Potential of long capillary monolithic columns for the analysis of protein digests

The gain in separation efficiency for protein digests using long monolithic columns has been evaluated for a LC‐MS system with capillary monolithic columns of different lengths (150 and 750 mm). A mixture of BSA, α‐casein and β‐casein tryptic digests was used as a test sample. Peak capacity and productivity (peak capacity per unit time) were determined from base peak chromatograms and MS/MS data were used for protein identification by MASCOT database searching. Peak capacity and protein identification scores were higher for the long column. Analyses with similar gradient slope for the two columns produced ratios of the peak capacities that were slightly higher than the expected value of the square root of the column length ratio. Peak capacity ratios varied from 2.7 to 4.0 for four different gradient slopes, while protein identification scores were 2–4 times higher for the long column. Similar values were obtained for the productivity of both columns and the highest productivity was obtained at gradient times of 45 and 75 min for the short and long column, respectively. The use of long monolithic columns improves peptide separation and increases reliability of protein identification for complex digests, especially if longer gradients are chosen.     

Potential of poly(styrene-<Emphasis Type="Italic">co</Emphasis>-divinylbenzene) monolithic columns for the LC-MS analysis of protein digests

Two polystyrene-based capillary monolithic columns of different length (50 and 250 mm) were used to evaluate the effects of column length on gradient separation of protein digests. A tryptic digest of a 9-protein mixture was used as a test sample. Peak capacities were determined from selected extracted ion chromatograms, and tandem mass spectrometry data were used for database matching using the MASCOT search engine. Peak capacities and protein identification scores were higher for the long column with all gradients. Peak capacities appear to approach a plateau for longer gradient times; maximum peak capacity was estimated to be 294 for the short column and 370 for the long column. Analyses with similar gradient slope produced a ratio of the peak capacities of 3.36 for the long and the short column, which is slightly higher than the expected value of the square root of the column length ratio. The use of a longer monolith improves peptide separation, as reflected by higher peak capacity, and also increases protein identification, as observed from higher identification scores and a larger number of identified peptides. Attention has also been paid to the peak production rate (PPR, peak capacity per unit time). For short analysis times, the short column produces a higher PPR, while for analysis times longer than 40 min, the PPR of the 250-mm column is higher.     

Methacrylate monolithic capillary columns for gradient peptide separations

For the separation of peptides with gradient-elution liquid chromatography a poly(butyl methacrylate-co-ethylene dimethacrylate) (BMA) monolithic capillary column was prepared and tested. The conditional peak capacity was used as a metric for the performance of this column, which was compared with a capillary column packed with C18-modified silica particles. The retention of the peptides was found to be smaller on the BMA column than on the particulate C18 column. To obtain the same retention in isocratic elution an approximately 15% (v/v) lower acetonitrile concentration had to be used in the mobile phase. The retention window in gradient elution was correspondingly smaller with the BMA column. The relation between peak width and retention under gradient conditions was studied in detail. It was found that in shallow gradients, with gradient times of 30min and more, the peak widths of the least retained compounds are strongly increased with the BMA column. This was attributed to the fact that these compounds migrate and elute with an unfavorable high retention factor. More retained compounds are eluted later in the gradient, but with a lower effective retention factor. With shallow gradients the peak capacity of the BMA column ( approximately 90) was clearly lower than that of a conventional packed column ( approximately 150). On the other hand, with steep gradients, when components elute with a low effective retention factor, the performance of the BMA column is relatively good. With a gradient time of 15min similar peak widths and thus similar peak capacities ( approximately 75) were found for the packed and the monolithic column. Two strategies were investigated to obtain higher peak capacities with methacrylate monolithic columns. The use of lauryl methacrylate (LMA) instead of butyl methacrylate (BMA) gave an increase in retention and narrower peaks for early eluting peptides. The peak capacity of the LMA column was approximately 125 in a 60min gradient. Another approach was to use a longer BMA column which resulted in a peak capacity of approximately 135 could be obtained in 60min.     

