A study of the effects of column porosity on gradient separations of proteins

The type of the stationary phase for reversed-phase liquid chromatography significantly affects the sample elution. Hydrodynamic properties, efficiency and gradient elution of proteins were investigated on five commercial C18 columns with wide-pore totally porous particles, with superficially porous layer particles, non-porous particles and a silica-based monolithic bed. The efficiency in the terms of reduced plate height is higher for low-molecular ethylbenzene than for proteins, but depends on the character of the pores in the individual columns tested. The superficially porous Poroshell and the non-porous Micra columns provide the best efficiency for proteins at high mobile phase flow rates, probably because of similar pore architecture in the stationary phase. The Zorbax column with similar pore architecture as the Poroshell active layer, i.e. narrow pore distribution of wider pores shows better efficiency than the packed column with narrow pores and broad pore distribution. The monolithic column shows lower efficiency for proteins at high flow rates, but it performs better than the broad-pore distribution totally porous particulate columns. Different pore architecture affects also the retention and selectivity for proteins on the individual columns. The retention times on all columns can be predicted using the model for reversed-phase gradient elution developed originally for low-molecular compounds. Consideration of the limited pore volume accessible to the biopolymers has negligible effect on the prediction of retention on the columns packed with non-porous or superficially porous particles, but improves the accuracy of the predicted data for the totally porous columns with broad pore distribution.     

Polymetacrylate and hybrid interparticle monolithic columns for fast separations of proteins by capillary liquid chromatography

Preparation of organic polymer monolithic columns in fused silica capillaries was aimed at fast gradient separation of proteins. For this purpose, polymerization in situ procedure was optimized, using ethylene dimetacrylate and butyl metacrylate monomers with azobisisobutyronitrile as initiator of the polymerization reaction in presence of non-aqueous porogen solvent mixtures composed of 1-propanol and 1,4-butanediol. The separation of proteins in totally monolithic capillary columns was compared with the chromatography on a new type of "hybrid interparticle monolithic" capillary columns, prepared by in situ polymerization in capillary packed with superficially porous spherical beds, 37-50 microm. The "hybrid" columns showed excellent stability and improved hydrodynamic flow properties with respect to the "totally" monolithic capillary columns. The separation selectivity is similar in the two types of columns. The nature of the superficially porous layer (bare silica or bonded C18 ligands) affects the separation selectivity less significantly than the porosity (density) of the monolithic moiety in the interparticle space, controlled by the composition of the polymerization mixture. The retention behaviour of proteins on all prepared columns is consistent with the reversed-phase gradient elution theory.     

Superficially porous silica microspheres for fast high-performance liquid chromatography of macromolecules

Very fast reversed-phase separations of biomacromolecules are performed using columns made with superficially porous silica microsphere column packings ("Poroshell"). These column packings consist of ultra-pure "biofriendly' silica microspheres composed of solid cores and thin outer shells with uniform pores. The excellent kinetic properties of these new column packings allow stable, high-resolution gradient chromatography of polypeptides, proteins, nucleic acids, DNA fragments, etc. in a fraction of the time required for conventional separations. Contrasted with <2-microm non-porous particles, Poroshell packings can be used optimally with existing equipment and greater sample loading capacities, while retaining kinetic (and separation speed) advantages over conventional totally porous particles.     

Highly efficient monolithic silica capillary columns modified with poly(acrylic acid) for hydrophilic interaction chromatography

Monolithic silica capillary columns for hydrophilic interaction liquid chromatography (HILIC) were prepared by on-column polymerization of acrylic acid on monolithic silica in a fused silica capillary modified with anchor groups. The products maintained the high permeability (K=5 x 10(-14)m(2)) and provided a plate height (H) of less than 10 microm at optimum linear velocity (u) and H below 20 microm at u=6mm/s for polar solutes including nucleosides and carbohydrates. The HILIC mode monolithic silica capillary column was able to produce 10000 theoretical plates (N) with column dead time (t(0)) of 20s at a pressure drop of 20 MPa or lower. The total performance was much higher than conventional particle-packed HILIC columns currently available. The gradient separations of peptides by a capillary LC-electrospray mass spectrometry system resulted in very different retention selectivity between reversed-phase mode separations and the HILIC mode separations with a peak capacity of ca. 100 in a 10 min gradient time in either mode. The high performance observed with the monolithic silica capillary column modified with poly(acrylic acid) suggests that the HILIC mode can be an alternative to the reversed-phase mode for a wide range of compounds, especially for those of high polarity in isocratic as well as gradient elution.     

