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Proteomix
Proteomix ion-exchange columns are specially designed for high resolution, high efficiency and high recovery separations of proteins, oligonucleotides, carbohydrates, and peptides. The packing support is composed of a rigid, spherical, highly cross-linked poly(styrene divinylbenzene) (PS/DVB) bead. Both porous and non-porous PS/DVB beads are provided. The porous PS/DVB resins have particle sizes of 5 and 10 mm with the pore size of 500 Å. The non-porous resins have particle sizes of 1.7, 3, 5 and 10 mm. The PS/DVB resin surface is grafted with a highly hydrophilic, neutral polymer layer with the thickness in the range of nanometer. The hydrophobic PS/DVB resin surface is totally covered by such a hydrophilic coating that eliminates non-specific bindings with biological analytes, leading to high efficiency and high recovery separations for biomolecules. On the top of the hydrophilic layer, a layer of ion-exchange functional groups is attached via chemical bonding. A proprietary chemistry was developed to synthesize a densely packed, uniform ion-exchange layer.
 The chemical structure of Proteomix ion-exchange phases is composed of a rigid PS/DVB core, a densely packed, nanometer thick, hydrophilic coating, and a uniform ion-exchange layer, as shown in Figure 1.
As shown in Figure 2, Proteomix ion-exchange phases are composed of SCX, WCX, SAX, and WAX, which are strong cation exchanger with sulfonate functional groups, weak cation exchanger with carboxylate functional groups, strong anion exchanger with quaternary ammonium functional groups, weak anion exchanger with tertiary amine functional, respectively. All ion-exchange functional groups are chemically bonded to the top of the hydrophilic coating for both porous and non-porous PS/DVB resins.
High Capacity of Proteomix Non-Porous Resins
It is well known that the non-porous resin maximizes the mass transfer and minimizes the lateral diffusion, resulting in high speed and high efficiency separations. However, due to the low surface area, the separation capacity is low. The low capacity issue creates a great challenge to the non-porous resins for many applications. The Proteomix non-porous ion-exchange resins developed by Sepax proprietary technologies have a breakthrough on the improvement of their capacities. This new technology enables the capacity of the non-porous resins increased to the level comparable to that of the porous resins. Figure 3 shows examples of dynamic binding capacities of the 3 mm non-porous Proteomix SAX and WAX resins: 35 and 26 mg/mL, respectively. Figure 4 shows an example of the dynamic binding capacity of 3 mm Proteomix SCX non-porous resin. Its dynamic binding capacity reaches as high as 53.5 mg/mL.
High dynamic binding capacity leads to high sample loading without deterioration of the separation efficiency and the resolution. Figure 5 shows the elution of cytochrome C with the loading up to 200 mg for a 1.7 mm Proteomix SCX non-porous column without any deterioration of the separation.
High Separation Efficiency, Resolution and Selectivity
Proteomix SCX-NP, WCX-NP, SAX-NP, and WAX-NP resins have three unique features. First, their nanometer thick hydrophilic layer completely eliminates the non-specific interactions with the biological analytes. Second, non-porous beads minimize the biological analytes’ lateral diffusion and suppress their diffusion into the chromatographic bed. Third, a uniform layer of ion-exchange functional groups synthesized by Sepax’s proprietary technology greatly improved their ion-exchange capacities. Such innovatively designed Proteomix SCX-NP, WCX-NP, SAX-NP, and WAX-NP phases result in the highest efficiency separations for proteins, oligonucleotides, carbohydrates, and peptides. Figure 6 is a typical test chromatogram for separation of three proteins: ribonuclease A, cytochrome C, and lysozyme by a 4.6 × 50 mm, Proteomix SCX-NP column (3 µm). The efficiency of lysozyme reaches 100,000 of plates with 5 cm long column. Such a high efficiency separation is unprecedented. The uniqueness of non-porous Proteomix SCX, WCX, SAX, and WAX phases offers highest resolution and selectivity for protein separations. Figure 7 shows an elution profile of lysozyme by a Proteomix SCX-NP3 phase (3 mm). Six peaks are well resolved with a short column (4.6 × 50 mm). Such a high selectivity and resolving power is unmatched with any other SCX phases.
Figure 8 is a typical test chromatogram for a 5 mm, Proteomix SAX-NP column for separation of a mixture of ovalbumin and BSA. High resolution and high selectivity of Proteomix SAX-NP phase can well separate the impurities contained in ovalbumin sample, as well as the BSA dimer from BSA.