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.     

Multidimensional LC x LC analysis of phenolic and flavone natural antioxidants with UV-electrochemical coulometric and MS detection

A comprehensive 2-D LC x LC system was developed for the separation of phenolic and flavone antioxidants, using a PEG-silica column in the first dimension and a C(18) column with porous-shell particles or a monolithic column in the second dimension. Combination of PEG and C18 or C8 stationary phase chemistries provide low selectivity correlations between the first dimension and the second dimension separation systems. This was evidenced by large differences in structural contributions to the retention by -COOH, -OH and other substituents on the basic phenol or flavone structure. Superficially porous columns with fused core particles or monolithic columns improve the resolution and speed of second dimension separation in comparison to a fully porous particle C(18) column. Increased peak capacity and high orthogonality in different 2-D setups was achieved by using gradients with matching profiles running in parallel in the two dimensions over the whole 2-D separation time range. Multi-dimensional set-up combining the LC x LC separation on-line with UV and multi-channel coulometric detection and off-line with MS/MS technique allowed positive peak identification. The Coularray software compensates for the effects of the baseline drift during the gradient elution and is compatible with parallel gradient comprehensive LC x LC technique. Furthermore, it provides significant improvement in the sensitivity and selectivity of detection in comparison to both UV and MS detection. The utility of these systems has been demonstrated in the analysis of beer samples.     

Implications of column peak capacity on the separation of complex peptide mixtures in single- and two-dimensional high-performance liquid chromatography

Column peak capacity was utilized as a measure of column efficiency for gradient elution conditions. Peak capacity was evaluated experimentally for reversed-phase (RP) and cation-exchange high-performance liquid chromatography (HPLC) columns, and compared to the values predicted from RP-HPLC gradient theory. The model was found to be useful for the prediction of peak capacity and productivity in single- and two-dimensional (2D) chromatography. Both theoretical prediction and experimental data suggest that the number of peaks separated in HPLC reaches an upper limit, despite using highly efficient columns or very shallow gradients. The practical peak capacity value is about several hundred for state-of-the-art RP-HPLC columns. Doubling the column length (efficiency) improves the peak capacity by only 40%, and proportionally increases both the separation time and the backpressure. Similarly, extremely shallow gradients have a positive effect on the peak capacity, but analysis becomes unacceptably long. The model predicts that a 2D-HPLC peak capacity of 15,000 can be achieved in 8 h using multiple fraction collection in the first dimension followed by fast RP-HPLC gradients employing short, but efficient columns in the second dimension.     

High peak capacity separations of peptides in reversed-phase gradient elution liquid chromatography on columns packed with porous shell particles

The performance of 5 and 15 cm long columns packed with shell particles (Halo, AMT) is compared in gradient elution separations of the tryptic digests of myoglobin and bovine serum albumin. The influences of the temperature and the mobile phase flow rate on the column efficiency for two peptides are discussed. The influences of this flow rate, of the temperature, and of the gradient slopes on the peak capacities are also considered. Peak capacities in excess of 400 were achieved in 6h with the longer column. Peak capacities of 200 could be achieved in 30 min with the shorter column.     

Methacrylate monolithic stationary phases for gradient elution separations in microfluidic devices

Methacrylate monolithic stationary phases were produced in fused-silica chips by UV initiation. Poly(butyl methacrylate-co-ethylene dimethacrylate) (BMA) and poly(lauryl methacrylate-co-ethylene dimethacrylate) (LMA) monoliths containing 30, 35 and 40% monomers were evaluated for the separation of peptides under gradient conditions. The peak capacity was used as an objective tool for the evaluation of the separation performance. LMA monoliths of the highest density gave the highest peak capacities (≈40) in gradients of 15 min and all LMA monoliths gave higher peak capacities than the BMA monoliths with the same percentage of monomers. Increasing the gradient duration to 30 min did not increase the peak capacity significantly. However, running fast (5 min) gradients provides moderate peak capacities (≈20) in a short time. Due to the system dead volume of 1 μL and the low bed volume of the chip, early eluting peptides migrated over a significant part of the column during the dwell time under isocratic conditions. It was shown that this could explain an increased band broadening on the monolithic stationary phase materials used. The effect is stronger with BMA monoliths, which partly explains the inferior performance of this material with respect to peak capacity. The configuration of the connections on the chip appeared to be critical when fast analyses were performed at pressures above 20 bar.     

