 |
SRT-SEC
Utilizing proprietary surface technologies, SRT SEC phases are made of uniform, hydrophilic, and neutral nanometer thick films chemically bonded onhigh purity and mechanically stabilizedsilica. The well-controlled surface chemistry results in excellent lot-to-lot reproducibility. Our unique bonding chemistry, coupled with the maximized bonding density, allows SRTSEC to provide high stability and negligible non-specific interactions. SRT SEC packings have large pore volume, resulting in high separation resolution. The narrowly dispersed, spherical silica particles of the SRT packings for SEC-100, SEC-150, SEC-300, SEC-500, SEC-1000 and SEC-2000 have nominal pore sizes at 100, 150, 300, 500, 1,000, and 2,000 Å, respectively. Typical applications for SRT SEC columns include separation and detection of biological molecules and water soluble polymers in aqueous buffers.
 As in size exclusion chromatography, peak capacity is primarily determined by the pore volume of the packing. The higher the pore volume, the higher peak capacity generated and better separation resolution. SRT packings are specially designed for achieving high pore volume, 1.3-1.5 mL/g for SRT SEC-150, 300 and 500 and 1.0-1.1 mL/g for SRT SEC-100, 1000 and 2000.
Compared to Vendor T SEC column, SRT SEC-150 demonstrates a number of benefits. First, SRT offers higher capacity, 6.7 mL for SRT vs. 6.17 mL for Vendor T, calculated from the total permeation peak (uracil) to total exclusion peak (thyroglobulin). Secondly SRT offers higher resolution than Vendor T. Poly-DL-alanine (from Sigma) is a peptide with the MW of1-5 kD. For size exclusion chromatography, an empirical rule is that a baseline separation can be achieved for two compounds if their MWs difference is two fold (2x). SRT SEC-150 column well separated ribonuclease A (13.7kD) and poly-DL-alanine (1-5 kDa), while Vendor T column did not achieve a baseline separation. Thirdly SRT column shows a good separation profile of Poly-DL-alanine, indicating SRT packing does not have non-specific interactions with Poly-DL-alanine. In contrast, a broad and tailing peak of Poly-DL-alanine from Vendor T column indicates some non-specific bindings between its packing and the peptide.
Figure 2 shows separations of 6 proteins and aggregates, 1 polypeptide, and a small molecule by SRT SEC-300 and Vendor T columns. Compared to Vendor T SEC column, SRT SEC-300 offers a number of advantages. First SRT offers higher capacity, calculating from the total permeation peak (uracil) to total exclusion peak (thyroglobulin aggregate). SRT has the capacity of 6.54 mL (thyroglobulin aggregate, 5.56 min; uracil, 12.10 min), while Vendor T column has the capacity of 6.08 mL (thyroglobulin aggregate, 5.23 min; uracil, 11.31 min). Secondly, in overall, SRT has higher resolution than Vendor T. Looking at the high molecular weight range, thyroglobulin and its aggregates were well separated by SRT SEC-300 column, but only partially separated by Vendor T column. Also in the low MW range, poly-DL-alanine (1-5 kD from Sigma) myoglobin (17.6 kD) were well separated by SRT SEC-300 column, but poorly separated by Vendor T column. Thirdly, SRT column shows a good separation profile of Poly-DL-alanine, indicating SRT packing does not have non-specific interactions with Poly-DL-alanine. In contrast, a broad and tailing peak profile from Vendor T column indicate some existence of non-specific bindingsbetween its packing and the peptide.
Figure 3 shows the separation profiles of four proteins (thyroglobulin, g-globulin, ovalbumin and myoglobulin) and vitamin B12 with the molecular weight in the range of 660,000 – 1,355. The peak capacity is the elution volume from the total exclusive peak of thyroglobulin aggregate to the total permeation peak of vitamin B12. The capacity of SRT SEC-500 is slightly larger than that of Vendor T column (4.9 mL vs 4.7 mL). The resolution and efficiency of SRT SEC-500 is better than that of Vendor T column.
High Robustness
SRT SEC packings have specially designed stationary phases that are densely bonded on the silica surface which enhances the stability of the column, resulting in high robustness at high flow rates.