Particle Size Impact on the Separation
The smaller the particle size, the better separation efficiency and higher separation speed. Proteomix ion-exchange resins have a wide range of the particle size selection from 1.0 to 10 mm for analytical and semi-preparative separation applications. Figure 9 is the separation of three proteins by four weak cation exchange resins with the particle size of 1.7, 3, 5, and 10 mm, separately. It is clearly seen that the smaller particle generates higher efficiency and resolution. One of the impurities in lysozyme labeled by Peak N is barely separated with10 mm WCX resin. For 5 mm WCX resin, it is almost a baseline separation. When the particle size is decreased to 3 mm, it becomes a baseline separation. With the particle size further decreased to 1.7 mm, the impurity is well separated. High Lot-to-Lot ReproducibilityWith well-controlled resin production and the surface chemistry, the manufacturing of the Proteomix ion-exchangers is highly reproducible. The typical variation of the retention time is less than 6% from batch to batch. One example is shown in Figure 11 for the production of three lots of 3 mm Proteomix SAX-NP resins.
 
Proteomix SCX-POR, WCX-POR, SAX-POR, and WAX-POR resins are based on porous PS/DVB resins that are coated with a proprietary hydrophilic layer and functionalized with a uniform ion-exchange layer. These phases have three unique features. First, the nanometer thick hydrophilic layer completely eliminates the non-specific interactions with biological analytes. Second, Sepax’s proprietary technology synthesizes a uniform and densely packed layer of ion-exchange functional groups. Third, porous beads provide high surface area, leading to high separation capacity. Such uniquely designed Proteomix SCX-POR, WCX-POR, SAX-POR, and WAX-POR phases offer high capacity and reasonably high resolution separations for proteins, oligonucleotides and peptides. Due to the porosity, Proteomix porous resins have less efficiency and lower resolution than Proteomix non-porous resins. Proteomix SCX-POR, WCX-POR, SAX-POR, and WAX-POR phases are well suited for semi-preparative and preparative separation and purification of biomolecules. Porous Proteomix SCX, WCX, SAX, and WAX columns are manufactured and tested under the strictest specifications, resulting in high column-to-column and lot-to-lot reproducibility.
The column dimensions of the Proteomix SCX, WCX, SAX, and WAX products are 0.75, 2.1, 3.0, 4.6, 7.8, 10, and 21.2 mm I.D., and 2, 3, 5, 10, 15, 25, and 30 cm length. Sepax also offers custom-made columns.
- Uniform polymer beads as the packing support
- Proprietary surface chemistry specially designed for elimination of non-specific bindings
- Unprecedented separation efficiency, selectivity and resolving power
- Complete selection for analytical, semi-preparative and preparative separations
- Wide particle size selection
- High stability
- High recovery
- Well suited for UPLC system
Proteomix ion-exchange phases have wide applications for separation, identification and purification of proteins, protein variants, peptide fragments, phosphorylated, sialylated, and other derivatized proteins. They are excellent for monitoring enzymatic reactions and protein-protein interactions. With extremely high separation efficiency, Proteomix phases are well designed for proteomics applications and 2D or multi-dimensional separations. One example is the high resolution of cell lysates by Proteomix SAX-NP phases. Proteomix SAX and WAX phases are suitable for separation of oligonucleotides. Porous Proteomix ion-exchange phases provide high resolution and high capacity separation for surface charged nanoparticles, nanorods and nanotubes.
Separation of Horse Serum, a Protein Mixture
Figure 12 shows the separation of a horse serum by using Proteomix SAX-NP5 and a commercial porous SAX column. Non-porous Proteomix SAX-NP5 has much high resolution than the porous SAX column.
Separation of Carbohydrates and Glycans
Figure 13 shows the separation of glucose oligomers and corn starch hydrolysate. ELSD is utilized to detect carbohydrate molecules. For ELSD detection, bleeding is a big problem for most columns, especially silica based amino columns. However, Proteomix SAX columns not only solved the bleeding problem, but also achieved excellent separation efficiency and resolution. Figure 14 is another example of high resolution separation of glycans and their isomers by non-porous Proteomix SAX column.
Separation of Peptides in Volatile Buffer
The peptides with different hydrophobicity can be resolved by reversed phases, such as C18. However, peptides with different charges may not be well separated on reversed phase mode if their hydrophobic properties are close. As an alternative method, ion exchange chromatography is recommended. With the net charge from +1 to +4, four peptides (C1, C2, C3 and C4 with their sequences listed in Figure 15) were well separated by Proteomix SCX-NP phase in a volatile buffer of ammonium acetate and acetonitrile, which makes it compatible for LC/MS application.