How to utilize the true performance of monolithic silica columns

Ways of utilizing the true separation efficiency of monolithic silica (MS) columns were studied. The true performance of MS columns, both regular-sized (rod-type clad with PEEK resin, 4.6 mm ID, 10 cm) and capillary sized (in 100 or 200 microm ID fused silica capillary, 25-140 cm) was evaluated by calculating the contribution of extra-column effects. HETP values of 7-9 microm were observed for solutes having retention factors (kvalues) of up to 4 for rod columns and up to 15 for a capillary column. The high permeability of MS columns allowed the use of long columns, with several connected together in the case of rod columns. Narrow-bore connectors gave good results. Peak variance caused by a column connector ranges from 50 to 70% of that caused by one rod-type column for up to three connectors or four columns in 80% methanol, but the addition of a 4th or 5th connector to add a 5th and 6th column, respectively, caused a much greater increase in peak variance, especially for long-retained solutes, which is greater than the variance caused by one rod column. Rod columns seem to show slightly lower efficiency at a pressure higher than 10 MPa or so. The use of acetonitrile-water as a mobile phase better preserved the ability of individual rod columns to generate up to 100,000 theoretical plates with 14 columns connected. Methods for eliminating extra-column effects in micro-HPLC were also studied. Split injection and on-column detection resulted in optimum performance. A long MS capillary measuring 140 cm produced 160,000 theoretical plates. The column efficiency of a capillary column was not affected by the pressure, showing advantages over the rod columns that exhibited peak broadening caused by connectors and pressure.     

Performance evaluation of long monolithic silica capillary columns in gradient liquid chromatography using peptide mixtures

A systematic study is reported on the performance of long monolithic capillary columns in gradient mode. Using a commercial nano-LC system, reversed-phase peptide separations obtained through UV-detection were conducted. The chromatographic performance, in terms of conditional peak capacity and peak productivity, was investigated for different gradient times (varying between 90 and 1320min) and different column lengths (0.25, 1, 2 and 4m) all originating from a single 4m long column. Peak capacities reaching values up to n=10(3) were measured in case of the 4m long column demonstrating the high potential of these long monoliths for the analysis of complex biological mixtures, amongst others. In addition, it was found that the different column fragments displayed similar flow resistance as well as consistent chromatographic performance in accordance with chromatographic theory indicating that the chromatographic bed of the original 4m long column possessed a structural homogeneity over its entire length.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

Simple 2D-HPLC using a monolithic silica column for peptide separation

Separation of peptides by fast and simple two-dimensional (2D)-HPLC was studied using a monolithic silica column as a second-dimension (2nd-D) column. Every fraction from the first column, 5 cm long (2.1 mm ID) packed with polymer-based cation exchange beads, was subjected to separation in the 2nd-D using an octadecylsilylated (C18) monolithic sillica column (4.6 mm ID, 2.5 cm). A capillary-type monolithic silica C18column (0.1 mm ID, 10 cm) was also employed as a 2nd-D column with split flow/injection. Effluentof the first dimension (1st-D) was directly loaded into an injector loop of 2nd-D HPLC. UV and MS detection were successfully carried out at high linear velocity of mobile phase at 2nd-D using flow splitting for the 4.6 mm ID 2nd-D column, or with directconnection of the capillary column to the MS interface. Two-minute fractionation inthe 1st-D, 118-second loading, and 2-second injection by the 2nd-D injector, allowed one minute for gradient separation in the 2nd-D, resulting in a maximum peak capacity of about 700 within 40 min. The use of a capillary column in solvent consumption and better MS detectability compared to a larger-sized column. This kind of fast and simple 2D-HPLC utilizing monolithic silica columns will be useful for the separation of complex mixtures in a short time.     