High Stability at pH 8.5
The proprietary stationary phases of SRT SEC packings utilize densely bonded chemistry on the silica surface, which greatly hinders the diffusion of the molecules that would attack the bond of silica-stationary phase layer, thus enabling high stability over a wide range of pH from 2 to 8.5. Figure 5 shows that SRT SEC-300 phase demonstrates negligible change after running 700 column volume of phosphate buffer at pH 8.5. SRT SEC phases are compatible with most aqueous buffers, such as ammonium acetate, phosphate, trizma and so on. When 150 mM phosphate buffer at pH 7.0 was used as the mobile phase, the average retention time change was within 5% after 100 injections over a time span of 45 days, as shown in Figure 6. SRT SEC phases can tolerate high concentration of salts, such as 2.0 M. Furthermore, SRT SEC columns are stable in both organic solvents, such as methanol, ethanol, THF, DMF, DMSO, and so on; as well as the mixture of water and organic solvents.
 
High Lot-to-Lot Reproducibility
The controlled surface chemistry used to synthesize SRT SEC phases makes the surface coating highly reproducible, leading to consistent column manufacturing. Separation variation from batch to batch is controlled to be within 5% for retention time. Figure 7 is a separation of the Sepax standard protein mixture by SRT SEC-300 columns from three different lots. The largest variation retention time for ribonuclease A is less than 2%.
High Protein Recovery
SRT SEC phases are hydrophilic and neutral. Proteins and other biological molecules have negligible nonspecific interactions with SRT stationary phases. The protein adsorption to the silica surface is suppressed, leading to high recovery of intact proteins, maintaining the protein activity after separation. More than 95% recovery is achieved for BSA and lysozyme, the representatives for acidic and basic proteins, respectively.
High Loading Capacity
Loading capacity is critical for size exclusion separation and purification. Figure 8 shows high loading capacity for BSA as one example (>500 µg for an analytical column).
 
Wide Pore Size Selection
Combining innovative surface chemistry with the widest selection of pore size from 100A to 2,000A, SRT SEC phases were designed to ensure highest resolution and maximum recovery for a broad range of sepatation applications. The applications cover large biological molecules (e.g. proteins and nucleic acids), small biological molecules (e.g. peptides and oligonucleotides), natural polymers (e.g. polysaccharides), synthetic polymers, biological cells (e.g. bacteria and virus), and nanomaterials (e.g. nanoparticles).
MW Calibration for Protein Separation
For size exclusion chromatography, individual pore size of packings determines the range of molecular weight for separation, while the pore volume controls the separation capacity and resolution. Six pore size SRT packings cover a wide range of separations of biological molecules. The protein calibration curves for SRT SEC-100, 150, 300, 500, and 1000 are shown in Figure 10.
Separation of Water Soluble Polymers
Benefiting from unique surface chemistry and wide pore size selection (100 – 2,000 Å), SRT SEC phases are ideal for separation and characterization of water soluble polymers. Even though Individual SRT SEC column is suitable for separation and characterization of water soluble polymers, two columns connected in series are often recommended for achieving highest resolution, efficiency and accuracy. SRT SEC-150 and SRT SEC-1000 are recommended for measurement of water soluble polymers. Figure 11 is the chromatogram of polysaccharide standards with the MW from 667 to 788,000 by using SRT SEC-1000 and SRT SEC-150 columns. Figure 12 is the calibration curve of MW vs. retention time.
Column Dimension Availability
Available SRT SEC column dimensions are 0.75, 1.0, 2.1, 3.0, 4.6, 7.8, 10, 21.2 and 30 mm I.D., and 20, 30, 50, 100, 150, 250, 300 and 600 mm length. Sepax also offers custom-made columns. Both stainless steel and PEEK tubes are available.
