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Porous silica particles were prepared by the sol-gel method with some modifications to obtain wide-pore particles. These particles were derivatized with N-phenylmaleimide-methylvinyl isocyanate (PMI) and styrene via reverse chain transfer-fragmentation (RAFT) polymerization to produce N-phenylmaleimide intercalated polyamides. Styrene (PMP) stationary phase. Narrow bore stainless steel columns (100 × 1.8 mm inner diameter) were packed with a slurry packing. The chromatographic performance of the PMP column was evaluated to separate a mixture of synthetic peptides consisting of five peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) and tryptic hydrolyzate of human serum albumin (HAS). Under optimal elution conditions, the theoretical number of plates with a mixture of peptides reached 280,000 plates/sq.m. Comparing the separation performance of the developed column with the commercial Ascentis Express RP-Amide column, it was observed that the separation efficiency of the PMP column was superior to the commercial column in terms of separation efficiency and resolution.
The biopharmaceutical industry has become an expanding global market with a significant increase in market share in recent years. With the explosive growth of the biopharmaceutical industry1,2,3 there is a great need for peptide and protein analysis. In addition to the target peptide, various impurities are formed during peptide synthesis, so chromatographic purification is required to obtain the desired purity of the peptide. 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 present in a single sample. Although mass spectrometry is an effective tool for sequencing peptides and proteins, if such samples are directly introduced into the mass spectrometer, the separation will be unsatisfactory. This problem can be solved by performing liquid chromatography (LC) before MS analysis, which will reduce the amount of analytes entering the mass spectrometer at a given time4,5,6. In addition, analytes can concentrate in a narrow region during liquid phase separation, thereby concentrating these analytes and increasing the sensitivity of MS detection. Liquid chromatography (LC) has advanced significantly over the past decade and has become a widely used method for proteomic analysis7,8,9,10.
Reverse-phase liquid chromatography (RP-LC) is widely used to purify and separate mixtures of peptides using octadecyl-modified silica (ODS) as the stationary phase11,12,13. However, due to their complex structure and amphoteric nature,14,15 RP stationary phases cannot provide a satisfactory separation of peptides and proteins. Therefore, the analysis of peptides and proteins with polar and non-polar fragments requires specially designed stationary phases to interact and retain these analytes16. Mixed chromatography, which offers multimodal interactions, can be an alternative to RP-LC for separating peptides, proteins, and other complex mixtures. Several mixed-type stationary phases were prepared and columns filled with these stationary phases were used to separate peptides and proteins17,18,19,20,21. Due to the presence of polar and non-polar groups, mixed mode stationary phases (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) are suitable for the separation of peptides and proteins22,23,24,25,26,27,28. , polar intercalated stationary phases with covalently bonded polar groups show good separation capabilities and unique selectivity for polar and non-polar analytes because separation depends on the interaction between the analyte and the stationary phase Multimodal interactions 29,30,31,32. Recently, Zhang et al. 30 obtained behenyl-terminated stationary phases of polyamines and successfully separated hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, and some other analytes. The polar embedded stationary material has both polar and non-polar groups, so it can be used to separate peptides and proteins into hydrophobic and hydrophilic parts. Polar inline columns (e.g., C18 columns with amide inline) are available under the trade name Ascentis Express RP-Amide columns, but these columns have only been used for the analysis of amine 33.
In the current study, a polar embedding stationary phase (N-phenylmaleimide, embedding polystyrene) was prepared and evaluated for peptide separation and tryptic HSA cleavage. The following strategy was used to prepare the stationary phase. Porous silica particles were prepared according to the procedures described in our previous publications, with some changes in preparation schemes 31, 34, 35, 36, 37, 38, 39. The ratios of urea, polyethylene glycol (PEG), TMOS and aqueous-acetic acid were adjusted to obtain silica particles with large pore sizes. Secondly, a new phenylmaleimide-methylvinyl isocyanate ligand was synthesized and its derivatized silica particles were used to prepare polar embedded stationary phases. The obtained stationary phase was packed into a stainless steel column (inner diameter 100 × 1.8 mm) according to an optimized packing scheme. The packing of the column is aided by mechanical vibration to ensure a uniform layer within the column. The packed column was evaluated for separation of a mixture of peptides consisting of five peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin peptide). and tryptic hydrolysates of human serum albumin (HSA). It was observed that the peptide mixture and the HSA tryptic digest separated with good resolution and efficiency. The separation efficiency of the PMP column was compared to that of the Ascentis Express RP-Amide column. It was observed that peptides and proteins have good resolution and high separation efficiency on the PMP column, and the separation efficiency of the PMP column is higher than that of the Ascentis Express RP-Amide column.
