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Utilizing proprietary surface technologies, SRT SEC phases are made of the uniform, hydrophilic, and neutral nanometer thick films chemically bonded on the high purity and enhanced mechanical stability silica. This proprietary surface technology results in excellent column-to-column reproducibility. The nature of the chemical bonding and the maximum bonding density of the thin film benefit SRT SEC phases with high stability and negligible non-specific interactions. Along with the widest pore size selection of 100, 150, 300, 500, 1000 and 2000Å, SRT SEC packings have highest capacity that enables highest resolution.
- Uniform, nanometer thick molecule layer
- Chemically bonded to silica surface
- Hydrophilic
- Maximum bonding density
Comparison of the separation patterns of a protein mixture by SRT SEC-150, SRT SEC-300 and SRT SEC-500 columns (5µm, 7.8×300mm). The separation conditions: mobile phase, 150 mM phosphate buffer, pH 7.0; flow rate, 1.0 mL/min; detection: UV 214 nm; temperature, ambient (23 °C); and injection volume, 10 µL. Sample: (1) Thyroglobulin (1.0 mg/mL), 670 kD; (2) BSA (1.0 mg/mL), 66 kD; (3) Ribonuclease A (1.0 mg/mL), 13.7 kD, and (4) Uracil (2.5 mg/mL), 120.
Comparison of the separation patterns of a protein mixture by SRT SEC-100, SRT SEC-150, SRT SEC-300, SRT SEC-500 and SRT SEC-1000 columns (5µm, 4.6×300mm). The separation conditions: mobile phase, 150 mM phosphate buffer, pH 7.0; flow rate, 0.35 mL/min; detection: UV 214 nm; temperature, ambient (23 °C); and injection volume, 3 µL. Sample: (1) Thyroglobulin (1.0 mg/mL), 670 kD; (2) BSA dimer, 132 kD; (3) BSA (1.0 mg/mL), 66 kD; (4) Ribonuclease A (1.0 mg/mL), 13.7 kD, and (5) Uracil (2.5 mg/mL), 120.
Separation of BSA dimer from BSA protein
The chromatograms show baseline separation of BSA dimer from BSA for all three SRT SEC-150, SRT SEC-300 and SRT SEC-500 columns. Due to the difference of pore sizes, the retention time for BSA and BSA dimer are different. The smaller the pore size, the shorter retention time for BSA and BSA dimmer. Since the MW of BSA (67K) and BSA dimmer are in the separation range for all those three columns, the resolution (separation factor) for BSA and BSA dimer is slightly varied.
Separation of the aggregates from thyroglobulin
Thyroglobulin is a huge protein (MW 670,000). The commercial sample of thyroglobulin contains the aggregate impurity, possibly dimer or tetramer. SRT SEC-300, SRT SEC-500 and SRT SEC-1000 provide a baseline separation of the aggregates from thyroglobulin, as shown in both chromatograms. Due to the small pore size of SRT SEC-100 and SRT SEC-150, both thyroglobulin and its aggregates are totally excluded, resulting in a sharp peak.
- Highest capacity and resolution
- High efficiency
- High reproducibility
- Widest selection of pore size from 100 Å to 2000 Å
- High protein recovery and maintaining biological activity
- pH range 2 - 8.5
High Efficiency Separation
For biological separations, nonspecific interactions with the packing materials are the major source for low separation efficiency, such as peak tailing, as well as low recovery. The unique surface chemistry of SRT SEC packings provides 1) uniform, neutral and hydrophilic coating that eliminates the hydrophobic interactions with proteins, and 2) maximum density of bonding that minimizes the electrostatic interactions with proteins. This specially designed coating allows SRT SEC columns to achieve high resolution and high efficiency separations. For example, uracil, as a test compound is measured at efficiency up to 90,000 plates per meter, as shown in Table 1.
Table 1. Column efficiencies (measured using uracil in 0.1 M phosphate buffer, pH 7.0).
