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Porous silica particles were prepared by a sol-gel method with some modifications to obtain macroporous particles.These particles were derivatized by reversible addition fragmentation chain transfer (RAFT) polymerization with N-phenylmaleimide-methylvinylisocyanate (PMI) and styrene to prepare N-phenylmaleimide intercalation of polystyrene (PMP) stationary phase.Narrow-bore stainless steel columns (100 × 1.8 mm id) were packed by slurry packing.Evaluated PMP column separation of a peptide mixture consisting of five peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) chromatographic performance) and trypsin digestion of human serum albumin (HAS).Under optimal elution conditions, the theoretical plate count of the peptide mixture is as high as 280,000 plates/m².Comparing the separation performance of the developed column with the commercial Ascentis Express RP-Amide column, it was observed that the separation performance of the PMP column was superior to the commercial column in terms of separation efficiency and resolution.
In recent years, the biopharmaceutical industry has become an expanding global market with a substantial increase in market share.With the explosive growth of the biopharmaceutical industry1,2,3, the analysis of peptides and proteins is highly desired.In addition to the target peptide, several impurities are generated during peptide synthesis, thus requiring chromatographic purification to obtain peptides of the desired purity.The analysis and characterization of proteins in body fluids, tissues and cells is an extremely challenging task due to the large number of potentially detectable species in a single sample.Although mass spectrometry is an effective tool for peptide and protein sequencing, if such samples are injected into the mass spectrometer in one pass, the separation will not be ideal.This problem can be mitigated by implementing liquid chromatography (LC) separations prior to MS analysis, which will reduce the number of analytes entering the mass spectrometer at a given time4,5,6.In addition, during liquid phase separation, analytes can be focused in narrow regions, thereby concentrating these analytes and improving MS detection sensitivity.Liquid chromatography (LC) has advanced significantly over the past decade and has become a popular technique in proteomic analysis7,8,9,10.
Reversed-phase liquid chromatography (RP-LC) is widely used for the purification and separation of peptide mixtures using octadecyl-modified silica (ODS) as the stationary phase11,12,13.However, RP stationary phases do not provide satisfactory separation of peptides and proteins due to their complex structure and amphiphilic nature 14,15 .Therefore, specially designed stationary phases are required to analyze peptides and proteins with polar and non-polar moieties to interact with and retain these analytes16.Mixed-mode chromatography, which provides multimodal interactions, can be an alternative to RP-LC for the separation of peptides, proteins, and other complex mixtures.Several mixed-mode stationary phases have been prepared, and columns packed with these phases have been used for peptide and protein separations17,18,19,20,21.Mixed-mode stationary phases (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) are suitable for peptide and protein separations due to the presence of both polar and non-polar groups22,23,24,25,26,27,28 .Similarly, polar intercalating stationary phases with covalently bonded polar groups show good separation power and unique selectivity for polar and non-polar analytes, as separation depends on the interaction between analyte and stationary phase. Multimodal interactions 29, 30, 31, 32.Recently, Zhang et al. 30 prepared a dodecyl-terminated polyamine stationary phase and successfully separated hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, and several other analytes.The polar intercalator has both polar and non-polar groups, so it can be used to separate peptides and proteins that have both hydrophobic and hydrophilic moieties.Polar-embedded columns (eg, amide-embedded C18 columns) are commercially available under the trade name Ascentis Express RP-Amide columns, but these columns are used for the analysis of amine 33 only.
In the current study, a polar-embedded stationary phase (N-phenylmaleimide-embedded polystyrene) was prepared and evaluated for separation of peptides and trypsin digests of HSA.The stationary phase was prepared using the following strategy.Porous silica particles were prepared according to the procedure given in our previous publication with some modifications to the preparation protocol.The ratio of urea, polyethylene glycol (PEG), TMOS, water acetic acid was adjusted to prepare silica particles with large pore size.Second, a new ligand, phenylmaleimide-methyl vinyl isocyanate, was synthesized and used to derivatize silica particles to prepare a polar embedded stationary phase.The resulting stationary phase was packed into a stainless steel column (100 × 1.8 mm id) using the optimized packing scheme.The column packing is assisted with mechanical vibration to ensure a homogeneous bed is formed within the column.Evaluate packed column separation of peptide mixtures consisting of five peptides; (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) and Trypsin digest of human serum albumin (HAS).The peptide mixture and trypsin digest of HSA were observed to separate with good resolution and efficiency.The separation performance of the PMP column was compared with that of the Ascentis Express RP-Amide column.Both peptides and proteins were observed to be well resolved and efficient on the PMP column, which was more efficient than the Ascentis Express RP-Amide column.