PEG (polyethylene glycol), urea, acetic acid, trimethoxyorthosilicate (TMOS), trimethylchlorosilane (TMCS), trypsin, human serum albumin (HSA), ammonium chloride, urea, hexamethylmethacryloyldisilazane (HMDS), methacryloyl chloride (MC), styrene, 4-hydroxy- TEMPO, benzoyl peroxide (BPO), acetonitrile (ACN) for HPLC, methanol, 2-propanol and acetone. Sigma-Aldrich Company (St. Louis, Missouri, 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 24 ml of TMOS was added thereto under ice-cooling. 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 decanted and the residue was dried at 70°C for 12 hours. The dried soft blocks were ground smoothly and calcined in an oven at 550°C for 12 hours. Three batches were prepared and characterized to test the reproducibility of particle sizes, pore size and surface area.
Polar group and stationary phase for polystyrene chains. The preparation procedure is described below.
N-phenylmaleimide (200 mg) and methyl vinyl isocyanate (100 mg) were dissolved in anhydrous toluene, and then 0.1 ml of 2,2′-azoisobutyronitrile (AIBN) was added to the reaction flask to obtain a copolymer of phenylmaleimide and methyl vinyl isocyanate (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 for 10 min in a 500 ml round bottom flask. PMCP (10 mg) was dissolved in toluene and added dropwise to the reaction flask via an addition funnel. The mixture was refluxed at 100°C for 8 hours, filtered, washed with acetone and dried at 60°C for 3 hours. Then, the silica particles associated with PMCP (100 g) were dissolved in toluene (200 ml), and 4-hydroxy-TEMPO (2 ml) was added thereto 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 silica particles attached to TEMPO-PMCP (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 overnight at 60°C. The general scheme of the reaction is shown in fig. one .
The samples were degassed at 393 K for 1 h until a residual pressure of less than 10–3 Torr was obtained. The amount of N2 adsorbed at relative pressure P/P0 = 0.99 was used to determine the total pore volume. The morphology of pure and ligand-bound silica particles was examined using a scanning electron microscope (Hitachi High Technologies, Tokyo, Japan). Dry samples (pure silica and ligand bound silica particles) were placed on aluminum rods using carbon tape. Gold was deposited on the sample using a Q150T sputtering device, and a 5 nm thick Au layer was deposited on the sample. This improves the efficiency of the low voltage process and provides fine cold spraying. Elemental analysis was performed using a Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental composition analyzer. A Malvern particle size analyzer (Worcestershire, UK) Mastersizer 2000 was used to obtain the particle size distribution. Uncoated silica particles and ligand-bound silica particles (5 mg each) were dispersed in 5 ml of isopropanol, sonicated for 10 minutes, agitated for 5 minutes, and placed on a Mastersizer optical bench. Thermogravimetric analysis is carried out at a rate of 5 °C per minute in the temperature range from 30 to 800 °C.
Glass fiber lined narrow bore stainless steel columns with dimensions (ID 100 × 1.8 mm) were packed by slurry filling method following the same procedure as in reference 31. Stainless steel column (glass lined, ID 100 × 1 .8 mm) and an outlet containing a 1 µm frit was connected to a slurry packaging machine (Alltech Deerfield, IL, USA). Prepare a suspension of the stationary phase by suspending 150 mg of the stationary phase in 1.2 ml of methanol and feeding it into a reservoir column. Methanol was used as the slurry solvent and control solvent. Pack the column by applying a pressure sequence of 100 MP for 10 min, 80 MP for 15 min, and 60 MP for 30 min. The packing process used two gas chromatography column vibrators (Alltech, Deerfield, IL, USA) for mechanical vibration to ensure uniform column packing. Close the slurry packer and release pressure slowly to prevent damage to the string. The column was disconnected from the slurry nozzle and another fitting was attached to the inlet and connected to the LC system to test its operation.
A custom MLC was constructed using an LC pump (10AD Shimadzu, Japan), a sampler with a 50 nL injection loop (Valco (USA) C14 W.05), a membrane degasser (Shimadzu DGU-14A), and a UV-VIS capillary window. Detector device (UV-2075) and enamelled microcolumn. Use very narrow and short connecting tubes to minimize the effect of additional column expansion. After filling the column, install a capillary (50 µm id 365) at the outlet of the 1/16″ reducing junction and install a capillary (50 µm) of the reducing junction. Data collection and chromatogram processing are performed using the Multichro 2000 software. At 254 nm, the UV absorbance of the subjects analytes were monitored at 0. Chromatographic data was analyzed using OriginPro8 (Northampton, MA).
Human serum albumin, 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 min and kept in a water bath at 37°C for 6 h, then quenched with 1 ml of 0.1% TFA. Filter the solution and store below 4°C.