On the optimization of the shell thickness of superficially porous particles

The thickness of the porous shells of superficially porous particles influences the separation power of columns packed with these packing materials. Models of the mass transfer kinetics across porous adsorbents permit the prediction of the HETP curves of columns packed with particles having shells of different thicknesses, for molecules of different sizes. Decreasing the thickness of the porous layer potentially results in lower values of the “C-term” of the HETP curve and of the minimum of these curves. The Poppe plots calculated under isocratic and gradient conditions show that the separation power of columns packed with superficially porous particles increases significantly with decreasing thickness of the porous layer but this increase is more important for larger than for smaller molecules. The resolution between pairs of compounds increases at constant values of their retention factors when the strength of the eluent must be reduced to compensate for the decrease of their retention that is caused by the reduction of the surface area of the stationary phase. Thus, the separation power of columns packed with superficially porous particles increases with decreasing shell thickness. In contrast, if analysts do not compensate for the retention decrease, the resolution between small molecular weight compounds becomes worse with thin than with thick superficially porous particles. Finally, the importance of using instruments providing low extra-column band broadening contributions is stressed.     

Effects of the gradient profile, sample volume and solvent on the separation in very fast gradients, with special attention to the second-dimension gradient in comprehensive two-dimensional liquid chromatography

Gradient elution provides significant improvement in peak capacity with respect to isocratic conditions and therefore should be used in comprehensive two-dimensional LC×LC, both in the first and in the second dimension, where, however, gradients are limited to a short time period available for separation, usually 1 min or less. Gradient conditions spanning over a broad mobile phase composition range in each second-dimension fraction analysis are used with generic "full in fraction" (FIF) gradients. "Segment in fraction" (SIF) gradients cover a limited gradient range adjusted independently to suit changing lipophilicity range of compounds transferred to the second dimension during the first-dimension gradient run and to provide regular coverage of the two-dimensional retention space. Optimization of the gradient profiles is important tool for achieving high two-dimensional peak capacity and savings of the separation time in comprehensive LC×LC. Calculations based on the well-established gradient-elution theory can be used to predict the elution times and bandwidths in fast gradients, taking into account increased probability of pre-gradient or post-gradient elution. The fraction volumes transferred into the second dimension may significantly affect the second-dimension bandwidths, especially at high elution strength of the fraction solvent, which may cause even band distortion or splitting in combined normal-phase (HILIC)-RP systems, but also in some two-dimensional RP-RP systems. In the present work, the effects of the fast gradient profile, of the sample volume and solvent on the elution time and bandwidths were investigated on a short column packed with fused-core porous-shell particles, providing narrow bandwidths and fast separations at moderate operating pressure.     

Morphology and efficiency of poly(styrene-co-divinylbenzene)-based monolithic capillary columns for the separation of small and large molecules

The morphology of organic monolithic stationary phases based on poly(styrene-divinylbenzene) was modified by changing the ratio of monomers to microporogen in order to make them also suitable for small molecule separations. The morphology of the columns was characterized by high-resolution scanning electron micrography, showing larger primary globules and larger macropores, as well as no mesopores >20 nm in the monolithic skeleton. The permeability of the modified monoliths was approximately three times higher than that of columns which have been optimized for large molecule separations, enabling operation of a 30 cm long column at pressures below 250 bar. In the isocratic separation of dansylated amino acids, plate counts of 50000–107000 m⁻¹ were achievable, which are equivalent to efficiencies obtained with 3.1 μm porous particles. The separation performance for small molecules in gradient elution was investigated using mixtures of dansylated amino acids, β-lactam antibiotics, and thyroid hormones. Finally, the modified monolithic capillary columns also proved to be highly efficient in the separation of biopolymers such as peptides and proteins, enabling peak width at half height of 3–8 s and peak capacities of 110–180 in 15–30 min gradient runs.     

