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• a dimensionless measure of the particle size gradient steepness was introduced. • about 15–20% gain in efficiency can be expected with particle size gradient. • too large particle size gradient can result in lower efficiencies than monodisperse packing. • large molecules (peptides, proteins) can in principle see the highest gain in efficiency. In this paper, the benefits of using columns packed with particles of decreasing size (particle size gradient) in liquid chromatography was investigated from a theoretical point of view. It is indeed well known that such columns may be useful in gradient elution, since the decrease of particle size along the chromatographic column can provide extra peak focusing effect. In the present contribution, several parameters (i.e., mobile phase gradient steepness, retention times and operating pressures) were considered and the kinetic performance of various types of columns packed with particle size gradient were evaluated. In the best case, about 15–20% gain in efficiency can be expected at a given retention time when utilizing a particle size gradient, compared to constant particle size. Conversely, when fixing efficiency, the analysis time can be decreased by about 15% with an optimal particle size gradient. However, it is also important to keep in mind that a too large a particle size gradient can result in lower efficiencies than a column packed with monodisperse packing. We have introduced the g d value, which is a dimensionless measure of the particle size gradient steepness that measures the relative variation of particle diameter throughout the column with respect to the average. We finally observed that g d = 0.3 - 0.4 provides the highest gain under practically useful conditions. [ABSTRACT FROM AUTHOR]

• Isomeric separation of underivatized N -glycans on nano ZIC-HILIC-LC-MS/MS. • Optimizing the chromatographic conditions improves separation of N -glycan isomers. • Lower retention of sialylated and oligomannose N -glycans at acidic mobile phase pH. • Lower resolution of isomers on ZIC-HILIC as mobile phase pH becomes more basic. • Higher temperature improves isomeric separation of native N -glycans on ZIC-HILIC. Changes in the expression of glycan isomers have been implicated in the development and progression of several diseases. However, the analysis of structurally diverse isomeric N- glycans by LC−MS/MS is still a major analytical challenge, particularly due to their large number of possible isomeric conformations. Common approaches derivatized the N -glycans to increase their hydrophobicity and to gain better detection in the MS system. Unfortunately, glycan derivatization is time-consuming and, in many cases, adds complexity because of the multiple reaction and cleaning steps, incomplete chemical labeling, possible degradation, and unwanted side reactions. Thus, analysis of native glycans, especially for samples with low abundance by LC−MS/MS, is desirable. Normal phase chromatography, which employs HILIC stationary phase, has been commonly employed for the identification and separation of labeled glycans. In this study, we focused on achieving efficient isomeric separation of native N -glycans using a nano ZIC-HILIC column commonly employed to separate labeled glycans and glycopeptides. Underivatized sialylated and oligomannose N -glycans derived from bovine fetuin and Ribonuclease B were initially utilized to optimize chromatographic conditions, including column temperature, pH of mobile phases, and gradient elution time. The optimized condition was then applied for the isomeric separation of native N -glycans derived from alpha-1 acid glycoprotein, as well as from biological samples. Finally, we confirmed the stability and reproducibility of the ZIC-HILIC column by performing run-to-run comparisons of the full width at half height (FWHM) and retention time on different N -glycans. The variability in FWHM was less than 0.5 min, while that of retention time was less than 1.0 min with %RSD less than 1.0%. [ABSTRACT FROM AUTHOR]