Ordering Information
|
Part Number
|
Particle Size
|
Pore Size
|
ID×Length
|
|
blank
|
|
215100-4605 *
|
5 µm
|
100Å
|
4.6×50mm
|
|
215100-4615
|
5 µm
|
100Å
|
4.6×150mm
|
|
215100-4625
|
5 µm
|
100Å
|
4.6×250mm
|
|
215100-4630
|
5 µm
|
100Å
|
4.6×300mm
|
|
215100-7805 *
|
5 µm
|
100Å
|
7.8×50mm
|
|
215100-7815
|
5 µm
|
100Å
|
7.8×150mm
|
|
215100-7825
|
5 µm
|
100Å
|
7.8×250mm
|
|
215100-7830
|
5 µm
|
100Å
|
7.8×300mm
|
* Guard column.
|
Part Number
|
Particle Size
|
Pore Size
|
ID×Length
|
|
Blank
|
|
215150-4605 *
|
5 µm
|
150Å
|
4.6×50mm
|
|
215150-4615
|
5 µm
|
150Å
|
4.6×150mm
|
|
215150-4625
|
5 µm
|
150Å
|
4.6×250mm
|
|
215150-4630
|
5 µm
|
150Å
|
4.6×300mm
|
|
215150-7805 *
|
5 µm
|
150Å
|
7.8×50mm
|
|
215150-7815
|
5 µm
|
150Å
|
7.8×150mm
|
|
215150-7825
|
5 µm
|
150Å
|
7.8×250mm
|
|
215150-7830
|
5 µm
|
150Å
|
7.8×300mm
|
* Guard column.
|
Part Number
|
Particle Size
|
Pore Size
|
ID×Length
|
|
blank
|
|
215300-4605 *
|
5 µm
|
300Å
|
4.6×50mm
|
|
215300-4615
|
5 µm
|
300Å
|
4.6×150mm
|
|
215300-4625
|
5 µm
|
300Å
|
4.6×250mm
|
|
215300-4630
|
5 µm
|
300Å
|
4.6×300mm
|
|
215300-7805 *
|
5 µm
|
300Å
|
7.8×50mm
|
|
215300-7815
|
5 µm
|
300Å
|
7.8×150mm
|
|
215300-7825
|
5 µm
|
300Å
|
7.8×250mm
|
|
215300-7830
|
5 µm
|
300Å
|
7.8×300mm
|
* Guard column.
|
Part Number
|
Particle Size
|
Pore Size
|
ID×Length
|
|
blank
|
|
215500-4605 *
|
5 µm
|
500Å
|
4.6×50mm
|
|
215500-4615
|
5 µm
|
500Å
|
4.6×150mm
|
|
215500-4625
|
5 µm
|
500Å
|
4.6×250mm
|
|
215500-4630
|
5 µm
|
500Å
|
4.6×300mm
|
|
215500-7805 *
|
5 µm
|
500Å
|
7.8×50mm
|
|
215500-7815
|
5 µm
|
500Å
|
7.8×150mm
|
|
215500-7825
|
5 µm
|
500Å
|
7.8×250mm
|
|
215500-7830
|
5 µm
|
500Å
|
7.8×300mm
|
* Guard column.
|
Part Number
|
Particle Size
|
Pore Size
|
ID×Length
|
|
blank
|
|
215950-4605 *
|
5 µm
|
1000Å
|
4.6×50mm
|
|
215950-4615
|
5 µm
|
1000Å
|
4.6×150mm
|
|
215950-4625
|
5 µm
|
1000Å
|
4.6×250mm
|
|
215950-4630
|
5 µm
|
1000Å
|
4.6×300mm
|
|
215950-7805 *
|
5 µm
|
1000Å
|
7.8×50mm
|
|
215950-7815
|
5 µm
|
1000Å
|
7.8×150mm
|
|
215950-7825
|
5 µm
|
1000Å
|
7.8×250mm
|
|
215950-7830
|
5 µm
|
1000Å
|
7.8×300mm
|
* Guard column.
|
Part Number
|
Particle Size
|
Pore Size
|
ID×Length
|
|
blank
|
|
215980-4605 *
|
5 µm
|
2000Å
|
4.6×50mm
|
|
215980-4615
|
5 µm
|
2000Å
|
4.6×150mm
|
|
215980-4625
|
5 µm
|
2000Å
|
4.6×250mm
|
|
215980-4630
|
5 µm
|
2000Å
|
4.6×300mm
|
|
215980-7805 *
|
5 µm
|
2000Å
|
7.8×50mm
|
|
215980-7815
|
5 µm
|
2000Å
|
7.8×150mm
|
|
215980-7825
|
5 µm
|
2000Å
|
7.8×250mm
|
|
215980-7830
|
5 µm
|
2000Å
|
7.8×300mm
|
* Guard column.
...waiting for catalog data – call us.