Isocratic and gradient elution chromatography: a comparison in terms of speed, retention reproducibility and quantitation

Chromatographers are cautioned to avoid gradient elution when isocratic elution will do. In this work, we compared the analytical properties of gradient and isocratic separations of a sample which can be done quite readily under isocratic conditions. We found that gradient elution gave a shorter overall analysis with similar resolution of the critical pair compared to isocratic elution without sacrificing repeatability in retention time, peak area and peak height or linearity of the calibration curve. We also obtained acceptable repeatability in peak area/height and linearity of calibrations curves for a sample that required gradient elution using a practical baseline subtraction technique. Based on these results and related work which show that columns can be reequilibrated by flushing with less than two column volumes of the initial eluent, we conclude that many of the reasons given to avoid gradient elution deserve serious reconsideration, especially for those samples which are easily separated isocratically. However, we believe isocratic elution will remain preferable when: (1) the sample contains less than 10 weakly retained components (i.e. the last peak elutes with k' < 5) or (2) the gradient baseline impedes trace analysis.     

Impact of pore structural parameters on column performance and resolution of reversed-phase monolithic silica columns for peptides and proteins

In this work, monolithic silica columns with the C4, C8, and C18 chemistry and having various macropore diameters and two different mesopore diameters are studied to access the differences in the column efficiency under isocratic elution conditions and the resolution of selected peptide pairs under reversed-phase gradient elution conditions for the separation of peptides and proteins. The columns with the pore structural characteristics that provided the most efficient separations are then employed to optimize the conditions of a gradient separation of a model mixture of peptides and proteins based on surface chemistry, gradient time, volumetric flow rate, and acetonitrile concentration. Both the mesopore and macropore diameters of the monolithic column are decisive for the column efficiency. As the diameter of the through-pores decreases, the column efficiency increases. The large set of mesopores studied with a nominal diameter of approximately 25 nm provided the most efficient column performance. The efficiency of the monolithic silica columns increase with decreasing n-alkyl chain length in the sequence of C18相似文献   

High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry

High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.     

Applicability of linear and nonlinear retention‐time models for reversed‐phase liquid chromatography separations of small molecules,peptides, and intact proteins

The applicability and predictive properties of the linear solvent strength model and two nonlinear retention‐time models, i.e., the quadratic model and the Neue model, were assessed for the separation of small molecules (phenol derivatives), peptides, and intact proteins. Retention‐time measurements were conducted in isocratic mode and gradient mode applying different gradient times and elution‐strength combinations. The quadratic model provided the most accurate retention‐factor predictions for small molecules (average absolute prediction error of 1.5%) and peptides separations (with a prediction error of 2.3%). An advantage of the Neue model is that it can provide accurate predictions based on only three gradient scouting runs, making tedious isocratic retention‐time measurements obsolete. For peptides, the use of gradient scouting runs in combination with the Neue model resulted in better prediction errors (<2.2%) compared to the use of isocratic runs. The applicability of the quadratic model is limited due to a complex combination of error and exponential functions. For protein separations, only a small elution window could be applied, which is due to the strong effect of the content of organic modifier on retention. Hence, the linear retention‐time behavior of intact proteins is well described by the linear solvent strength model. Prediction errors using gradient scouting runs were significantly lower (2.2%) than when using isocratic scouting runs (3.2%).     

Peak capacity in gradient reversed-phase liquid chromatography of biopolymers. Theoretical and practical implications for the separation of oligonucleotides

Reversed-phase ultra-performance liquid chromatography was used for biopolymer separations in isocratic and gradient mode. The gradient elution mode was employed to estimate the optimal mobile phase flow rate to obtain the best column efficiency and the peak capacity for three classes of analytes: peptides, oligonucleotides and proteins. The results indicate that the flow rate of the Van Deemter optimum for 2.1 mm I.D. columns packed with a porous 1.7 microm C18 sorbent is below 0.2 mL/min for our analytes. However, the maximum peak capacity is achieved at flow rates between 0.15 and 1.0 mL/min, depending on the molecular weight of the analyte. The isocratic separation mode was utilized to measure the dependence of the retention factor on the mobile phase composition. Constants derived from isocratic experiments were utilized in a mathematical model based on gradient theory. Column peak capacity was predicted as a function of flow rate, gradient slope and column length. Predicted peak capacity trends were compared to experimental results.     