Highlights • Theory was developed to predict the peak widths for coupled column systems. • In gradient elution mode, the order of the columns impacts the apparent peak width. • “Peak sharpening” may occur when the most efficient column is the last in the assembly. • Theory and predictions have been experimentally verified. • Limits of apparent efficiency is discussed assuming infinite plate count for one column. Abstract The goal of this work was to understand the variation of apparent efficiency when serially coupling columns with identical stationary phase chemistries, but with differences in their kinetic performance. For this purpose, a mathematical treatment was developed both for isocratic and gradient modes to assess the change in plate numbers and peak widths when coupling arbitrary several columns. To validate the theory, experiments were also carried out using various mixtures of compounds, on columns packed with different particle sizes, to mimic highly efficient (new, not used) and poorly efficient columns (used one with many injections). Excellent agreement was found between measured and calculated peak widths. The average error in prediction was about 5% (which may be explained by the additional volume of the coupling tubes). In isocratic mode, the plate numbers are not additive when the coupled columns possess different efficiencies, and a limiting plate count value can be calculated depending on the efficiency and length of the individual columns. Theoretical efficiency limit can also be determined assuming one column in the row with infinite efficiency. In gradient elution mode, the columns’ order has a role (non-symmetrical system). When the last column has high enough efficiency, the gradient band compression effect may outperform the competing band broadening caused by dispersive and diffusive processes (peak sharpening). Therefore, in gradient mode, the columns should generally be sequentially placed according to their increasing efficiency. [ABSTRACT FROM AUTHOR]

• BEH HILIC, BEH Amide, and BEH Z-HILIC Surface Chemistries are studied • Retention and mass transfer properties of these three columns are compared • Guo's method is validated to measure the water layer volume in HILIC columns • Retention and particle diffusivity are directly correlated to this water volume • Retention increases from BEH HILIC to BEH Amide to BEH Z-HILIC Columns This work investigates the link between the retentivity and the stationary phase to mobile phase mass transfer resistance of hydrophilic interaction liquid chromatography (HILIC) columns packed with the same base ethylene-bridged hybrid particles (BEH). The retention volumes, the plate heights, and the volume of the adsorbed water layer were measured for the ACQUITY T M UPLC T M BEH T M 130 Å HILIC Column (unbonded BEH), ACQUITY UPLC BEH 130 Å Amide Column (amide group attached), and Atlantis T M Premier BEH 95 Å Z-HILIC (zwitterionic group attached) Column. The method of Guo (toluene retention volumes in pure acetonitrile and in the HILIC eluent) was validated from the UNIFAC group-contribution method and applied to measure accurately the water layer volumes in these columns. A strong correlation was found between the retention volumes of most neutral polar analytes and the volume of the water layer adsorbed in the HILIC column. The fraction of the pore volume occupied by the water layer increases significantly from the BEH HILIC Column to the BEH Amide Column, and to the BEH Z-HILIC Column. This is explained by the water solvation of the attached ligands in the pore volume of the BEH Particles and to the smaller average mesopore size of the BEH Z-HILIC Particles. A second and strong correlation is also observed between the water content in the HILIC particle and the stationary phase to mobile phase mass transfer resistance of the HILIC columns at high mobile phase linear velocities. The measured intra-particle diffusivity normalized to the bulk diffusion coefficient decreased from 0.33 (BEH HILIC Column) to 0.10 (BEH Amide Column) and to only 0.03 (BEH Z-HILIC Column) for comparable retention of cytosine. These results are fully consistent with the higher viscosity of the internal eluent (higher water content) and higher internal obstruction for diffusion (smaller mesopores and internal porosity) in the BEH Z-HILIC Particles. Still, in gradient elution mode, the peak capacity was found to be 18% higher for the BEH Z-HILIC Column than that on the BEH Amide Column because the retention factors at elution were smaller when maintaining the same analysis time and starting eluent composition. [ABSTRACT FROM AUTHOR]