SRT-SEC
Utilizing proprietary surface technologies, SRT SEC phases are made of uniform, hydrophilic, and neutral nanometer thick films chemically bonded onhigh purity and mechanically stabilizedsilica. The well-controlled surface chemistry results in excellent lot-to-lot reproducibility. Our unique bonding chemistry, coupled with the maximized bonding density, allows SRTSEC to provide high stability and negligible non-specific interactions. SRT SEC packings have large pore volume, resulting in high separation resolution. The narrowly dispersed, spherical silica particles of the SRT packings for SEC-100, SEC-150, SEC-300, SEC-500, SEC-1000 and SEC-2000 have nominal pore sizes at 100, 150, 300, 500, 1,000, and 2,000 Å, respectively. Typical applications for SRT SEC columns include separation and detection of biological molecules and water soluble polymers in aqueous buffers.
As in size exclusion chromatography, peak capacity is primarily determined by the pore volume of the packing. The higher the pore volume, the higher peak capacity generated and better separation resolution. SRT packings are specially designed for achieving high pore volume, 1.3-1.5 mL/g for SRT SEC-150, 300 and 500 and 1.0-1.1 mL/g for SRT SEC-100, 1000 and 2000.
Compared to Vendor T SEC column, SRT SEC-150 demonstrates a number of benefits. First, SRT offers higher capacity, 6.7 mL for SRT vs. 6.17 mL for Vendor T, calculated from the total permeation peak (uracil) to total exclusion peak (thyroglobulin). Secondly SRT offers higher resolution than Vendor T. Poly-DL-alanine (from Sigma) is a peptide with the MW of1-5 kD. For size exclusion chromatography, an empirical rule is that a baseline separation can be achieved for two compounds if their MWs difference is two fold (2x). SRT SEC-150 column well separated ribonuclease A (13.7kD) and poly-DL-alanine (1-5 kDa), while Vendor T column did not achieve a baseline separation. Thirdly SRT column shows a good separation profile of Poly-DL-alanine, indicating SRT packing does not have non-specific interactions with Poly-DL-alanine. In contrast, a broad and tailing peak of Poly-DL-alanine from Vendor T column indicates some non-specific bindings between its packing and the peptide.
Figure 2 shows separations of 6 proteins and aggregates, 1 polypeptide, and a small molecule by SRT SEC-300 and Vendor T columns. Compared to Vendor T SEC column, SRT SEC-300 offers a number of advantages. First SRT offers higher capacity, calculating from the total permeation peak (uracil) to total exclusion peak (thyroglobulin aggregate). SRT has the capacity of 6.54 mL (thyroglobulin aggregate, 5.56 min; uracil, 12.10 min), while Vendor T column has the capacity of 6.08 mL (thyroglobulin aggregate, 5.23 min; uracil, 11.31 min). Secondly, in overall, SRT has higher resolution than Vendor T. Looking at the high molecular weight range, thyroglobulin and its aggregates were well separated by SRT SEC-300 column, but only partially separated by Vendor T column. Also in the low MW range, poly-DL-alanine (1-5 kD from Sigma) myoglobin (17.6 kD) were well separated by SRT SEC-300 column, but poorly separated by Vendor T column. Thirdly, SRT column shows a good separation profile of Poly-DL-alanine, indicating SRT packing does not have non-specific interactions with Poly-DL-alanine. In contrast, a broad and tailing peak profile from Vendor T column indicate some existence of non-specific bindingsbetween its packing and the peptide.
Figure 3 shows the separation profiles of four proteins (thyroglobulin, g-globulin, ovalbumin and myoglobulin) and vitamin B12 with the molecular weight in the range of 660,000 – 1,355. The peak capacity is the elution volume from the total exclusive peak of thyroglobulin aggregate to the total permeation peak of vitamin B12. The capacity of SRT SEC-500 is slightly larger than that of Vendor T column (4.9 mL vs 4.7 mL). The resolution and efficiency of SRT SEC-500 is better than that of Vendor T column.
High Robustness
SRT SEC packings have specially designed stationary phases that are densely bonded on the silica surface which enhances the stability of the column, resulting in high robustness at high flow rates.
