluent, the kinetics of diffusion are enhanced at the interface of the solute and stationary phase and results in additional mass transfer effects referred to as 

 (28), which increases with stationary-phase surface area. Surface diffusion is minimal in core–shell particles by virtue of the presence of the impermeable solid core particles; thus the 

 term is smaller for the core–shell particles and at zero to very low flow rates. The 

 term can be measured experimentally by the peak parking method (3) and the same method (after subtle modification) has also been reported to estimate the bulk molecular diffusivity, 

Trans-Particle and Film Mass Transfer Resistance

: The improvement in trans-particle mass transfer resistance (the 

 term) in columns packed with core–shell particles is not significantly greater than it is with the fully porous particles for the separation of small molecules. This is because of the larger contribution of surface diffusion (28) found in fully porous particles (which have a larger specific surface area in comparison to core–shell particles of the same pore size), which increases the particle diffusivity, 

 (a contribution of both pore and surface diffusion) for molecules with faster 

. With large molecules, however, the pore size of the core–shell particles greatly improves 

 term for large molecules. The Aeris WidePore core–shell particles serve this purpose, allowing even faster separation of macro-molecule and proteins without loss of efficiency (30). 

The film mass transfer resistance coefficient is larger for the new generation of core–shell particles, especially for large molecules, as a result of the particles' surface roughness, a property that fully porous particles, with their smooth surfaces, lack (17). A substantial increase in pore size has been reported to alleviate the film mass transfer resistance (18); this is the advantage of the Aeris core–shell particles that are designed with  large pores. 

: The frictional heat effect of columns packed with core–shell particles is only severe with sub-2-μm particles. However, in comparison to the fully porous sub-2-μm particles, the solid core present in the core–shell particles promotes better heat conductivity and frictional heating become preponderant on the fully porous sub-2-μm particles, especially when operated at high linear velocity (31). The low permeability of columns packed with sub-2-μm particles inherently causes large heat generation that changes the properties of the mobile phase that carries the solute through the columns. The sub-3-μm core–shell particles are now the solution to heat effect when very fast analysis is desired. 

A brief summary of the relationship of the physicochemical and mass transfer kinetic properties of the recently evolved core–shell particles has been given, based on recent scientific reports. The superior performance of sub-3-μm core–shell particles is largely attributed to the significant improvement in their manufacture, resulting in improvement in their physicochemical properties compared to the early day core–shell particles. The advantage of the lower permeability brought by the sub-3-μm core–shell particles greatly surpasses those of the sub-2-μm fully porous particles to allow a significant increase in speed of analysis. The future of core–shell particles will see further dramatic changes in porous shell design and configuration, including possible decreases in particle diameter to increase the speed of analysis even further (11). 

J.O.O. would like to thank Elaine Stack, a PhD student working in the ISSC Cork laboratory, for producing the HETP data shown in Figure 3(e). Special thanks to Science Foundation Ireland for the financial support (Grant Number 08/SRC/B1412) for research funding of the Irish Separation Science Cluster (ISSC) under the Strategic Research Cluster Programme.  

 are with the Irish Separation Science Cluster (ISSC) at Dublin City University in Glasnevin, Dublin, Ireland.  

are with the Irish Separation Science Cluster (ISSC) and Analytical and Biological Chemistry Research Facility (ABCRF) in the Department of Chemistry at the University College Cork, in Cork. Ireland. Please direct correspondence to: 

(1) K.K. Unger, R. Skudas and M.M. Schulte, 

(2) F. Gritti, I. Leonardis, D. Shock, P. Stevenson, A. Shalliker and G. Guiochon, 

(3) F. Gritti, I. Leonardis, J. Abia and G. Guiochon, 

(6) C.G. Horvath, B.A. Preiss and S.R. Lipsky, 

(8) J.O. Omamogho, J.P. Hanrahan, J. Tobin and J.D. Glennon, 

http://www.phenomenex.com/webdocument/aeris_bro.pdf

(14) S. Fekete, J. Fekete and K. Ganzler,

(15) E. Oláh, S. Fekete, J. Fekete and K. Ganzler, 

A brief review of the physicochmical properties of core-shell particles and their chromatographic efficiencies compared with those of sub-2-?m fully porous particles.

Next-generation stationary phases: Properties and performance of core-shell columns

The last two years have seen a continuous introduction of new high performance liquid chromatography (HPLC) and ultrahigh-pressure liquid chromatography (UHPLC) packing materials, including a range of sub-2-µm particles, superficially porous particles and second-generation monoliths. Here, we look at these introductions and their impact on instrumentation and laboratory productivity. In addition, we discuss future directions in column technology.

Every two years, we publish a special supplement on high performance liquid chromatography (HPLC) column technology in 

that brings readers up to date on the latest improvements in this important area. This is the first time this supplement has been published in

 and we have a total of four articles (in addition to this one), which discuss hybrid packings, superficially porous (core–shell) packings and hydrophilic interaction liquid chromatography (HILIC) (1–6). The last time I gave a general overview on columns was in the 2008 supplement (7). In the 2010 supplement (8), I focused on improvements and phases for reversed-phase chromatography, the most frequently used mode. This article will focus on an overview of column developments and discuss future directions in this area. Since the 2008 article, there has been a continuing interest in column developments with improvements in small porous particle technology, monoliths (both polymer- and silica-based), superficially porous particles (SPP) and a tremendous focus on HILIC. For handling complex samples, multidimensional and comprehensive LC×LC are still getting attention. In this update on column technology, I will cover some of the packing material and column developments and highlight observations made in the past four years. Other articles in this special supplement will expand on changes that have occurred in stationary phases in the various modes. 

