Made of 3 µm silica particles, Zenix SEC packing is a breakthrough technology for size exclusion chromatography. Combination of 3 µm silica particle size and proprietary surface technology enables highest separation efficiency and resolution for biological molecules and water soluble polymers. Available pore sizes of Zenix packings are 100, 150 and 300Å.
"Tough samples" such as hydrophobic proteins like insulin, membrane protein monoclonal antibodies derivatized with polymer branches, e.g. polypeptide, PEG.
Zenix SEC Technical Specifications
Phase
Zenix SEC-80
Zenix SEC-100
Zenix SEC-150
Zenix SEC-300
Material
Neutral, hydrophilic film bonded silica
Particle size
3 µm
Pore size
~ 80 Å
~ 100 Å
~ 150 Å
~ 300 Å
Protein MW range (native)
100 - 50,000
100 - 66,000
500 - 150,000
5,000 - 1,250,000
pH stability
2.5 – 7.5 (up to pH 8.5 can be tolerated temporarily*)
Available Zenix 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.
High Separation Efficiency
The advantages of developing small particle size are higher efficiency and higher resolution. When particle size is decreased to 3 µm from 5 µm, the column efficiency is almost doubled. As shown in Table 1, the plate numbers of BSA dimmer, BSA, ribonuclease A increased from 2720 to 4600, 6590 to 13090, 11160 to 22000 when the particle size decreased from 5 µm to 3 µm. Fig. 2 and Fig. 3 further show that high efficiency has been achieved by 3 µm Zenix columns with various proteins. The efficiency of p-aminobenzoic acid reached to the plate number of 40,000 for 30 cm long Zenix column.
Figure 1. Separation of protein mixture A by Zenix SEC-300 and SRT SEC-300 columns.
Conditions
Column:
Zenix SEC-300 and SRT SEC-300
Mobile phase:
150 mM phosphate buffer, pH 7.0
Flow rate:
1.0 mL/min for 7.8×300mm, 0.35 mL/min for 4.6×300mm
Table 3. Efficiency of 4.6×300 mm Zenix SEC-150 and 300 columns.
Peak
Protein
Zenix 150
Zenix 300
1
Thyroglobulin
6020
2410
2
γ-Globulin
2560
3000
3
Ovalbumin
6030
10260
4
Ribonuclease A
13350
17020
5
p-Aminobenzoic acid
35500
33480
High Loading Capacity
Loading capacity is critical for size exclusion separation and purification. Figure 4 shows high loading capacity for BSA as one example (>500 µg for an analytical column).
Figure 4. BSA loading test on a Zenix SEC-150 column.
Conditions
Column:
Zenix SEC-150 (3 µm, 7.8×300 mm)
Mobile phase:
150 mM phosphate buffer, pH 7.0
Flow rate:
1.0 mL/min
Temperature:
Ambient
Detector:
UV 214 nm
Injection volume:
10 µL
Figure 5. BSA loading test on a Zenix SEC-300 column.
Conditions
Column:
Zenix SEC-300 (3 µm, 7.8×300 mm)
Mobile phase:
150 mM phosphate buffer, pH 7.0
Flow rate:
1.0 mL/min
Temperature:
Ambient
Detector:
UV 214 nm
Injection volume:
10 µL
High Stability
The proprietary stationary phases of Zenix 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.
Conditions
Column:
Zenix 3 µm, 7.8×300 mm
Mobile phase:
150 mM phosphate buffer, pH 7.0
Flow rate:
1.0 mL/min
Temperature:
Ambient
Injection volume:
10 µL
Sample:
A) BSA
B) Thyroglobulin (1.0 mg/mL)
BSA (1.0 mg/mL)
Ribonuclease A (1.0 mg/mL)
Uracil (2.5 mg/mL)
Mobile Phase Compatibility
Zenix SEC phases are compatible with most aqueous buffers, such as ammonium acetate, phosphate, trizma and so on. Zenix SEC phases can tolerate high concentration of salts, such as 2.0 M. Furthermore, Zenix 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.