High Stability at pH 8.5
The proprietary stationary phases of SRT SEC packings utilize densely bonded chemistry on the silica surface, which greatly hinders the diffusion of the molecules that would attack the bond of silica-stationary phase layer, thus enabling high stability over a wide range of pH from 2 to 8.5. Figure 5 shows that SRT SEC-300 phase demonstrates negligible change after running 700 column volume of phosphate buffer at pH 8.5. SRT SEC phases are compatible with most aqueous buffers, such as ammonium acetate, phosphate, trizma and so on. When 150 mM phosphate buffer at pH 7.0 was used as the mobile phase, the average retention time change was within 5% after 100 injections over a time span of 45 days, as shown in Figure 6. SRT SEC phases can tolerate high concentration of salts, such as 2.0 M. Furthermore, SRT SEC columns are stable in both organic solvents, such as methanol, ethanol, THF, DMF, DMSO, and so on; as well as the mixture of water and organic solvents.
High Lot-to-Lot Reproducibility
The controlled surface chemistry used to synthesize SRT SEC phases makes the surface coating highly reproducible, leading to consistent column manufacturing. Separation variation from batch to batch is controlled to be within 5% for retention time. Figure 7 is a separation of the Sepax standard protein mixture by SRT SEC-300 columns from three different lots. The largest variation retention time for ribonuclease A is less than 2%.
High Protein Recovery
SRT SEC phases are hydrophilic and neutral. Proteins and other biological molecules have negligible nonspecific interactions with SRT stationary phases. The protein adsorption to the silica surface is suppressed, leading to high recovery of intact proteins, maintaining the protein activity after separation. More than 95% recovery is achieved for BSA and lysozyme, the representatives for acidic and basic proteins, respectively.
High Loading Capacity
Loading capacity is critical for size exclusion separation and purification. Figure 8 shows high loading capacity for BSA as one example (>500 µg for an analytical column).
Wide Pore Size Selection
Combining innovative surface chemistry with the widest selection of pore size from 100A to 2,000A, SRT SEC phases were designed to ensure highest resolution and maximum recovery for a broad range of sepatation applications. The applications cover large biological molecules (e.g. proteins and nucleic acids), small biological molecules (e.g. peptides and oligonucleotides), natural polymers (e.g. polysaccharides), synthetic polymers, biological cells (e.g. bacteria and virus), and nanomaterials (e.g. nanoparticles).
MW Calibration for Protein Separation
For size exclusion chromatography, individual pore size of packings determines the range of molecular weight for separation, while the pore volume controls the separation capacity and resolution. Six pore size SRT packings cover a wide range of separations of biological molecules. The protein calibration curves for SRT SEC-100, 150, 300, 500, and 1000 are shown in Figure 10.
Separation of Water Soluble Polymers
Benefiting from unique surface chemistry and wide pore size selection (100 – 2,000 Å), SRT SEC phases are ideal for separation and characterization of water soluble polymers. Even though Individual SRT SEC column is suitable for separation and characterization of water soluble polymers, two columns connected in series are often recommended for achieving highest resolution, efficiency and accuracy. SRT SEC-150 and SRT SEC-1000 are recommended for measurement of water soluble polymers. Figure 11 is the chromatogram of polysaccharide standards with the MW from 667 to 788,000 by using SRT SEC-1000 and SRT SEC-150 columns. Figure 12 is the calibration curve of MW vs. retention time.
Column Dimension Availability
Available SRT SEC column dimensions are 0.75, 1.0, 2.1, 3.0, 4.6, 7.8, 10, 21.2 and 30 mm I.D., and 20, 30, 50, 100, 150, 250, 300 and 600 mm length. Sepax also offers custom-made columns. Both stainless steel and PEEK tubes are available.