High Protein Loading Separation
Ribonuclease A was separated by a non-porous SCX column. When the injection amount of Ribonuclease A was increased from 50 µg to 5 mg, the separation resolution had negligible change and each of the impurities was well separated, as shown in Figure 16. The combination of high loading capacity and high resolution separation enables non-porous Proteomix ion-exchange columns very well suited for both analytical and preparative separations of biological molecules.
Separation of Oligonucleotides
High resolution separation is critical for DNA analysis and purification. The non-porous Proteomix SAX resins offer high separation efficiency with high capacity. Figure 17 shows the separation of a 50-mer oligonucleotide synthesized by ABI synthesizer. The largest peak is the modified 50-mer oligo and the others are failures or the impurities. As a comparison, the same sample was separated with a commercial porous SAX column. The results showed similar retention time for the 50-mer oligonucleotide from both non-porous and porous SAX columns, which indicated that their capacities are close to each other. However, the non-porous SAX generated much higher efficiency and resolution. Figure 18 is another example of high resolution separation of oligonucleotides of Mw 12196 from its degraded fragments. At least 35 species of those degraded oligo fragments were well resolved within 22 minutes.
Separation and Analysis of Cell Lysates
The key issue for proteome studies is to separate and identify a large number of biological species in a cell, such as proteins, nucleotides, peptides and others. The demand for separation is unprecedented. With the uniqueness of high resolution and high capacity, the non-porous Proteomix ion-exchange resins are very much suitable for separating cell lysates. Figure 19(a) showed the separation profiles of E. coli lysate with 3, 5, and 10 µm non-porous Proteomix SAX particles. The minimum number of resolved peaks increased from 40 to 60 to 75 when the particle size decreased from 10 to 5 to 3 µm. To better view the separation performance, the elution profiles in the range of 10-22 minutes were shown in Figure 19(b). At least 45, 38, 27 peaks were resolved at the retention time of 10-22 minutes for 3, 5, and 10 µm SAX columns, respectively. Figure 20 showed various sample loading for a 3 µm, 4.6x50 mm non-porous SAX column. When the amount of E. coli lysate increased from 25 µg to 50 µg to 125 µg, the separation efficiency and resolution remained consistent.
Proteomix
Proteomix ion-exchange columns are specially designed for high resolution, high efficiency and high recovery separations of proteins, oligonucleotides, carbohydrates, and peptides. The packing support is composed of a rigid, spherical, highly cross-linked poly(styrene divinylbenzene) (PS/DVB) bead. Both porous and non-porous PS/DVB beads are provided. The porous PS/DVB resins have particle sizes of 5 and 10 mm with the pore size of 500 Å. The non-porous resins have particle sizes of 1.7, 3, 5 and 10 mm. The PS/DVB resin surface is grafted with a highly hydrophilic, neutral polymer layer with the thickness in the range of nanometer. The hydrophobic PS/DVB resin surface is totally covered by such a hydrophilic coating that eliminates non-specific bindings with biological analytes, leading to high efficiency and high recovery separations for biomolecules. On the top of the hydrophilic layer, a layer of ion-exchange functional groups is attached via chemical bonding. A proprietary chemistry was developed to synthesize a densely packed, uniform ion-exchange layer.
The chemical structure of Proteomix ion-exchange phases is composed of a rigid PS/DVB core, a densely packed, nanometer thick, hydrophilic coating, and a uniform ion-exchange layer, as shown in Figure 1.
As shown in Figure 2, Proteomix ion-exchange phases are composed of SCX, WCX, SAX, and WAX, which are strong cation exchanger with sulfonate functional groups, weak cation exchanger with carboxylate functional groups, strong anion exchanger with quaternary ammonium functional groups, weak anion exchanger with tertiary amine functional, respectively. All ion-exchange functional groups are chemically bonded to the top of the hydrophilic coating for both porous and non-porous PS/DVB resins.
High Capacity of Proteomix Non-Porous Resins
It is well known that the non-porous resin maximizes the mass transfer and minimizes the lateral diffusion, resulting in high speed and high efficiency separations. However, due to the low surface area, the separation capacity is low. The low capacity issue creates a great challenge to the non-porous resins for many applications. The Proteomix non-porous ion-exchange resins developed by Sepax proprietary technologies have a breakthrough on the improvement of their capacities. This new technology enables the capacity of the non-porous resins increased to the level comparable to that of the porous resins. Figure 3 shows examples of dynamic binding capacities of the 3 mm non-porous Proteomix SAX and WAX resins: 35 and 26 mg/mL, respectively. Figure 4 shows an example of the dynamic binding capacity of 3 mm Proteomix SCX non-porous resin. Its dynamic binding capacity reaches as high as 53.5 mg/mL.