• Automation and control for a multi-step polishing process has been demonstrated. • Customized Python control layer was developed to schedule and automate BioSMB. • Inline conditioning achieved using inbuilt pumps and sensors available on BioSMB. • pH, conductivity inputs are used for real-time automated control of pumps and valves. • Complex polishing step combinations were carried out in continuous processing trains. We propose a strategy for automation and control of multi-step polishing chromatography in integrated continuous manufacturing of monoclonal antibodies. The strategy is demonstrated for a multi-step polishing process consisting of cation exchange chromatography in bind-and-elute mode followed by mixed-mode chromatography in flowthrough mode. A BioSMB system with a customized Python control layer is used for automation and scheduling of both the chromatography steps. Further, the BioSMB valve manifold is leveraged for in-line conditioning between the two steps, as tight control of pH and conductivity is essential when operating with multimodal resins because even slight fluctuations in load conditions adversely affect the chromatography performance. The pH and conductivity of the load to the multimodal chromatography columns is consistent, despite the elution gradient of the preceding cation exchange chromatography step. Inputs from the BioSMB pH and conductivity sensors are used for real-time control of the 7 pumps and 240 valves to achieve in-line conditioning inside the BioSMB manifold in a fully automated manner. This is confirmed by showcasing different elution strategies in cation exchange chromatography, including linear gradient, step gradient and process deviations like tubing leakage. In all the above cases, the model was able to maintain the pH and conductivity of multimodal chromatography load within the range of 6 ± 0.1 pH and 7 ± 0.3 mS/cm conductivity. The strategy eliminates the need for using multiple BioSMB units or integrating external pumps, valves, mixers, surge tanks, or sensors between the two steps as is currently the standard approach, thus offering a simple and robust structure for integrating multiple polishing chromatography steps in continuous downstream monoclonal antibody purification trains. [ABSTRACT FROM AUTHOR]

• A method of column preparation for high-performance solid phase extraction chromatography has been developed. • This allows for flexible, reversible and repeatable column impregnation. • The method is developed and demonstrated for the extractants HDEHP and HEHEHP. • Resulting column ability to separate rare earth elements is verified. • The importance of the water washing stage after impregnation is highlighted. The flexible and reversible preparation of columns for use in high-performance solid phase extraction chromatography by physisorption of organophosphorus acid extractants has been investigated in detail. Two extractants have been evaluated, bis (2-ethyl-1-hexyl) phosphoric acid (HDEHP) and 2-ethyl-1-hexyl (2-ethyl-1-hexyl) phosphonic acid (HEHEHP), but the developed procedure should be broadly applicable to other extractants. The liquid-liquid solubility of the extractants in feed solvents consisting of aqueous ethanol solutions of varying composition has been determined. The total amount of adsorbed extractant has been quantified by complete desorption and elution with ethanol followed by acid-base titrimetry. Column impregnation with feed solutions of varying concentration in the undersaturated region has been systematically evaluated, and the influence of a subsequent water wash step has been explored. It is shown that to achieve a robust and reproducible physisorption, the adsorbed amount of extractant should be determined after the wash step, and care must be taken when using indirect methods of measurement. Equilibrium Langmuir-type adsorption isotherms as a function of the extractant concentration in the feed solution have been determined. Adsorption of HEHEHP is higher than HDEHP for equal feed compositions, but the solubility of HEHEHP is lower, resulting in approximately identical maximum coverage levels. The ability of the resulting columns to separate rare earth elements have been verified for a mixture of eight metals using a combined isocratic and gradient elution of nitric acid. [ABSTRACT FROM AUTHOR]

We report on the results of an experimental and theoretical study of the effect of the extra-column band broadening (ECBB) on the performance of narrow-bore columns filled with the smallest particles that are currently commercially available. Emphasis is on the difference between the effect of ECBB under gradient and isocratic conditions, as well as on the ability to model and predict the ECBB effects using well-established band broadening expressions available from the theory of chromatography. The fine details and assumptions that need to be taken into account when using these expressions are discussed. The experiments showed that, the steeper the gradient, the more pronounced the extra-column band broadening losses become. Whereas the pre-column band broadening can in both isocratic and gradient elution be avoided by playing on the possibilities to focus the analytes on top of the column (e.g. by using the POISe injection method when running isocratic separations), the post-column extra-column band broadening is inescapable in both cases. Inducing extra-column band broadening by changing the inner diameter of the post-column tubing from 65 to 250 μm, we found that all peaks in the chromatogram are strongly affected (around a factor of 1.9 increase in relative peak width) when running steep gradients, while usually only the first eluting peak was affected in the isocratic mode or when running shallow gradients (factor 1.6–1.8 increase in relative peak width for the first eluting analyte). [ABSTRACT FROM AUTHOR]