Separation of a mixture of peptides and tryptic digest HSA on a PMP column was evaluated separately. Check the tryptic hydrolysis of a mixture of peptides and HSA separated by a PMP column and compare the results with an Ascentis Express RP-Amide column. The number of theoretical plates is calculated using the following equation:
SEM images of pure silica particles and ligand bound silica particles are shown in Figure 2. SEM images of pure silica particles (A, B) show a spherical shape in which the particles are elongated or have irregular symmetry compared to our previous studies. The surface of the silica particles bound by the ligand (C, D) is smoother than that of pure silica particles, which may be due to the polystyrene chains covering the surface of the silica particles.
Scanning electron micrographs of pure silica particles (A, B) and ligand bound silica particles (C, D).
The particle size distribution of pure silica particles and ligand-bound silica particles is shown in Fig. 2. 3(A). Volumetric particle size distribution curves showed that the silica particle size increased after chemical modification (Fig. 3A). The silica particle size distribution data from the current study and the previous study are compared in Table 1(A). The volumetric particle size d(0.5) of PMP was 3.36 µm, compared to a d(0.5) value of 3.05 µm in our previous study (polystyrene bonded silica particles)34. Due to the change in the ratio of PEG, urea, TMOS and acetic acid in the reaction mixture, the particle size distribution of this batch was narrower compared to our previous study. The particle size of the PMP phase is slightly larger than that of the polystyrene bound silica particle phase that we studied earlier. This means that the surface functionalization of silica particles with styrene deposited only a polystyrene layer (0.97 µm) on the silica surface, while in the PMP phase the layer thickness was 1.38 µm.
Particle size distribution (A) and pore size distribution (B) of pure silica particles and ligand bound silica particles.
The pore size, pore volume, and surface area of ​​the silica particles used in this study are shown in Table 1 (B). The PSD profiles of pure silica particles and ligand-bound silica particles are shown in Figs. 3(B). The results were comparable to our previous study34. The pore sizes of pure and ligand-bound silica particles were 310 Å and 241 Å, respectively, indicating that after chemical modification, the pore size decreased by 69 Å, as shown in Table 1 (B), and the shift curve is shown in Fig. The specific surface area of ​​silica particles in the current study 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 silica particles after chemical modification also decreased from 116 m2/g to 105 m2/g.
The results of elemental analysis of the stationary phase are presented in Table. 2. The carbon content of the current stationary phase is 6.35%, which is lower than in our previous study (silica particles associated with polystyrene, 7.93%35 and 10.21%, respectively) 42. The carbon content of the current stationary phase below, since some polar ligands such as phenylmaleimide methyl vinyl isocyanate (PCMP) and 4-hydroxy-TEMPO have been used in addition to styrene in the preparation of SP. The weight percentage of nitrogen in the current stationary phase is 2.21% compared to 0.1735 and 0.85% in previous studies42. This means that the current stationary phase has a high weight percentage of nitrogen due to the phenylmaleimide. Similarly, products (4) and (5) have a carbon content of 2.7% and 2.9%, respectively, while the final product (6) has a carbon content of 6.35%, as shown in Table 2. Thermogravimetric analysis (TGA) was used on the stationary phase of PMP to test for weight loss, 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 content (6.35%), since the ligands contain not only C , but also N, O and H.
The ligand phenylmaleimide-methylvinyl isocyanate was chosen to modify the surface of the silica particles because of its polar phenylmaleimide 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 moderate interactions with the analyte and no strong electrostatic interactions 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 the free radical polymerization. The final step of the reaction (free radical polymerization) is critical as it changes the polarity of the stationary phase. Elemental analysis was carried out to check the carbon content in these stationary phases. It has been observed that increasing the amount of styrene and the reaction time increases the carbon content of the stationary phase and vice versa. SPs prepared with different concentrations of styrene have different carbon loads. Similarly, these stationary phases were placed on stainless steel columns and their chromatographic characteristics (selectivity, resolution, N value, etc.) were checked. Based on these experiments, an optimized composition for the preparation of the PMP stationary phase was chosen to provide controlled polarity and good retention of the analyte.
The PMP column was also evaluated for the analysis of five mixtures of peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin) using the capacity of the mobile phase. 60/40 (v/v) ACN/water (0.1% TFA) at a flow rate of 80 µl/min. Under optimal elution conditions (200,000 plates/m), the number of theoretical plates (N) per column (100 × 1.8 mm) is 20,000 ± 100. The N values ​​for the three PMP columns are shown in Table 3 and the chromatograms are shown in Figure 5A . Fast analysis at high flow rate (700 µl/min) on a PMP column, five peptides eluted within one minute, excellent N value of 13,500 ± 330 per column (100 x 1.8 mm diameter), equivalent to 135,000 plates/ m (Fig. 5B). Three columns of the same size (inner diameter 100 x 1.8 mm) were filled with three different batches of PMP stationary phase to test reproducibility. Analytes were recorded for each column by separating the same test mixture on each column using optimal elution conditions, number of theoretical plates N, and retention time. The reproducibility data for the PMP columns are shown in Table 4. The reproducibility of the PMP column correlated well with very low %RSD values ​​as shown in Table 3.