Methodology for porting retention prediction data from old to new columns and from conventional-scale to miniaturised ion chromatography systems

Several procedures are available for simulating and optimising separations in ion chromatography (IC), based on the application of retention models to an extensive database of analyte retention times on a wide range of columns. These procedures are subject to errors arising from batch-to-batch variability in the synthesis of stationary phases, or when using a column having a different diameter to that used when the database was acquired originally. Approaches are described in which the retention database can be recalibrated to accommodate changes in the stationary phase (ion-exchange selectivity coefficient and ion-exchange capacity) or in the column diameter which lead to changes in phase ratio. The entire database can be recalibrated for all analytes on a particular column by performing three isocratic separations with two analyte ions. The retention data so obtained are then used to derive a "porting" equation which is employed to generate the required simulated separation. Accurate prediction of retention times is demonstrated for both anions and cations on 2mm and 0.4mm diameter columns under elution conditions which consist of up to five sequential isocratic or linear gradient elution steps. The proposed approach gives average errors in retention time prediction of less than 3% and the correlation coefficient was 0.9849 between predicted and observed retention times for 344 data points comprising 33 anionic or cationic analytes, 5 column internal diameters and 8 complex elution profiles.     

Steroid profiles determined by capillary electrochromatography, laser-induced fluorescence detection and electrospray-mass spectrometry

Macroporous, monolithic capillary electrochromatography (CEC) columns, featuring a hydrophobic stationary phase, have been applied to the separations of steroids with good column efficiency. Using isocratic and gradient elution runs, mixtures of neutral or conjugated steroids could be resolved. While dansylated ketosteroids were detectable through laser-induced fluorescence at attomole levels, the CEC columns coupled to electrospray-ion-trap mass spectrometry featured femtomole detection limits.     

A practical approach to maximizing peak capacity by using long columns packed with pellicular stationary phases for proteomic research

Maximization of peak capacity is a very important step in developing one-dimensional separations of complex samples. In recent work, it was shown that the use of small particles in combination with the new technique of ultrahigh pressure liquid chromatography (UHPLC) was able to generate very high peak capacities. Here we show the ability of conventional HPLC instrumentation to give comparable peak capacities to those obtained in UHPLC for the important case of complex mixtures of peptides but at much lower pressures by using a 60 cm long set of columns packed with 5 microm pellicular (superficially porous) particles. We first show, in complete agreement with the well known results of the theory of isocratic separations that, when time is not limiting, the best peak capacities in gradient elution chromatography are obtained by using large particles and the longest column that can be operated at the pump's pressure limit. Two different types of 5 microm particles (superficially porous and totally porous) were compared for their efficiency in gradient chromatography of peptides. We find that the pellicular material gave about 50% higher peak capacity compared to the analogous porous material. A 60 cm column set packed with pellicular particles was made by connecting short columns in series; a peak capacity of about 460 was obtained in 4 h at room temperature. Increasing the column temperature to 70 degrees C reduced the analysis time to 2 h and further increased the peak capacity to more than 500. The number of peaks observed in the separation of bovine serum albumin tryptic peptides was greatly increased and the separation quality was significantly improved.     

Capillary electrochromatography of proteins with polymer-based strong-cation-exchanger microspheres

Monodisperse poly(glycidyl methacrylate-divinylbenzene) microspheres were functionalized with propyl sulfonic acid moieties to obtain beads negatively charged in a wide pH range. They were packed into fused-silica capillary of 50 micro, I.D. in order to separate proteins by capillary electrochromatography (CEC). Baseline separation of four basic proteins as well as three cytochrome c variants with an average column efficiency of 60,000 theoretical plates was obtained under isocratic elution conditions. The high efficiency is attributed to the uniformity of the column packing and the hydrophilic surface coverage of the polymer beads derived from the functionalization process. The effect of pH and salt concentration on protein separations was investigated and the results showed that the CEC separation mechanism is the combination of chromatographic retention and electrophoretic migration. Moreover, the column packed with the strongly acidic poly(glycidyl methacrylate-divinylbenzene) beads was also suitable for protein separations by micro-HPLC with a salt gradient. The comparison between the two kinds of elution modes shows that the column described here exhibited higher peak efficiency with isocratic elution in CEC than with gradient elution in micro-HPLC.     