PEG (Polyethylene Glycol), Urea, Acetic Acid, Trimethoxy Orthosilicate (TMOS), Trimethyl Chlorosilane (TMCS), Trypsin, Human Serum Albumin (HSA), Ammonium Chloride, Urea, Hexane Methyldisilazane (HMDS), Methacryloyl Chloride (MC), Styrene, 4-Hydroxy-TEMPO, Benzoyl Peroxide (BPO), HPLC Grade Acetonitrile (ACN), Methanol, 2-Propanol, and Acetone Purchased from Sigma-Aldrich (St. Louis, MO, USA).
A mixture of urea (8 g), polyethylene glycol (8 g), and 8 mL of 0.01 N acetic acid was stirred for 10 minutes, and then 24 mL of TMOS was added thereto under ice-cold conditions.The reaction mixture was heated at 40°C for 6 hours and then at 120°C for 8 hours in a stainless steel autoclave.The water was poured off and the residual material was dried at 70°C for 12 hours.The dried soft mass was smooth ground in an oven and calcined at 550°C for 12 hours.Three batches were prepared and characterized to examine reproducibility in particle size, pore size and surface area.
By surface modification of silica particles with pre-synthesized ligand phenylmaleimide-methylvinylisocyanate (PCMP) followed by radial polymerization with styrene, a polar group-containing compound was prepared. Stationary phase for aggregates and polystyrene chains.The preparation process is described below.
N-phenylmaleimide (200 mg) and methyl vinyl isocyanate (100 mg) were dissolved in dry toluene, and 0.1 mL of 2,2′-azoisobutyronitrile ( AIBN) was added to the reaction flask to prepare phenylmaleimide-methyl vinyl isocyanate copolymer (PMCP).. The mixture was heated at 60°C for 3 hours, filtered and dried in an oven at 40°C for 3 hours.
Dried silica particles (2 g) were dispersed in dry toluene (100 mL), stirred and sonicated in a 500 mL round bottom flask for 10 min.PMCP (10 mg) was dissolved in toluene and added dropwise to the reaction flask via a dropping funnel.The mixture was refluxed at 100°C for 8 hours, filtered and washed with acetone and dried at 60°C for 3 hours.Then, PMCP-bonded silica particles (100 g) were dissolved in toluene (200 ml) and 4-hydroxy-TEMPO (2 mL) was added in the presence of 100 µL of dibutyltin dilaurate as a catalyst.The mixture was stirred at 50°C for 8 hours, filtered and dried at 50°C for 3 hours.
Styrene (1 mL), benzoyl peroxide BPO (0.5 mL), and TEMPO-PMCP-attached silica particles (1.5 g) were dispersed in toluene and purged with nitrogen.The polymerization of styrene was carried out at 100°C for 12 hours.The resulting product was washed with methanol and dried at 60°C overnight.The overall reaction scheme is shown in Figure 1.
The samples were degassed at 393 K for 1 hour to obtain a residual pressure of less than 10-3 Torr.The amount of N2 adsorbed at a relative pressure of P/P0 = 0.99 was used to determine the total pore volume.The morphology of bare and ligand-bonded silica particles was examined with scanning electron microscopy (Hitachi High Technologies, Tokyo, Japan).Dried samples (bare silica and ligand-bonded silica particles) were placed on an aluminum column using adhesive carbon tape.Gold was plated on the samples using a Q150T sputter coater, and a 5 nm Au layer was deposited on the samples.This improves process efficiency using low voltages and provides fine grain, cold sputtering.A Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental analyzer was used for elemental analysis.A Malvern (Worcestershire, UK) Mastersizer 2000 particle size analyzer was used to obtain the particle size distribution.Naked silica particles and ligand-bonded silica particles (5 mg each) were dispersed in 5 mL of isopropanol, sonicated for 10 min, vortexed for 5 min, and placed on the optical bench of the Mastersizer.Thermogravimetric analysis was performed at a rate of 5 °C per minute over a temperature range of 30 to 800 °C.