High dynamic binding capacity leads to high sample loading without deterioration of the separation efficiency and the resolution. Figure 5 shows the elution of cytochrome C with the loading up to 200 mg for a 1.7 mm Proteomix SCX non-porous column without any deterioration of the separation.
High Separation Efficiency, Resolution and Selectivity
Proteomix SCX-NP, WCX-NP, SAX-NP, and WAX-NP resins have three unique features. First, their nanometer thick hydrophilic layer completely eliminates the non-specific interactions with the biological analytes. Second, non-porous beads minimize the biological analytes’ lateral diffusion and suppress their diffusion into the chromatographic bed. Third, a uniform layer of ion-exchange functional groups synthesized by Sepax’s proprietary technology greatly improved their ion-exchange capacities. Such innovatively designed Proteomix SCX-NP, WCX-NP, SAX-NP, and WAX-NP phases result in the highest efficiency separations for proteins, oligonucleotides, carbohydrates, and peptides. Figure 6 is a typical test chromatogram for separation of three proteins: ribonuclease A, cytochrome C, and lysozyme by a 4.6 × 50 mm, Proteomix SCX-NP column (3 µm). The efficiency of lysozyme reaches 100,000 of plates with 5 cm long column. Such a high efficiency separation is unprecedented. The uniqueness of non-porous Proteomix SCX, WCX, SAX, and WAX phases offers highest resolution and selectivity for protein separations. Figure 7 shows an elution profile of lysozyme by a Proteomix SCX-NP3 phase (3 mm). Six peaks are well resolved with a short column (4.6 × 50 mm). Such a high selectivity and resolving power is unmatched with any other SCX phases.
Figure 8 is a typical test chromatogram for a 5 mm, Proteomix SAX-NP column for separation of a mixture of ovalbumin and BSA. High resolution and high selectivity of Proteomix SAX-NP phase can well separate the impurities contained in ovalbumin sample, as well as the BSA dimer from BSA.
Particle Size Impact on the Separation
The smaller the particle size, the better separation efficiency and higher separation speed. Proteomix ion-exchange resins have a wide range of the particle size selection from 1.0 to 10 mm for analytical and semi-preparative separation applications. Figure 9 is the separation of three proteins by four weak cation exchange resins with the particle size of 1.7, 3, 5, and 10 mm, separately. It is clearly seen that the smaller particle generates higher efficiency and resolution. One of the impurities in lysozyme labeled by Peak N is barely separated with10 mm WCX resin. For 5 mm WCX resin, it is almost a baseline separation. When the particle size is decreased to 3 mm, it becomes a baseline separation. With the particle size further decreased to 1.7 mm, the impurity is well separated. High Lot-to-Lot ReproducibilityWith well-controlled resin production and the surface chemistry, the manufacturing of the Proteomix ion-exchangers is highly reproducible. The typical variation of the retention time is less than 6% from batch to batch. One example is shown in Figure 11 for the production of three lots of 3 mm Proteomix SAX-NP resins.
Proteomix SCX-POR, WCX-POR, SAX-POR, and WAX-POR resins are based on porous PS/DVB resins that are coated with a proprietary hydrophilic layer and functionalized with a uniform ion-exchange layer. These phases have three unique features. First, the nanometer thick hydrophilic layer completely eliminates the non-specific interactions with biological analytes. Second, Sepax’s proprietary technology synthesizes a uniform and densely packed layer of ion-exchange functional groups. Third, porous beads provide high surface area, leading to high separation capacity. Such uniquely designed Proteomix SCX-POR, WCX-POR, SAX-POR, and WAX-POR phases offer high capacity and reasonably high resolution separations for proteins, oligonucleotides and peptides. Due to the porosity, Proteomix porous resins have less efficiency and lower resolution than Proteomix non-porous resins. Proteomix SCX-POR, WCX-POR, SAX-POR, and WAX-POR phases are well suited for semi-preparative and preparative separation and purification of biomolecules. Porous Proteomix SCX, WCX, SAX, and WAX columns are manufactured and tested under the strictest specifications, resulting in high column-to-column and lot-to-lot reproducibility.
The column dimensions of the Proteomix SCX, WCX, SAX, and WAX products are 0.75, 2.1, 3.0, 4.6, 7.8, 10, and 21.2 mm I.D., and 2, 3, 5, 10, 15, 25, and 30 cm length. Sepax also offers custom-made columns.