Ordering Information
|
Part Number
|
Particle Size
|
Pore Size
|
ID×Length
|
|
blank
|
|
215100-4605 *
|
5 µm
|
100Å
|
4.6×50mm
|
|
215100-4615
|
5 µm
|
100Å
|
4.6×150mm
|
|
215100-4625
|
5 µm
|
100Å
|
4.6×250mm
|
|
215100-4630
|
5 µm
|
100Å
|
4.6×300mm
|
|
215100-7805 *
|
5 µm
|
100Å
|
7.8×50mm
|
|
215100-7815
|
5 µm
|
100Å
|
7.8×150mm
|
|
215100-7825
|
5 µm
|
100Å
|
7.8×250mm
|
|
215100-7830
|
5 µm
|
100Å
|
7.8×300mm
|
* Guard column.
|
Part Number
|
Particle Size
|
Pore Size
|
ID×Length
|
|
Blank
|
|
215150-4605 *
|
5 µm
|
150Å
|
4.6×50mm
|
|
215150-4615
|
5 µm
|
150Å
|
4.6×150mm
|
|
215150-4625
|
5 µm
|
150Å
|
4.6×250mm
|
|
215150-4630
|
5 µm
|
150Å
|
4.6×300mm
|
|
215150-7805 *
|
5 µm
|
150Å
|
7.8×50mm
|
|
215150-7815
|
5 µm
|
150Å
|
7.8×150mm
|
|
215150-7825
|
5 µm
|
150Å
|
7.8×250mm
|
|
215150-7830
|
5 µm
|
150Å
|
7.8×300mm
|
* Guard column.
|
Part Number
|
Particle Size
|
Pore Size
|
ID×Length
|
|
blank
|
|
215300-4605 *
|
5 µm
|
300Å
|
4.6×50mm
|
|
215300-4615
|
5 µm
|
300Å
|
4.6×150mm
|
|
215300-4625
|
5 µm
|
300Å
|
4.6×250mm
|
|
215300-4630
|
5 µm
|
300Å
|
4.6×300mm
|
|
215300-7805 *
|
5 µm
|
300Å
|
7.8×50mm
|
|
215300-7815
|
5 µm
|
300Å
|
7.8×150mm
|
|
215300-7825
|
5 µm
|
300Å
|
7.8×250mm
|
|
215300-7830
|
5 µm
|
300Å
|
7.8×300mm
|
* Guard column.
|
Part Number
|
Particle Size
|
Pore Size
|
ID×Length
|
|
blank
|
|
215500-4605 *
|
5 µm
|
500Å
|
4.6×50mm
|
|
215500-4615
|
5 µm
|
500Å
|
4.6×150mm
|
|
215500-4625
|
5 µm
|
500Å
|
4.6×250mm
|
|
215500-4630
|
5 µm
|
500Å
|
4.6×300mm
|
|
215500-7805 *
|
5 µm
|
500Å
|
7.8×50mm
|
|
215500-7815
|
5 µm
|
500Å
|
7.8×150mm
|
|
215500-7825
|
5 µm
|
500Å
|
7.8×250mm
|
|
215500-7830
|
5 µm
|
500Å
|
7.8×300mm
|
* Guard column.
|
Part Number
|
Particle Size
|
Pore Size
|
ID×Length
|
|
blank
|
|
215950-4605 *
|
5 µm
|
1000Å
|
4.6×50mm
|
|
215950-4615
|
5 µm
|
1000Å
|
4.6×150mm
|
|
215950-4625
|
5 µm
|
1000Å
|
4.6×250mm
|
|
215950-4630
|
5 µm
|
1000Å
|
4.6×300mm
|
|
215950-7805 *
|
5 µm
|
1000Å
|
7.8×50mm
|
|
215950-7815
|
5 µm
|
1000Å
|
7.8×150mm
|
|
215950-7825
|
5 µm
|
1000Å
|
7.8×250mm
|
|
215950-7830
|
5 µm
|
1000Å
|
7.8×300mm
|
* Guard column.
|
Part Number
|
Particle Size
|
Pore Size
|
ID×Length
|
|
blank
|
|
215980-4605 *
|
5 µm
|
2000Å
|
4.6×50mm
|
|
215980-4615
|
5 µm
|
2000Å
|
4.6×150mm
|
|
215980-4625
|
5 µm
|
2000Å
|
4.6×250mm
|
|
215980-4630
|
5 µm
|
2000Å
|
4.6×300mm
|
|
215980-7805 *
|
5 µm
|
2000Å
|
7.8×50mm
|
|
215980-7815
|
5 µm
|
2000Å
|
7.8×150mm
|
|
215980-7825
|
5 µm
|
2000Å
|
7.8×250mm
|
|
215980-7830
|
5 µm
|
2000Å
|
7.8×300mm
|
* Guard column.
...waiting for catalog data – call us.
|
 |
|