Glass-lined stainless steel narrow-bore columns of dimensions (100 × 1.8 mm id) were packed using the slurry packing method, applying the same procedure used in Ref. 31.A stainless steel column (glass-lined, 100 × 1.8 mm id) with an outlet fitting containing a 1 µm frit was connected to a slurry packer (Alltech Deerfield, IL, USA).Prepare a stationary phase slurry by suspending 150 mg of stationary phase in 1.2 mL of methanol and send it to the storage column.Methanol was used as the slurry solvent as well as the propelling solvent.Fill the column sequentially by applying pressures of 100 MP for 10 minutes, 80 MP for 15 minutes, and 60 MP for 30 minutes.During packing, mechanical vibration was applied with two GC column shakers (Alltech, Deerfield, IL, USA) to ensure uniform packing of the column.Close the slurry packer and release the pressure slowly to prevent any damage within the column.Disconnect the column from the slurry packing unit and connect another fitting to the inlet and to the LC system to check its performance.
An LC pump (10AD Shimadzu, Japan), injector (Valco (USA) C14 W.05) with 50nL injection loop, membrane degasser (Shimadzu DGU-14A), UV-VIS capillary window was constructed Special µLC device detector (UV-2075) and glass-lined microcolumns.Use very narrow and short connecting tubing to minimize the effect of extra column band broadening.After packaging, capillaries (50 μm id 365 and reducing union capillaries (50 μm) were installed at the 1/16″ outlet of the reducing union. Data collection and chromatographic processing were done using Multichro 2000 software. Monitoring at 254 nm Analytes were tested for UV absorbance. Chromatographic data were analyzed by OriginPro8 (Northampton, MA).
Albumin from human serum, lyophilized powder, ≥ 96% (agarose gel electrophoresis) 3 mg mixed with trypsin (1.5 mg), 4.0 M urea (1 mL), and 0.2 M ammonium bicarbonate (1 mL).The solution was stirred for 10 minutes and kept in a water bath at 37°C for 6 hours, then quenched with 1 mL of 0.1% TFA.Filter the solution and store below 4 °C.
Separation of peptide mixtures and HSA trypsin digests were evaluated separately on PMP columns.Check the separation of the peptide mixture and trypsin digest of HSA by the PMP column and compare the results to the Ascentis Express RP-Amide column.The theoretical plate number is calculated as follows:
SEM images of bare silica particles and ligand-bonded silica particles are shown in FIG. 2 .SEM images of bare silica particles (A, B) show that, in contrast to our previous studies, these particles are spherical in which the particles are elongated or have irregular symmetry.The surface of the ligand-bonded silica particles (C, D) is smoother than that of the bare silica particles, which may be due to the coating of polystyrene chains on the surface of the silica particles.
Scanning electron microscope images of bare silica particles (A, B) and ligand-bonded silica particles (C, D).
The particle size distributions of bare silica particles and ligand-bonded silica particles are shown in Figure 3(A).Volume-based particle size distribution curves showed that the size of the silica particles increased after chemical modification (Fig. 3A).The particle size distribution data of the silica particles from the current study and the previous study are compared in Table 1(A).The volume-based particle size, d(0.5), of PMP is 3.36 μm, compared to our previous study with a d(0.5) value of 3.05 μm (polystyrene-bound silica particles)34.This batch had a narrower particle size distribution compared to our previous study due to the varying ratios of PEG, urea, TMOS, and acetic acid in the reaction mixture.The particle size of the PMP phase is slightly larger than that of the polystyrene-bound silica particle phase we previously studied.This means that surface functionalization of silica particles with styrene only deposited a polystyrene layer (0.97 µm) on the silica surface, whereas in the PMP phase the layer thickness was 1.38 µm.
Particle size distribution (A) and pore size distribution (B) of bare silica particles and ligand-bound silica particles.
The pore size, pore volume and surface area of ​​the silica particles of the current study are given in Table 1(B).The PSD profiles of bare silica particles and ligand-bonded silica particles are shown in Figure 3(B).The results are comparable to our previous study.The pore sizes of the bare and ligand-bound silica particles are 310 and 241, respectively, which indicates that the pore size decreases by 69 after chemical modification, as shown in Table 1(B), and the change of the curve is shown in Fig. 3(B) .Similarly, the pore volume of the silica particles decreased from 0.67 to 0.58 cm3/g after chemical modification.The specific surface area of ​​the currently studied silica particles is 116 m2/g, which is comparable to our previous study (124 m2/g).As shown in Table 1(B), the surface area (m2/g) of the silica particles also decreased from 116 m2/g to 105 m2/g after chemical modification.