| SRT SEC-100 (5 µm) | >100,000 plates/meter |
| SRT SEC-150 (5 µm) | >90,000 plates/meter |
| SRT SEC-300 (5 µm) | >90,000 plates/meter |
| SRT SEC-500 (5 µm) | >85,000 plates/meter |
| SRT SEC-1000 (5 µm) | >90,000 plates/meter |
| SRT SEC-2000 (5 µm) | >85,000 plates/meter |
The Impact of Pore Structure on Separation
The pore size of the SEC packings plays a critical role in protein separations. The selection of an SRT SEC phase for a particular molecular weight range protein sample is based on the exclusion limit and linear molecular weight range of the SRT packings:
| SRT packings | Protein MW exclusion limit |
| SEC-100 | 100,000 |
| SEC-150 | 150,000 |
| SEC-300 | 1,250,000 |
| SEC-500 | 5,000,000 |
| SEC-1000 | 7,500,000 |
| SEC-2000 | >10,000,000 |
High Protein Recovery and Maintaining Activity
SRT SEC phases are composed of a hydrophilic, neutral thin film that is covalently bonded on the silica surface with the maximum density. Proteins and other biological molecules have negligible nonspecific interactions with such stationary phases. The protein adsorption to the silica surface is suppressed, leading to high recovery and maintaining the activity after the separation. Table below shows the recovery results of BSA and lysozyme, the representatives for acidic and basic proteins, respectively.
Recovery of proteins from SRT SEC columns (measured with a 4.6x300 mm column in 0.15 M phosphate buffer, pH 7.0)
| SRT SEC-100 | BSA | >95% |
| Lysozyme | >95% |
| SRT SEC-150 | BSA | >95% |
| Lysozyme | >95% |
| SRT SEC-300 | BSA | >95% |
| Lysozyme | >95% |
| SRT SEC-500 | BSA | >95% |
| Lysozyme | >95% |
| SRT SEC-1000 | BSA | >95% |
| Lysozyme | >95% |
High Stability of SRT SEC Phases
SRT SEC columns have a stationary phase that is densely bonded on the silica surface which greatly hinders the diffusion of any molecules that would attack the bond of silica-stationary phase layer, thus enabling high stability. 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 in 45 days. SRT SEC phases can tolerate very high concentration of salts, such as 1.0 M. SRT SEC columns are stable in both organic solvents, such as methanol, ethanol, THF, DMF, DMSO, and so on, and the mixture of water and organic solvents.
Stability test for retention time of proteins and a small molecule.
| Conditions | |
| Column: | SRT SEC-150 (5 µm, 7.8×300mm) |
| Mobile phase: | 150 mM phosphate buffer, pH 7.0 |
| Flow rate: | 1.0 mL/min |
| Temperature: | Ambient |
| Injection volume: | 10 mL |
| Sample: | 1. Thyroglobulin (1.0 mg/mL)
2. BSA (1.0 mg/mL)
3. Ribonuclease A (1.0 mg/mL)
4. Uracil (50 mg/mL) |
SRT SEC columns are stable over a wide range of pH from 2 to 8.5. In addition, they can even tolerate higher pH, such as pH 8.5-10 for temporary usage.
The temperature tolerance for SRT SEC phases is also excellent. The working temperature can go as high as 80°C.
Reproducibility of the SRT SEC columns
The controlled surface chemistry used to synthesize SRT SEC phases makes the surface coating very reproducible, leading to very consistent columns. Separation variation from batch to batch is within 5% for retention time. Figure below is a separation of the Sepax standard protein mixture by the SRT SEC-150 columns from three different lots. The largest variation retention time for ribonuclease A is less than 2%.
Comparison of the separations by three different batches of SRT SEC-150 (5 µm), 4.6×300 mm columns. The separation conditions: mobile phase, 150 mM phosphate buffer, pH 7.0; flow rate, 0.35 mL/min; detection: UV 214 nm; temperature, ambient (23 °C); and injection volume, 5 µL. Sample: (1) Thyroglobulin (1.0 mg/mL), (2) BSA (1.0 mg/mL), (3) Ribonuclease A (1.0 mg/mL), and (4) Uracil (2.5 mg/mL).
MW Calibration for Protein Separation
For size exclusion chromatography, individual pore size packings determine the range of molecular weight for separation, while the pore volume controls the separation capacity. SRT¡¯s six pore size packings cover the separation of a wide range of biological molecules. The protein calibration curves for SRT SEC-150, SRT SEC-300, and SRT SEC-500 are shown in figure below.
Protein MW calibration with SRT SEC-150, SRT SEC ¨C300 and SRT SEC¨C500 phases.
| Conditions | |
| Columns: | 7.8x300 mm, 5 ¦Ìm |
| Mobile phase: | 150 mM phosphate buffer, pH 7.0 |
| Flow rate: | 1.0 mL/min |
| Detector: | UV 214 nm |
| Injection volume: | 10 ¦ÌL |
| Sample: | 1. Thyroglobulin, 670 kD
2. g-Globulin, 158 kD
3. BSA dimer, 132 kD
4. BSA, 66 kD
5. Ovalbumin, 44 kD
6. Myoglobin, 17.6 kD
7. Ribonuclease A, 13.7 kD
8. B12, 1.35 kD
9. Uracil, 120 |
Another widely used method to characterize the SEC phases is the calibration curve between protein MW and the distribution coefficient (Kd). Kd value is defined by the equation below:
Kd = (Ve-Vo)/(VT-Vo)
Where Ve, VT, and Vo are the sample elution volume, the column total liquid volume, and the column void volume, respectively. The linear region between the protein MW and Kd is frequently used as the reference for the selection of the SEC phases.