Polymethacrylate monolithic and hybrid particle-monolithic columns for reversed-phase and hydrophilic interaction capillary liquid chromatography

We prepared hybrid particle-monolithic polymethacrylate columns for micro-HPLC by in situ polymerization in fused silica capillaries pre-packed with 3–5 μm C₁₈ and aminopropyl silica bonded particles, using polymerization mixtures based on laurylmethacrylate–ethylene dimethacrylate (co)polymers for the reversed-phase (RP) mode and [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl) zwitterionic (co)polymers for the hydrophilic interaction (HILIC) mode. The hybrid particle-monolithic columns showed reduced porosity and hold-up volumes, approximately 2–2.5 times lower in comparison to the pure monolithic columns prepared in the whole volume of empty capillaries. The elution volumes of sample compounds are also generally lower in comparison to packed or pure monolithic columns. The efficiency and permeability of the hybrid columns are intermediate in between the properties of the reference pure monolithic and particle-packed columns. The chemistries of the embedded solid particles and of the interparticle monolithic moiety in the hybrid capillary columns contribute to the retention to various degrees, affecting the selectivity of separation. Some hybrid columns provided improved separations of proteins in comparison to the reference particle-packed columns in the reversed-phase mode. Zwitterionic hybrid particle-monolithic columns show dual mode retention HILIC/RP behaviour depending on the composition of the mobile phase and allow separations of polar compounds such as phenolic acids in the HILIC mode at lower concentrations of acetonitrile and, often in shorter analysis time in comparison to particle-packed and full-volume monolithic columns.     

Relationship between isocratic and gradient retention times in the high-performance ion-exchange chromatography of proteins. Theory and experiment

Using isocratic retention parameters, the gradient elution retention time for several proteins has been calculated. The gradient retention time calculation is based on fitting the isocratic retention data to an equation of the form: log k' = m log (1/[Ca2+]) + log K and on applying well-established principles of gradient elution. A good correlation between the observed and calculated retention times for several test proteins was obtained at various total gradient times and column flow-rates. Conversely, isocratic retention parameters characterizing protein retention can be calculated from gradient elution retention data. However, even with retention data of high quality, small errors are amplified by the log-log nature of the ion-exchange isocratic retention model employed. Based on the close correlation between predicted and observed gradient retention times, no evidence for protein denaturation resulting from immobilization of the protein at high initial k' values at or near the column inlet was observed.     

Oil-In-Water Microemulsion LC Determination of Pharmaceuticals Using Gradient Elution

Efficient and novel oil-in-water microemulsion HPLC (MELC) separations of a range of solutes have been performed on conventional reversed-phase HPLC columns using gradient elution. This work follows previous successful separations using isocratic oil-in-water MELC [1]. It was found that by changing certain variables, peak-peak resolution, separation selectivity, efficiency and solute retention could be manipulated. The method was compatible with very low UV detection wavelengths. A robust separation method was developed for the quantitative analysis of 2 steroids in a combination-inhaled product for asthma. The method offered similar chromatography and run time when compared with conventional HPLC modes, thus demonstrating its potential for routine use. Stability-indicating methods were developed to separate synthetic and degradative impurities from the main component peaks in 4 pharmaceutical products. The methods offered quicker analysis times and equivalent selectivity to conventional HPLC modes. In developing the separations the effect on the chromatography of varying the operating parameters was studied.     

New trends in fast and high-resolution liquid chromatography: a critical comparison of existing approaches

Recent developments in chromatographic supports and instrumentation for liquid chromatography (LC) are enabling rapid and highly efficient separations. Various analytical strategies have been proposed, for example the use of silica-based monolithic supports, elevated mobile phase temperatures, and columns packed with sub-3 μm superficially porous particles (fused core) or with sub-2 μm porous particles for use in ultra-high-pressure LC (UHPLC). The purpose of this review is to describe and compare these approaches in terms of throughput and resolving power, using kinetic data gathered for compounds with molecular weights ranging between 200 and 1300 g mol⁻¹ in isocratic and gradient modes. This study demonstrates that the best analytical strategy should be selected on the basis of the analytical problem (e.g., isocratic vs. gradient, throughput vs. efficiency) and the properties of the analyte. UHPLC and fused-core technologies are quite promising for small-molecular-weight compounds, but increasing the mobile phase temperature is useful for larger molecules, for example peptides.