Comparison of separation power of ultra performance liquid chromatography and comprehensive two-dimensional liquid chromatography in the separation of phenolic compounds in beverages

In this study, 1-D and 2-D liquid chromatographic systems, namely, conventional HPLC, UPLC, HPLC x HPLC and HPLC x UPLC systems were developed and evaluated for the separation of phenolic acids in wine and juices. In the LC x LC studies, the first dimension separation was based on RPLC and the second dimension was performed with ion-pair chromatography. Three different columns, namely two short columns packed with either 2.5 or 1.7 microm particles and a monolithic column, were tested for the fast second dimension separation. The best results were obtained when the monolithic column was applied for the second dimension separation. The peak capacities for comprehensive 2-D systems varied from 330 to 616.     

Ultra-fast tandem mass spectrometry scanning combined with monolithic column liquid chromatography increases throughput in proteomic analysis

Liquid chromatography combined with electrospray ionization mass spectrometry (LC/ESI-MS) has been used successfully for the characterization of biomolecules in proteomics in the last few years. This methodology relied largely on the use of reversed-phase chromatography, in particular C18-based resins, which are suitable for separation of peptides. Here we show that polymeric [polystyrene divinylbenzene] monolithic columns can be used to separate peptide mixtures faster and at a higher resolution. For 500 fmol bovine serum albumin, up to 68% sequence coverage and Mascot Mowse scores of >2000 were obtained using a 9 min gradient on a monolithic column coupled to an ion trap mass spectrometer with ultra-fast MS/MS scan rates. In order to achieve similar results using C18 columns, it was necessary to extend gradient times to 30 min. In addition, we demonstrate the utility of this approach for the analysis of whole Escherichia coli cell lysates by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) in combination with LC/MS/MS using 4 min gradients on monolithic columns. Our results indicate higher throughput capabilities of monolithic columns (3-fold gain in time or more) for conventional proteomics applications, such as protein identification and high sequence coverage usually required for detection of post-translational modifications (PTMs). Further optimization of sensitivity and quality of sequence information is discussed, in particular when combined with mass spectrometers that have very fast MS-MS/MS switching and scanning capabilities.     

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-resolution separations of protein isoforms with liquid chromatography time-of-flight mass spectrometry using polymer monolithic capillary columns

The separation of intact proteins, including protein isoforms arising from various amino-acid modifications, employing a poly(styrene-co-divinylbenzene) monolithic capillary column in high-performance liquid chromatography coupled on-line to a time-of-flight mass spectrometer (MS) is described. Using a 250 mm × 0.2 mm monolithic capillary column high-sensitivity separations yielding peak capacities of >600 were achieved with a 2h linear gradient and formic acid added in the mobile phase as ion-pairing agent. The combination of high-resolution chromatography with high-accuracy MS allowed to distinguish protein isoforms that differ only in their oxidation and biotinylation state allowing the separation between structural isoforms. Finally, the potential to separate proteins isoforms due to glycosylation is discussed.     

High-efficiency liquid chromatography–mass spectrometry separations with 50 mm, 250 mm,and 1 m long polymer-based monolithic capillary columns for the characterization of complex proteolytic digests