The results of elemental analysis of the stationary phase are shown in Table 2.The carbon loading of the current stationary phase is 6.35%, which is lower than the carbon loading of our previous study (polystyrene bonded silica particles, 7.93%35 and 10.21%, respectively) 42. The carbon loading of the current stationary phase is low, Because in the preparation of the current SP, in addition to styrene, some polar ligands such as phenylmaleimide-methylvinylisocyanate (PCMP) and 4-hydroxy-TEMPO were used.The nitrogen weight percent of the current stationary phase is 2.21%, compared to 0.1735 and 0.85% by weight of nitrogen in previous studies, respectively.This means that the wt % of nitrogen is higher in the current stationary phase due to phenylmaleimide.Similarly, the carbon loadings of products (4) and (5) were 2.7% and 2.9%, respectively, while the carbon loading of the final product (6) was 6.35%, as shown in Table 2.The weight loss was checked with PMP stationary phase, and the TGA curve is shown in Figure 4.The TGA curve shows a weight loss of 8.6%, which is in good agreement with the carbon loading (6.35%) because the ligands contain not only C but also N, O, and H.
The phenylmaleimide-methylvinylisocyanate ligand was chosen for surface modification of silica particles because it has polar phenylmaleimide groups and vinylisocyanate groups.Vinyl isocyanate groups can further react with styrene by living radical polymerization.The second reason is to insert a group that has a moderate interaction with the analyte and no strong electrostatic interaction between the analyte and the stationary phase, since the phenylmaleimide moiety has no virtual charge at normal pH.The polarity of the stationary phase can be controlled by the optimal amount of styrene and the reaction time of free radical polymerization.The last step of the reaction (free-radical polymerization) is critical and can change the polarity of the stationary phase.Elemental analysis was performed to check the carbon loading of these stationary phases.It was observed that increasing the amount of styrene and the reaction time increased the carbon loading of the stationary phase and vice versa.SPs prepared with different concentrations of styrene have different carbon loadings.Again, load these stationary phases into stainless steel columns and check their chromatographic performance (selectivity, resolution, N value, etc.).Based on these experiments, an optimized formulation was selected to prepare the PMP stationary phase to ensure controlled polarity and good analyte retention.
Five peptide mixtures (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) were also evaluated using a PMP column using a mobile phase; 60/40 (v/v) acetonitrile/water (0.1% TFA) at a flow rate of 80 μL/min.Under optimal elution conditions, the theoretical plate number (N) per column (100 × 1.8 mm id) is 20,000 ± 100 (200,000 plates/m²).Table 3 gives the N values ​​for the three PMP columns and the chromatograms are shown in Figure 5A.Fast analysis on a PMP column at high flow rate (700 μL/min), five peptides were eluted within one minute, N values ​​were very good, 13,500 ± 330 per column (100 × 1.8 mm id), Corresponds to 135,000 plates/m (Figure 5B).Three identically sized columns (100 × 1.8 mm id) were packed with three different lots of PMP stationary phase to check reproducibility.The analyte concentration for each column was recorded using the optimal elution conditions and the number of theoretical plates N and retention time to separate the same test mixture on each column.The reproducibility data for the PMP columns are shown in Table 4.The reproducibility of the PMP column correlates well with very low %RSD values, as shown in Table 3.
Separation of peptide mixture on PMP column (B) and Ascentis Express RP-Amide column (A); mobile phase 60/40 ACN/H2O (TFA 0.1%), PMP column dimensions (100 × 1.8 mm id); analytical The elution order of the compounds: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) and 5 (leucine) acid enkephalin)).
A PMP column (100 × 1.8 mm id) was evaluated for separation of tryptic digests of human serum albumin in high performance liquid chromatography.The chromatogram in Figure 6 shows that the sample is well separated and the resolution is very good.HSA digests were analyzed using a flow rate of 100 µL/min, mobile phase 70/30 acetonitrile/water and 0.1% TFA.As shown in the chromatogram (Figure 6), the HSA digestion has been split into 17 peaks corresponding to 17 peptides.The separation efficiency of each peak in the HSA digest was calculated and the values ​​are given in Table 5.