- Uniform polymer beads as the packing support
- Proprietary surface chemistry specially designed for elimination of non-specific bindings
- Unprecedented separation efficiency, selectivity and resolving power
- Complete selection for analytical, semi-preparative and preparative separations
- Wide particle size selection
- High stability
- High recovery
- Well suited for UPLC system
Proteomix ion-exchange phases have wide applications for separation, identification and purification of proteins, protein variants, peptide fragments, phosphorylated, sialylated, and other derivatized proteins. They are excellent for monitoring enzymatic reactions and protein-protein interactions. With extremely high separation efficiency, Proteomix phases are well designed for proteomics applications and 2D or multi-dimensional separations. One example is the high resolution of cell lysates by Proteomix SAX-NP phases. Proteomix SAX and WAX phases are suitable for separation of oligonucleotides. Porous Proteomix ion-exchange phases provide high resolution and high capacity separation for surface charged nanoparticles, nanorods and nanotubes.
Separation of Horse Serum, a Protein Mixture
Figure 12 shows the separation of a horse serum by using Proteomix SAX-NP5 and a commercial porous SAX column. Non-porous Proteomix SAX-NP5 has much high resolution than the porous SAX column.
Separation of Carbohydrates and Glycans
Figure 13 shows the separation of glucose oligomers and corn starch hydrolysate. ELSD is utilized to detect carbohydrate molecules. For ELSD detection, bleeding is a big problem for most columns, especially silica based amino columns. However, Proteomix SAX columns not only solved the bleeding problem, but also achieved excellent separation efficiency and resolution. Figure 14 is another example of high resolution separation of glycans and their isomers by non-porous Proteomix SAX column.
Separation of Peptides in Volatile Buffer
The peptides with different hydrophobicity can be resolved by reversed phases, such as C18. However, peptides with different charges may not be well separated on reversed phase mode if their hydrophobic properties are close. As an alternative method, ion exchange chromatography is recommended. With the net charge from +1 to +4, four peptides (C1, C2, C3 and C4 with their sequences listed in Figure 15) were well separated by Proteomix SCX-NP phase in a volatile buffer of ammonium acetate and acetonitrile, which makes it compatible for LC/MS application.
High Protein Loading Separation
Ribonuclease A was separated by a non-porous SCX column. When the injection amount of Ribonuclease A was increased from 50 µg to 5 mg, the separation resolution had negligible change and each of the impurities was well separated, as shown in Figure 16. The combination of high loading capacity and high resolution separation enables non-porous Proteomix ion-exchange columns very well suited for both analytical and preparative separations of biological molecules.
Separation of Oligonucleotides
High resolution separation is critical for DNA analysis and purification. The non-porous Proteomix SAX resins offer high separation efficiency with high capacity. Figure 17 shows the separation of a 50-mer oligonucleotide synthesized by ABI synthesizer. The largest peak is the modified 50-mer oligo and the others are failures or the impurities. As a comparison, the same sample was separated with a commercial porous SAX column. The results showed similar retention time for the 50-mer oligonucleotide from both non-porous and porous SAX columns, which indicated that their capacities are close to each other. However, the non-porous SAX generated much higher efficiency and resolution. Figure 18 is another example of high resolution separation of oligonucleotides of Mw 12196 from its degraded fragments. At least 35 species of those degraded oligo fragments were well resolved within 22 minutes.
Separation and Analysis of Cell Lysates
The key issue for proteome studies is to separate and identify a large number of biological species in a cell, such as proteins, nucleotides, peptides and others. The demand for separation is unprecedented. With the uniqueness of high resolution and high capacity, the non-porous Proteomix ion-exchange resins are very much suitable for separating cell lysates. Figure 19(a) showed the separation profiles of E. coli lysate with 3, 5, and 10 µm non-porous Proteomix SAX particles. The minimum number of resolved peaks increased from 40 to 60 to 75 when the particle size decreased from 10 to 5 to 3 µm. To better view the separation performance, the elution profiles in the range of 10-22 minutes were shown in Figure 19(b). At least 45, 38, 27 peaks were resolved at the retention time of 10-22 minutes for 3, 5, and 10 µm SAX columns, respectively. Figure 20 showed various sample loading for a 3 µm, 4.6x50 mm non-porous SAX column. When the amount of E. coli lysate increased from 25 µg to 50 µg to 125 µg, the separation efficiency and resolution remained consistent.
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