In this study, high-efficiency LC–MS/MS separations of complex proteolytic digests are demonstrated using 50 mm, 250 mm, and 1 m long poly(styrene-co-divinylbenzene) monolithic capillary columns. The chromatographic performance of the 50 and 250 mm monoliths was compared at the same gradient steepness for gradient durations between 5 and 150 min. The maximum peak capacity of 400 obtained with a 50 mm column, increased to 485 when using the 250 mm long column and scaling the gradient duration according column length. With a 5-fold increase in column length only a 20% increase in peak capacity was observed, which could be explained by the larger macropore size of the 250 mm long monolith. When taking into account the total analysis time, including the dwell time, gradient time and column equilibration time, the 50 mm long monolith yielded better peptide separations than the 250 mm long monolithic column for gradient times below 80 min (n_c = 370). For more demanding separation the 250 mm long monolith provided the highest peak production rate and consequently higher sequence coverage. For the analysis of a proteolytic digest of Escherichia coli proteins a monolithic capillary column of 1 m in length was used, yielding a peak capacity of 1038 when applying a 600 min gradient.     

Optimizing the peak capacity per unit time in one-dimensional and off-line two-dimensional liquid chromatography for the separation of complex peptide samples

To obtain the best compromise between peak capacity and analysis time in one-dimensional and two-dimensional (2D) liquid chromatography (LC), column technology and operating conditions were optimized. The effects of gradient time, flow rate, column temperature, and column length were investigated in one-dimensional reversed-phase (RP) gradient nano-LC, with the aim of maximizing the peak per unit time for peptide separations. An off-line two-dimensional LC approach was developed using a micro-fractionation option of the autosampler, which allowed automatic fractionation of peptides after a first-dimension ion-exchange separation and re-injection of the fractions onto a second-dimension RP nano-LC column. Under the applied conditions, which included a preconcentration/desalting time of 5 min, and a column equilibration time of 12.5 min, the highest peak capacity per unit time in the 2D-LC mode was obtained when applying a short (10 min) first-dimension gradient and second-dimension RP gradients of 20 min duration. For separations requiring a maximum peak capacity of 375, one-dimensional LC was found to be superior to the off-line strong cation-exchange/×/RPLC approach in terms of analysis time. Although a peak capacity of 450 could be obtained in one-dimensional LC when applying 120-min gradients on 500-mm long columns packed with 3-μm particles, for separations requiring a peak capacity higher than 375 2D-LC experiments provide a higher peak capacity per unit time. Finally, the potential of off-line 2D-LC coupled to tandem mass spectrometry detection is demonstrated with the analysis of a tryptic digest of a mixture of nine proteins and an Escherichia coli digest.     

Recent advances in micro-scale and nano-scale high-performance liquid-phase chromatography for proteome research

High-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS–MS) is regarded as one of the most powerful techniques for separation and identification of proteins. Recently, much effort has been made to improve the separation capacity, detection sensitivity, and analysis throughput of micro- and nano-HPLC, by increasing column length, reducing column internal diameter, and using integrated techniques. Development of HPLC columns has also been rapid, as a result of the use of submicrometer packing materials and monolithic columns. All these innovations result in clearly improved performance of micro- and nano-HPLC for proteome research.     

Parameters affecting the separation of intact proteins in gradient-elution reversed-phase chromatography using poly(styrene-co-divinylbenzene) monolithic capillary columns

An experimental study was performed to investigate the effects of column parameters and gradient conditions on the separation of intact proteins using styrene-based monolithic columns. The effect of flow rate on peak width was investigated at constant gradient steepness by normalizing the gradient time for the column hold-up time. When operating the column at a temperature of 60 °C a small C-term effect was observed in a flow rate range of 1–4 μL/min. However, the C-term effect on peak width is not as strong as the decrease in peak width due to increasing flow rate. The peak capacity increased according to the square root of the column length. Decreasing the macropore size of the polymer monolith while maintaining the column length constant, resulted in an increase in peak capacity. A trade-off between peak capacity and total analysis time was made for 50, 100, and 250 mm long monolithic columns and a microparticulate column packed with 5 μm porous silica particles while operating at a flow rate of 2 μL/min. The peak capacity per unit time of the 50 mm long monolithic column with small pore size was superior when the total analysis time is below 120 min, yielding a maximum peak capacity of 380. For more demanding separations the 250 mm long monolith provided the highest peak capacity in the shortest possible time frame.