Separation and MW Calibration of Water Soluble Polymers
Benefiting from unique surface bonding and wide pore size selection (100 ¨C 2,000 Å), SRT SEC phases are ideal for separation and characterization of water soluble polymers. Even though each individual SRT SEC column is suitable for separation and characterization of water soluble polymers, two columns connected in series are recommended for achieving highest resolution, efficiency and accuracy. SRT SEC-150 and SRT SEC-1000 are recommended for measurement of water soluble polymers. Figure below is the chromatogram of polysaccharide standards with the MW from 667 to 788,000 by using SRT SEC-1000 and SRT SEC-150 columns.
Separation of polysaccharide standards by using a SRT SEC-1000 column (5 µm, 4.6×300 mm) and a SRT SEC-150 column (5 µm, 4.6×300 mm). (Courtesy of Dr. Kihong Park, PolysisLab)
| Conditions | |
| Mobile phase: | 0.2 M phosphate buffer, pH 7.0 |
| Flow rate: | 0.35 mL/min |
| Detector: | Refractive index |
| Injection volume: | 20 µL |
| Sample: | Polysaccharides (1.0 mg/mL)
Polysaccharide R (Mp 788,000, 112,000, 11,800)
Polysaccharide G (Mp 404,000, 47,300, 5,900)
Polysaccharide Y (Mp 212,000, 22,800, 667) |
Calibration curve of polysaccharide MW vs. retention time by using a SRT SEC-1000 column (5 µm, 4.6×300 mm) and a SRT SEC-150 column (5 µm, 4.6×300 mm). The separation conditions are the same as those in figure above. (Courtesy of Dr. Kihong Park, PolysisLab)
Sepax SRT SEC-100 | Part Number | Particle Size | Pore Size | ID×Length | Price |
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| | 215100-4605 [1] | 5 µm | 100Å | 4.6×50mm | $395.00 | | 215100-4615 | 5 µm | 100Å | 4.6×150mm | $810.00 | | 215100-4625 | 5 µm | 100Å | 4.6×250mm | $940.00 | | 215100-4630 | 5 µm | 100Å | 4.6×300mm | $1,000.00 | | 215100-7805 [1] | 5 µm | 100Å | 7.8×50mm | $395.00 | | 215100-7815 | 5 µm | 100Å | 7.8×150mm | $870.00 | | 215100-7825 | 5 µm | 100Å | 7.8×250mm | $1,000.00 | | 215100-7830 | 5 µm | 100Å | 7.8×300mm | $1,210.00 |
[1] Guard column. Sepax SRT SEC-150 | Part Number | Particle Size | Pore Size | ID×Length | Price |
|---|
| | 215150-4605 [1] | 5 µm | 150Å | 4.6×50mm | $395.00 | | 215150-4615 | 5 µm | 150Å | 4.6×150mm | $810.00 | | 215150-4625 | 5 µm | 150Å | 4.6×250mm | $940.00 | | 215150-4630 | 5 µm | 150Å | 4.6×300mm | $1,000.00 | | 215150-7805 [1] | 5 µm | 150Å | 7.8×50mm | $395.00 | | 215150-7815 | 5 µm | 150Å | 7.8×150mm | $870.00 | | 215150-7825 | 5 µm | 150Å | 7.8×250mm | $1,000.00 | | 215150-7830 | 5 µm | 150Å | 7.8×300mm | $1,210.00 |
[1] Guard column. Sepax SRT SEC-300 | Part Number | Particle Size | Pore Size | ID×Length | Price |
|---|
| | 215300-4605 [1] | 5 µm | 300Å | 4.6×50mm | $395.00 | | 215300-4615 | 5 µm | 300Å | 4.6×150mm | $810.00 | | 215300-4625 | 5 µm | 300Å | 4.6×250mm | $940.00 | | 215300-4630 | 5 µm | 300Å | 4.6×300mm | $1,000.00 | | 215300-7805 [1] | 5 µm | 300Å | 7.8×50mm | $395.00 | | 215300-7815 | 5 µm | 300Å | 7.8×150mm | $870.00 | | 215300-7825 | 5 µm | 300Å | 7.8×250mm | $1,000.00 | | 215300-7830 | 5 µm | 300Å | 7.8×300mm | $1,210.00 |