Separation of peptide mixtures on a PMP column (B) and an Ascentis Express RP-Amide column (A), mobile phase 60/40 ACN/H2O (TFA 0.1%), PMP column dimensions (100 x 1.8 mm id) , analysis Elution order of compounds: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) and 5 ( leucic acid enkephalin).
A PMP column (inner diameter 100 x 1.8 mm) was evaluated for the separation of the tryptic hydrolyzate of human serum albumin by HPLC. The chromatogram in Figure 6 shows that the samples are well separated with very good resolution. HSA solutions were analyzed using a flow rate of 100 μl/min, a mobile phase of 70/30 acetonitrile/water and 0.1% TFA. Cleavage of HSA was divided into 17 peaks, as shown in the chromatogram (Fig. 6), corresponding to 17 peptides. Separation efficiencies of individual peaks from the HSA hydrolyzate were calculated and the values ​​are shown in Table 5.
HSA tryptic hydrolysates were separated on a PMP column (inner diameter 100 x 1.8 mm), flow rate (100 μl/min), mobile phase 60/40 acetonitrile/water, and 0.1% TFA.
where L is the column length, η is the viscosity of the mobile phase, ΔP is the back pressure of the column, 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, 60/40 v/v was used. ACN/water. The permeability of the PMP column (ID 100 × 1.8 mm) was similar to that of our previous Ref.34 study. The permeability of a column filled with superficially porous particles is 1.7×10 .6 µm, 2.5×10-14 m2 for 5 µm particles43. Therefore, the permeability of the PMP phase is similar to the permeability of core-shell particles with a size of 5 μm.
where Wx is the mass of the column filled with chloroform, Wy is the mass of the column filled with methanol, and ρ is the density of the solvent. Density of methanol (ρ = 0.7866) and chloroform (ρ = 1.484). The total porosity of the silica-C18 particle column (100 × 1.8 mm ID)34 and our previously studied C18-urea31 column was 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 (inner diameter 100 × 1.8 mm) is 0.60. PMP columns are less permeable than columns packed with C18 bound silica particles because in C18 type stationary phases the C18 ligands are attached to the silica particles in linear chains, while in polystyrene type stationary phases a relatively thick polymer is formed around the particles. layer A. In a typical experiment, column porosity is calculated as follows:
On fig. 7A, B show Van Deemter plots for a PMP column (id 100 x 1.8 mm) and an Ascentis Express RP-Amide column (id 100 x 1.8 mm) under the same elution conditions, 60/40 ACN/H2O and 0 .1% TFA 20 µl/min to 800 µl/min on both columns. The minimum HETP values ​​at the optimal flow rate (80 µl/min) were 2.6 µm and 3.9 µm for the PMP column and the Ascentis Express RP-Amide column, respectively. The HETP values ​​show that the separation efficiency of the PMP column (100 x 1.8 mm id) is much higher than that of the commercially available Ascentis Express RP-Amide column (100 x 1.8 mm id). The van Deemter graph in Fig. 7(A) shows that the decrease in N value is not significantly higher with increasing flow compared to our previous study. The higher separation efficiency of the PMP column (id 100 × 1.8 mm) compared to the Ascentis Express RP-Amide column is based on the improved particle shape and size and the sophisticated column packing procedure used in current work34.
(A) Van Deemter plot (HETP vs. mobile phase linear velocity) obtained on a PMP column (id 100 x 1.8 mm) in 60/40 ACN/H2O with 0.1% TFA. (B) Van Deemter plot (HETP versus mobile phase linear velocity) obtained on an Ascentis Express RP-Amide column (id 100 x 1.8 mm) in 60/40 ACN/H2O with 0.1% TFA.
A polar stationary phase of intercalated polystyrene was prepared and evaluated for the separation of a mixture of synthetic peptides and tryptic hydrolyzate of human serum albumin (HSA) in high performance liquid chromatography. The chromatographic performance of PMP columns for peptide mixtures is excellent in terms of separation efficiency and resolution. The improved separation efficiency of PMP columns is due to several reasons such as silica particle size and pore size, controlled synthesis of stationary phases, and complex column packing materials. In addition to the high separation efficiency, another advantage of this stationary phase is the low column back pressure at high flow rates. PMP columns are highly reproducible and can be used to analyze mixtures of peptides and tryptic digestion of various proteins. We intend to use this column for the separation of bioactive compounds from natural products, extracts of medicinal plants and mushrooms in liquid chromatography. In the future, PMP columns will also be evaluated for the separation of proteins and monoclonal antibodies.
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