Abstract: A new mode of gradient elution in liquid chromatography is proposed. It is derived from simple theoretical considerations and is particularly suitable for applications to fast, very-high pressure gradients. It is designed to improve the injection-to-injection repeatability of chromatographic runs at either constant flow (cF, cooling case scenario) or constant pressure (cP, heating case scenario). The purpose of this original gradient mode is to minimize the variations of the temperatures and the pressures across and along the column during the gradient time. These variations are caused by the heat generated in the column due to the friction of the eluent percolating the bed. Any temperature fluctuation affects to some extent the precision of the measurements of retention times and bandwidths of eluted compounds. The minimization of this effect was achieved by maintaining constant the frictional heat power (i.e., the product of the flow rate by the column pressure drop) generated during the gradient run, the washing step, and the re-equilibration time. The eluent temperature was recorded at the column outlet. One useful application of gradient chromatography at constant frictional heat (cFH) is illustrated for a 50–100% volume gradient of acetonitrile in water using a 4.6mm × 150mm column packed with 3.5μm BEH-C18 particles and operated with an Agilent 1290 Infinity liquid chromatograph. The reproducibility (eleven consecutive injections) of the retention times and peak variances of nine small molecules (RPLC check-out sample mixture) using cF, cP, and cFH gradients were compared for the same amount of heat produced (403J) during each run time. The RSDs of the retention times and the peak variances for four consecutive injections were systematically below 0.035 and 0.50% in constant frictional heat gradient chromatography, after three initial injections. These RSDs were markedly higher for cF (0.050 and 0.90%) and cP (0.070 and 1.80%) gradients. [Copyright &y& Elsevier]

• A kinetic model of protein adsorption on ion exchange media was derived. • The model is able to capture complex elution profiles in preparative CEX processes. • The formation of a peak shoulder can be observed at high protein load densities. • The shoulder is formed during salt and pH gradient elution and on different CEX media. A fundamental understanding of the protein retention mechanism in preparative ion exchange (IEX) chromatography columns is essential for a model-based process development approach. For the past three decades, the mechanistic description of protein retention has been based predominantly on the steric mass action (SMA) model. In recent years, however, retention profiles of proteins have been reported more frequently for preparative processes that are not consistent with the mechanistic understanding relying on the SMA model. In this work, complex elution behavior of proteins in preparative IEX processes is analyzed using a colloidal particle adsorption (CPA) model. The CPA model is found to be capable of reproducing elution profiles that cannot be described by the traditional SMA model. According to the CPA model, the reported complex behavior can be ascribed to a strong compression and concentration of the elution front in the lower unsaturated part of the chromatography column. As the unsaturated part of the column decreases with increasing protein load density, exceeding a critical load density can lead to the formation of a shoulder in the peak front. The general applicability of the model in describing preparative IEX processes is demonstrated using several industrial case studies including multiple monoclonal antibodies on different IEX adsorber systems. In this context, the work covers both salt controlled and pH-controlled protein elution. [ABSTRACT FROM AUTHOR]

• Efficiency of non-uniform columns was studied by theoretical approach. • In isocratic elution mode, particle size gradient did not offer any advantage. • In gradient elution mode, the final physical bandwidth decreased by 1--4%. • By further optimization, 5--8% extra bandwidth compression was achieved. Modern analytical applications of liquid chromatography require more and more efficient columns. In this work, the possibility of utilizing particle size gradient in the chromatographic column was studied by a theoretical approach. In the course of our work three different scenarios of particle size gradients were considered with different shapes (linear, convex and concave). The evolution of bandwidth inside the column was plotted for each scenario. As a reference point, the bandwidth of the uniform column was used, which had the same pressure drop as the non-uniform column. According to our calculations, in isocratic elution mode, the non-uniform column does not offer any advantage compared to the uniform column, regardless the type of the particle size gradient. In gradient elution mode, however, extra band compression occurs was found. For negative particle size gradients, the final physical bandwidth was found to be approximately 1–4 % smaller than for uniform columns. This slight gain in efficiency in terms of bandwidth compression can be expanded to 5–8 % by the optimization of the limiting particle sizes. These optimized results are obtained when the final particle size is approximately 40% of the initial particle diameter. [ABSTRACT FROM AUTHOR]