[1] Guard column. Sepax SRT SEC-500 | Part Number | Particle Size | Pore Size | ID×Length | Price |
|---|
| | 215500-4605 [1] | 5 µm | 500Å | 4.6×50mm | $395.00 | | 215500-4615 | 5 µm | 500Å | 4.6×150mm | $810.00 | | 215500-4625 | 5 µm | 500Å | 4.6×250mm | $940.00 | | 215500-4630 | 5 µm | 500Å | 4.6×300mm | $1,040.00 | | 215500-7805 [1] | 5 µm | 500Å | 7.8×50mm | $415.00 | | 215500-7815 | 5 µm | 500Å | 7.8×150mm | $970.00 | | 215500-7825 | 5 µm | 500Å | 7.8×250mm | $1,155.00 | | 215500-7830 | 5 µm | 500Å | 7.8×300mm | $1,270.00 |
[1] Guard column. Sepax SRT SEC-1000 | Part Number | Particle Size | Pore Size | ID×Length | Price |
|---|
| | 215950-4605 [1] | 5 µm | 1000Å | 4.6×50mm | $395.00 | | 215950-4615 | 5 µm | 1000Å | 4.6×150mm | $810.00 | | 215950-4625 | 5 µm | 1000Å | 4.6×250mm | $940.00 | | 215950-4630 | 5 µm | 1000Å | 4.6×300mm | $1,040.00 | | 215950-7805 [1] | 5 µm | 1000Å | 7.8×50mm | $415.00 | | 215950-7815 | 5 µm | 1000Å | 7.8×150mm | $970.00 | | 215950-7825 | 5 µm | 1000Å | 7.8×250mm | $1,155.00 | | 215950-7830 | 5 µm | 1000Å | 7.8×300mm | $1,270.00 |
[1] Guard column. Sepax SRT SEC-2000 | Part Number | Particle Size | Pore Size | ID×Length | Price |
|---|
| | 215980-4605 [1] | 5 µm | 2000Å | 4.6×50mm | $395.00 | | 215980-4615 | 5 µm | 2000Å | 4.6×150mm | $810.00 | | 215980-4625 | 5 µm | 2000Å | 4.6×250mm | $940.00 | | 215980-4630 | 5 µm | 2000Å | 4.6×300mm | $1,040.00 | | 215980-7805 [1] | 5 µm | 2000Å | 7.8×50mm | $415.00 | | 215980-7815 | 5 µm | 2000Å | 7.8×150mm | $970.00 | | 215980-7825 | 5 µm | 2000Å | 7.8×250mm | $1,155.00 | | 215980-7830 | 5 µm | 2000Å | 7.8×300mm | $1,270.00 |
[1] Guard column. Sepax SRT SEC-100 | Part Number | Particle Size | Pore Size | ID×Length | Price |
|---|
| | 215100-10005 [1] | 5 µm | 100Å | 10.0×50mm | $660.00 | | 215100-10010 | 5 µm | 100Å | 10.0×100mm | $1,275.00 | | 215100-10015 | 5 µm | 100Å | 10.0×150mm | $1,495.00 | | 215100-10025 | 5 µm | 100Å | 10.0×250mm | $1,935.00 | | 215100-10030 | 5 µm | 100Å | 10.0×300mm | $2,150.00 | | 215100-21205 [1] | 5 µm | 100Å | 21.2×50mm | $1,500.00 | | 215100-21225 | 5 µm | 100Å | 21.2×250mm | $4,915.00 | | 215100-21230 | 5 µm | 100Å | 21.2×300mm | $5,955.00 |
[1] Guard column. Sepax SRT SEC-150 | Part Number | Particle Size | Pore Size | ID×Length | Price |
|---|
| | 215150-10005 [1] | 5 µm | 150Å | 10.0×50mm | $660.00 | | 215150-10010 | 5 µm | 150Å | 10.0×100mm | $1,275.00 | | 215150-10015 | 5 µm | 150Å | 10.0×150mm | $1,495.00 | | 215150-10025 | 5 µm | 150Å | 10.0×250mm | $1,935.00 | | 215150-10030 | 5 µm | 150Å | 10.0×300mm | $2,150.00 | | 215150-21205 [1] | 5 µm | 150Å | 21.2×50mm | $1,500.00 | | 215150-21225 | 5 µm | 150Å | 21.2×250mm | $4,915.00 | | 215150-21230 | 5 µm | 150Å | 21.2×300mm | $5,955.00 |