 Figure 1: Comparison of diffusion distances for (a) 5-Âµm totally porous particles (TPP), with a sub-2-Âµm particle below the arrow, (b) 5-Âµm superficially porous particles (SPP) and (c) 2.7-Âµm SPP.			

High-Throughput and Productivity: Still a Driving Force in Liquid-Phase Separations

One of the driving forces for continued improvements in HPLC column technology is the requirement for higher throughput without sacrificing analytical performance. One of the trends noted in the last several years is the strong interest in smaller totally porous particles (TPP), especially those with average particle diameters less than 2 µm [Figure 1(a)]. In 2012, at least 25 companies are now promoting the virtues of sub-2-µm columns (Table 1). New companies are being added each year. It is almost expected now for the introduction of new columns to provide at least one sub-2 µm particle size in the portfolio. 

Table 1: Commercial 2- and sub-2-Âµm totally porous HPLC columns.*			

When sub-2-µm porous particles are packed into short columns, separations can be performed faster, sometimes in just a minute or two, than longer columns packed with larger particles (~5 µm) without sacrificing chromatographic resolution. The flat van Deemter curves noted for sub2µm columns allow (and perhaps demand) relatively high flow rates to be used, in the range of 1.5–2 mL/min, if necessary. Even though the column pressure increases with the inverse square of the average particle diameter, these shorter columns (usually 50 mm and under) can be used with most conventional HPLC pumping systems, even at these increased flow rates. 

For more demanding separations, longer columns of 100- and 150-mm lengths may be required. With such lengths, column back pressures may increase beyond the capabilities of conventional pumping systems (~400 bar upper limit). Thus, in recent years, pumping systems capable of operation at pressures as high as 20000 psi (1330 bar) have come onto the market. The advent of the term 

 (UHPLC) has somewhat mesmerized the chromatography world into thinking that an entirely new technology has arisen. However, it is rare to see a separation run at such high pressures, even at 1000 bar. User concerns about stress on instrument hardware and on the columns themselves have limited widespread applications at these high pressures. The demands of sample cleanliness and easier "pluggability" of the small porosity frits terminating the ends of sub-2-µm columns also have made some users cautious about jumping into their routine application. Nevertheless, these columns have proven to be rugged, even at extremely high pressures, if used properly by ensuring particle-free samples are injected. With the introductions of zero- or low-dead-volume, high-pressure guard columns and zero-dead-volume in-line filters to help protect the analytical column, liquid chromatographers are becoming more comfortable using these columns and many publications are now seen employing sub-2-µm columns, even in routine environments.  

What the advent of UHPLC has done is make instrument manufacturers more aware of the need to provide systems with minimal extra column effects (that is, band broadening outside of the column itself). These small-particle columns are so efficient (~250000 plates/m) that any unswept and unnecessary volumes and connections in the flow path have to be minimized. In addition to the high-pressure capability required for the pump and other hardware components (such as injector valves, mixers, and fittings), attention also is paid to the dwell volume now (also referred to as the 

 — the volume from the point of mixing of the solvents to the head of the column). When attempting to develop 1–2 min separations, liquid chromatographers can no longer wait for a gradient that takes several minutes to reach the column because ballistic gradients are now the norm. 

Some column and instrument suppliers have held off on joining the sub-2-µm bandwagon and have made more moderate reductions in the particle size from the popular 3–3.5 µm columns. The particle diameter is larger for packed columns in the range of 2–3 µm than the sub-2-µm particles, so the pressure drop is lower but efficiency is better than the more popular 3–3.5 µm particles. The arguments for using packings in the 2–3 µm range revolve around considering the entire separation cycle time: Higher temperatures improve efficiency and reduce back pressure, improved LC system hydraulics decrease band dispersion and gradient re-equilibration time, and faster autosamplers, detectors and data systems increase overall system efficiency. The use of lower pressures also places less stress on the instrumentation and column materials.  

Interestingly, I have not seen even smaller totally porous particle columns coming to the market in the last four years. Currently, the smallest totally porous particle size that is commercially available is 1.5 µm and this diameter has been available for many years. However, there are other competing technologies now on the market (and coming onto the market) that do not expose the column and instrumentation to such dramatic pressure conditions: SPP columns and monoliths. 

A discussion of new column packing materials– including sub-2-?m particles, superficially porous particles and second-generation monoliths– and their impact on instrumentation and laboratory productivity.

Special Issues-10-01-2012 | LCGC Supplements

Synthesis and applications of BEH particles in liquid chromatography

Advances in the understanding of stationary phases for HILIC

Recent progress in chiral stationary phase development and current chiral applications

Next-generation stationary phases: Properties and performance of core-shell columns

Developments in HPLC/UHPLC Column Technology