A tryptic digest of HSA (100 × 1.8 mm id) was separated on a PMP column; flow rate (100 µL/min), mobile phase 60/40 acetonitrile/water with 0.1% TFA.
where L is the column length, η is the viscosity of the mobile phase, ΔP is the column back pressure, and u is the linear velocity of the mobile phase.The permeability of the PMP column was 2.5 × 10-14 m2, the flow rate was 25 μL/min, and 60/40 v/v ACN/water was used.The permeability of the PMP column (100 × 1.8 mm id) was similar to that of our previous study Ref.34.The permeability of the column packed with superficially porous particles is: 1.7 × 10-15 for 1.3 μm particles, 3.1 × 10-15 for 1.7 μm particles, 5.2 × 10-15 and 2.5 × 10-14 m2 for 2.6 μm particles For 5 μm particles 43.Therefore, the permeability of the PMP phase is similar to that of 5 μm core-shell particles.
where Wx is the weight of the column packed with chloroform, Wy is the weight of the column packed with methanol, and ρ is the density of the solvent.Densities of methanol (ρ = 0.7866) and chloroform (ρ = 1.484).The total porosity of SILICA PARTICLES-C18 columns (100 × 1.8 mm id) 34 and C18-Urea columns 31 that we previously studied were 0.63 and 0.55, respectively.This means that the presence of urea ligands reduces the permeability of the stationary phase.On the other hand, the total porosity of the PMP column (100 × 1.8 mm id) is 0.60.The permeability of PMP columns is lower than that of columns packed with C18-bonded silica particles because in C18-type stationary phases the C18 ligands are attached to the silica particles as linear chains, while in polystyrene-type stationary phases, the A relatively thick polymer layer is formed around it.In a typical experiment, the column porosity is calculated as:
Figure 7A,B show the PMP column (100 × 1.8 mm id) and Ascentis Express RP-Amide column (100 × 1.8 mm id) using the same elution conditions (ie, 60/40 ACN/H2O and 0.1% TFA). ) of the van Deemter plot. Selected peptide mixtures (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) were prepared in 20 µL/ The minimum flow rate for both columns is 800 µL/min.The minimum HETP values ​​at the optimum flow rate (80 µL/min) for the PMP column and the Ascentis Express RP-Amide column were 2.6 µm and 3.9 µm, respectively.The HETP values ​​indicate that the separation efficiency of the PMP column (100 × 1.8 mm id) is much better than the commercially available Ascentis Express RP-Amide column (100 × 1.8 mm id).The van Deemter plot in Fig. 7(A) shows that the decrease in N value with increasing flow is not significant compared to our previous study.The higher separation efficiency of the PMP column (100 × 1.8 mm id) compared to the Ascentis Express RP-Amide column is based on improvements in particle shape, size, and complex column packing procedures used in the current work34.
(A) van Deemter plot (HETP versus mobile phase linear velocity) obtained using a PMP column (100 × 1.8 mm id) in 60/40 ACN/H2O with 0.1% TFA.(B) van Deemter plot (HETP versus mobile phase linear velocity) obtained using an Ascentis Express RP-Amide column (100 × 1.8 mm id) in 60/40 ACN/H2O with 0.1% TFA.
A polar-embedded polystyrene stationary phase was prepared and evaluated for separation of synthetic peptide mixtures and trypsin digests of human serum albumin (HAS) in high performance liquid chromatography.The chromatographic performance of PMP columns for peptide mixtures is excellent in separation efficiency and resolution.The improved separation performance of PMP columns is due to a variety of reasons, such as the particle size and pore size of the silica particles, controlled synthesis of the stationary phase, and complex column packing.In addition to high separation efficiency, low column back pressure at high flow rates is another advantage of this stationary phase.PMP columns exhibit good reproducibility and can be used for the analysis of peptide mixtures and trypsin digestion of various proteins.We intend to use this column for the separation of natural products, bioactive compounds from medicinal plants and fungal extracts in liquid chromatography.In the future, PMP columns will also be evaluated for the separation of proteins and monoclonal antibodies.
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