[1] Guard column. Sepax SRT SEC-300 | Part Number | Particle Size | Pore Size | ID×Length | Price |
|---|
| | 215300-10005 [1] | 5 µm | 300Å | 10.0×50mm | $660.00 | | 215300-10010 | 5 µm | 300Å | 10.0×100mm | $1,275.00 | | 215300-10015 | 5 µm | 300Å | 10.0×150mm | $1,495.00 | | 215300-10025 | 5 µm | 300Å | 10.0×250mm | $1,935.00 | | 215300-10030 | 5 µm | 300Å | 10.0×300mm | $2,150.00 | | 215300-21205 [1] | 5 µm | 300Å | 21.2×50mm | $1,500.00 | | 215300-21225 | 5 µm | 300Å | 21.2×250mm | $4,915.00 | | 215300-21230 | 5 µm | 300Å | 21.2×300mm | $5,955.00 | | 215300-30025 | 5 µm | 300Å | 30.0×250mm | Inquire | | 215300-30030 | 5 µm | 300Å | 30.0×300mm | Inquire | | 225300-10025 | 10 µm | 300Å | 10.0×250mm | Inquire | | 225300-10030 | 10 µm | 300Å | 10.0×300mm | Inquire | | 225300-21225 | 10 µm | 300Å | 21.2×250mm | Inquire | | 225300-21230 | 10 µm | 300Å | 21.2×300mm | Inquire | | 225300-30025 | 10 µm | 300Å | 30.0×250mm | Inquire | | 225300-30030 | 10 µm | 300Å | 30.0×300mm | Inquire |
[1] Guard column. Sepax SRT SEC-500 | Part Number | Particle Size | Pore Size | ID×Length | Price |
|---|
| | 215500-10005 [1] | 5 µm | 500Å | 10.0×50mm | $700.00 | | 215500-10010 | 5 µm | 500Å | 10.0×100mm | $1,375.00 | | 215500-10015 | 5 µm | 500Å | 10.0×150mm | $1,570.00 | | 215500-10025 | 5 µm | 500Å | 10.0×250mm | $2,040.00 | | 215500-10030 | 5 µm | 500Å | 10.0×300mm | $2,375.00 | | 215500-21205 [1] | 5 µm | 500Å | 21.2×50mm | $1,580.00 | | 215500-21225 | 5 µm | 500Å | 21.2×250mm | $5,320.00 | | 215500-21230 | 5 µm | 500Å | 21.2×300mm | $6,440.00 |
[1] Guard column. Sepax SRT SEC-1000 | Part Number | Particle Size | Pore Size | ID×Length | Price |
|---|
| | 215950-10005 [1] | 5 µm | 1000Å | 10.0×50mm | $700.00 | | 215950-10010 | 5 µm | 1000Å | 10.0×100mm | $1,375.00 | | 215950-10015 | 5 µm | 1000Å | 10.0×150mm | $1,570.00 | | 215950-10025 | 5 µm | 1000Å | 10.0×250mm | $2,040.00 | | 215950-10030 | 5 µm | 1000Å | 10.0×300mm | $2,375.00 | | 215950-21205 [1] | 5 µm | 1000Å | 21.2×50mm | $1,580.00 | | 215950-21225 | 5 µm | 1000Å | 21.2×250mm | $5,320.00 | | 215950-21230 | 5 µm | 1000Å | 21.2×300mm | $6,440.00 |
[1] Guard column. Sepax SRT SEC-2000 | Part Number | Particle Size | Pore Size | ID×Length | Price |
|---|
| | 215980-10005 [1] | 5 µm | 2000Å | 10.0×50mm | $700.00 | | 215980-10010 | 5 µm | 2000Å | 10.0×100mm | $1,375.00 | | 215980-10015 | 5 µm | 2000Å | 10.0×150mm | $1,570.00 | | 215980-10025 | 5 µm | 2000Å | 10.0×250mm | $2,040.00 | | 215980-10030 | 5 µm | 2000Å | 10.0×300mm | $2,375.00 | | 215980-21205 [1] | 5 µm | 2000Å | 21.2×50mm | $1,730.00 | | 215980-21225 | 5 µm | 2000Å | 21.2×250mm | $6,070.00 | | 215980-21230 | 5 µm | 2000Å | 21.2×300mm | $7,340.00 |
[1] Guard column. |
SRT SEC-100 SRT SEC-150 SRT SEC-300 SRT SEC-500 SRT SEC-1000 SRT SEC-2000
SRT SEC-100 SRT SEC-150 SRT SEC-300 SRT SEC-500 SRT SEC-1000 SRT SEC-2000
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Analytical LC Columns