• Influence of injection volume based on comprehensive data. • Differentiation between isocratic and gradient elution. • Utilization of sub 2 µm reverse phase material. • Significant volume overloading possible without sacrificing efficiency. The injection volume and the associated column volume overload is one of the most common issues in miniaturized chromatography. The injection volume should not exceed 10% of the effective column volume. A further reduction of the injection volume leads to an increase in chromatographic efficiency. However, the signal intensity must be above a certain threshold to generate a chromatographic peak that can be detected. Therefore, the injection volume has to be optimized to reach the ideal balance between chromatographic efficiency and sensitivity. This study examined the general influence of the injection volume for both isocratic and gradient elution, depending on the retention factor and peak standard deviation. For this purpose, substances of different polarity were selected to represent a broad elution spectrum. Besides the model analyte naphthalene, these were mainly pharmaceuticals. For all measurements a microbore column with an ID of 300 µm and packed with 1.9 μm fully porous particles was used. For isocratic elution, the injection volume was varied between 4 and 16% of the effective column volume. The retention factors were adjusted between 2 and 10. For gradient elution, the injection volume was varied between 4 and 160% of the effective column volume. The observed effects were further investigated using the gradient kinetic plot theory. In isocratic elution, a loss in plate height up to 50% was observed for components that elute near the void time. A significant reduction of the chromatographic efficiency was noticed up to a retention factor of 4. In gradient elution, a reduction in peak capacity could only be observed if the injection volume exceeded 40% of the effective column volume. For some substances, only a slight loss in peak capacity was noticed even with a volume overload of 160%. [ABSTRACT FROM AUTHOR]

• Theoretical investigations show 2 µm open tube capillaries produce high performance. • The performance is compared with normal, slip-flow boundaries and electrochromatography. • These techniques are suggested to not be worth the effort, compared to gradient elution operation. • The zone standard deviations are shown to be similar for these different flow profiles. • The number of plates for the different flow profiles are also shown to be similar. Modifications to the flow profile used in open tube capillary liquid chromatography (OT-CLC) include using slip-flow walls and using electroosmosis as a fluid pump as practiced in electrochromatography. These modifications are implemented experimentally by changing the capillary surface and solvent conditions which results in the change of boundary conditions at the capillary wall. In this paper we employ a theory-based study and compare the zone broadening of simple solutes using parabolic flow from a liquid pump, slip-flow from a highly hydrophobic inner surface with water eluent, and electroosmosis for the conditions of pure water and dilute salt utilizing 2 µm inner diameter OT capillaries. In general, the two types of flow other than parabolic exhibit thin zones in the early part of the chromatogram, consistent with previous studies of slip-flow and electroosmotic flow used in electrochromatography. Electrochromatography is shown to yield higher efficiency and less zone broadening than parabolic and slip-flow conditions used in this study. Nonetheless, it is found that the zone standard deviations are shown to be similar for these flow profiles as is the number of plates for these different flow profiles under the conditions utilized here. It is revealed that these modifications do not warrant the effort to maintain the special solvent conditions when compared to gradient elution OT-CLC, which gives a nearly constant peak width throughout the chromatogram, is easiest to implement, and is the method of choice for complex analysis. [ABSTRACT FROM AUTHOR]

• A radical new way to prepare aqueous buffers for column chromatography • The solid compounds is directly dosed to the aqueous solution • Buffers can be prepared on demand • The method is suited for linear and step gradients Buffer preparation and storage requires a significant facility footprint in large scale bioprocessing and together with the costs of supply chain management can have a substantial economic impact. In-line buffer mixing in chromatography is commonly performed by blending different buffer solutions using at least two pumps and a static or dynamic mixer. We developed a device for an in-line gradient delivery of buffering agents directly from solids to be applied for chromatographic separation processes. A solid feeding device with a screw conveyor and a hold tank for the solids was designed and a miniaturized system was 3D printed. The coefficient of variation for the precision of the solid feeding of 5 different buffering agents was below 5% even for very small solid flow rates necessary for lab-scale chromatography. Stability was demonstrated by a constant linear solid feed at a very low dosing rate of 0.05 g.min−1 over 24 hours. We demonstrated the suitability for chromatography by directly connecting the system to a standard chromatography workstation for protein chromatography. The solids were fed into a miniaturized continuously stirred tank reactor connected to an ÄKTA purification system. The performance of the in-line gradient delivery of buffering agents directly from solids was compared to conventional in-line buffer mixing. We were able to achieve highly linear gradients for elution using only one pump of a chromatographic system, generating the gradient by the direct addition of solids avoiding the necessity of additional pumps and hold tanks. By direct conditioning of buffers and the addition of solids a simple, just in time, at site preparation of buffers was possible. The design of the feeding unit for solid addition for buffer preparation is easily scalable and adaptable to work with or as a replacement for already existing in-line dilution or conditioning units. [ABSTRACT FROM AUTHOR]

• The advantage of UHT-PGC column had high peak capacity and selectivity for native glycans. • UHT-PGC-LC-MSMS provided the RIR of diagnostic ions to predict terminal linkage and arm specificity of biantennary fucosylated N -glycans. • The neutral N -glycans without reduction can be directly analyzed by UHT-PGC-LCMS. Due to the heterogeneous and isomeric nature of glycans, the development of an advanced separation of distinct glycan isomers is essential for glycan research and application. In this study, we utilized porous graphite carbon (PGC) chromatography for the separation of isomeric oligosaccharides without reduction or chemical derivatization at 190 °C in a custom-built heating oven. Furthermore, the fine structures of glycan isomers could be identified by using ultrahigh temperature PGC liquid chromatography mass spectrometry (UHT-PGC-LCMS). A nonreduced hydrolyzed dextran was applied to verify the performance of UHT-PGC. When the temperature of the PGC column was increased from 25 to 190 °C, the liquid chromatography separation power of the nonreduced dextran ladder significantly increased. The advantage of the UHT-PGC column was its high peak capacity with gradient elution in 10 min at 190 °C, 6700 psi, and a 250 μL/min flow rate for native glycan analysis. Four synthetic Lewis antigen isomers were used to elucidate the separation effectiveness in UHT-PGC. Moreover, mass spectrometry-based sequencing to generate specific diagnostic ions from the four synthetic Lewis antigens was used to predict isomeric glycans based on the relative intensity ratio (RIR) of diagnostic ions. The intensities of the diagnostic ions of synthetic isomers were used to identify each isomer of the fucosylated glycan. The results clearly showed that terminal Lewis A and X residues were in the 3- and 6-arms of N -glycan, respectively. [ABSTRACT FROM AUTHOR]

• Bispecific antibodies exhibit reversible, three-peak elution on HIC columns. • Elution behavior caused by interconversion of three molecular forms. • Phenomenological column model predicts gradient elution behavior. • BiSAb multiple-peak elution on HIC and CEX are compared. The elution behavior of bivalent bispecific antibodies (BiSAb) comprising an immunoglobulin G framework genetically fused to a pair of single chain variable fragments (scFvs) was studied on hydrophobic interaction chromatography (HIC) columns using ammonium sulfate gradients. Each of the BiSAb molecules studied exhibited a three-peak elution behavior regardless of the location of scFv attachment to the framework IgG. Collecting and re-injecting each of the isolated peaks and eluting with the same gradient resulted in the same three-peak profile indicating that the behavior is reversible. Analogous behavior was observed for HIC resins with different functional ligands, matrix structures, and particle sizes. Residence time, operating temperature, and hold time were shown to affect the elution behavior. While three peaks were obtained at short residence times and room temperature, residence times longer than about 27 min or operating at 45 °C resulted in a single merged peak indicating that the underlying mechanism occurs on time scales comparable to that of chromatographic separation. Holding the protein on the resins prior to elution enriched the late eluting peak indicating that multiple binding states formed on the chromatographic surface are responsible for this behavior. Tryptophan auto-fluorescence measurements show that stronger binding forms have increased solvent exposure indicating that surface-catalyzed conformational changes play a role. A model was developed to describe the interplay of chromatographic separation and slow conformational changes. [ABSTRACT FROM AUTHOR]

• Monolithic silica columns with sub-2 µm domain size have been synthesized. • The produced monolith columns provide a high column efficiency (H = ~4 µm). • Peak capacities (n p) are 30‒40% higher than in a commercial particle column. • A high repeatability of the performance of the fabricated columns is demonstrated. We report on a direct comparison of the separation performance in capillary nano-LC between commercial packed bed columns and the small-domain silica monoliths in applications. Octadecylsilylated monolithic silica capillary columns with a 50 and 100 µm inner diameter (i.d.) were prepared with a procedure providing domain sizes in the sub-2 µm range. The fabricated monolith columns could provide plate heights (H) of 4.0‒4.2 µm for hexylbenzene (retention factor (k) = 3.6) at an optimal linear velocity range under an isocratic condition, while showing a column permeability (K v0 = 1.6‒1.8 × 10−14 m2) comparable to that of a column packed with 3‒3.5 µm particles. When the peak capacity (n p) for a cytochrome C digest was compared for variable gradient times (t G = 15, 30, 60, and 120 min) and constant gradient steepness (b '), the present monolith columns could show a 30‒40% higher n p -value than the packed capillary column with 2 µm particles (e.g. n p = 180 versus n p = 259 at t G = 30 min). The produced monolith columns showed a high chromatographic repeatability for both isocratic and gradient elution (e.g. relative standard deviation (n = 3, RSD (%)) = 0.5% for H , 2,6% for k , and 5.6% for K v0 in the isocratic mode using the 100 µm i.d.-columns). The present results show that the domain sizes which can now be achieved for capillary silica monoliths are sufficiently small to result in separation efficiencies that can successfully compete with the commercial packed bed columns available for use in nano-LC applications. [ABSTRACT FROM AUTHOR]

• Repeatable partial gradient elution equilibrium shown for different HILIC columns. • Rapid partial equilibration independent of stationary phase and gradient slope. • Full equilibration time depends on water layer thickness on stationary phase. • Full equilibration faster at elevated temperature and increased column pore size. • Full equilibration achieved on some columns with passing only 12 column volumes. For a wide variety of hydrophilic interaction chromatography stationary phases, a repeatable partial equilibration was demonstrated in gradient elution after purging with as little as 12 column volumes of mobile phase. Relative standard deviations of retention time of on average ~0.15% could be obtained after 1 or 2 conditioning (blank) runs. The equilibration period must be kept strictly constant, otherwise selectivity changes occur, but this is not problematic on modern instruments. Partial equilibration was largely independent of stationary phase or gradient slope. Alternatively, full column equilibration is favoured for stationary phases that do not trap extensive water layers, and for materials with a wider pore size that have a lower surface area. Temperatures somewhat above ambient also shorten the equilibration time. Some stationary phases under optimum conditions can achieve full column equilibration using purging with ~12 column volumes, which is useful for rapid set-up of isocratic separations or for conventional gradient analysis. [ABSTRACT FROM AUTHOR]