Lot-to-Lot Reproducibility
The controlled surface chemistry used to synthesize Zenix 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.
Zenix SEC phases are hydrophilic and neutral. Proteins and other biological molecules have negligible nonspecific interactions with Zenix 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.
[1] Guard column [2] Sepax Protein standard for size exclusion chromatography and used for general correlation of elution time vs. protein MW [3] One-piece PEEK coupler, recommended to connect columns in series. [4] PEEK/Stainless Steel coupler, recommended to connect columns of IDs > 7.8 mm in series. [5] Column coupler with 10cm length flexible tubing,10-32 fitting with 1/16 OD PEEK tubing. Recommended to connect analytical columns in series [6] Includes one ZenixTM 300 Å, and one ZenixTM-C 300 Å, columns (7.8 x 300mm). [7] Includes one ZenixTM 300 Å, and one ZenixTM-C 300 Å, columns (4.6 x 300mm). [8] 0.5 µm PEEK frit for column with particle size 3 µm or less [9] Intended for use with column IDs ≥ 21.2 mm [10] For Precolumn Filter (0.5 µm) 5 units/pk [11] SS Refill Frits for 102020-21200, 5 units/pk
[1] Guard column [2] Column packed with PEEK tubing. [3] Sepax Protein standard for size exclusion chromatography and used for general correlation of elution time vs. protein MW [4] One-piece PEEK coupler, recommended to connect columns in series. [5] PEEK/Stainless Steel coupler, recommended to connect columns of IDs > 7.8 mm in series. [6] Column coupler with 10cm length flexible tubing,10-32 fitting with 1/16 OD PEEK tubing. Recommended to connect analytical columns in series [7] Includes one ZenixTM 300 Å, and one ZenixTM-C 300 Å, columns (7.8 x 300mm). [8] Includes one ZenixTM 300 Å, and one ZenixTM-C 300 Å, columns (4.6 x 300mm). [9] 0.5 µm PEEK frit for column with particle size 3 µm or less [10] Intended for use with column IDs ≥ 21.2 mm [11] For Precolumn Filter (0.5 µm) 5 units/pk [12] SS Refill Frits for 102020-21200, 5 units/pk
[1] Guard column [2] Column packed with PEEK tubing. [3] Sepax Protein standard for size exclusion chromatography and used for general correlation of elution time vs. protein MW [4] One-piece PEEK coupler, recommended to connect columns in series. [5] PEEK/Stainless Steel coupler, recommended to connect columns of IDs > 7.8 mm in series. [6] Column coupler with 10cm length flexible tubing,10-32 fitting with 1/16 OD PEEK tubing. Recommended to connect analytical columns in series [7] Includes one ZenixTM 300 Å, and one ZenixTM-C 300 Å, columns (7.8 x 300mm). [8] Includes one ZenixTM 300 Å, and one ZenixTM-C 300 Å, columns (4.6 x 300mm). [9] 0.5 µm PEEK frit for column with particle size 3 µm or less [10] Intended for use with column IDs ≥ 21.2 mm [11] For Precolumn Filter (0.5 µm) 5 units/pk [12] SS Refill Frits for 102020-21200, 5 units/pk
[1] Guard column [2] Column packed with PEEK tubing. [3] Sepax Protein standard for size exclusion chromatography and used for general correlation of elution time vs. protein MW [4] One-piece PEEK coupler, recommended to connect columns in series. [5] PEEK/Stainless Steel coupler, recommended to connect columns of IDs > 7.8 mm in series. [6] Column coupler with 10cm length flexible tubing,10-32 fitting with 1/16 OD PEEK tubing. Recommended to connect analytical columns in series [7] Includes one ZenixTM 300 Å, and one ZenixTM-C 300 Å, columns (7.8 x 300mm). [8] Includes one ZenixTM 300 Å, and one ZenixTM-C 300 Å, columns (4.6 x 300mm). [9] 0.5 µm PEEK frit for column with particle size 3 µm or less [10] Intended for use with column IDs ≥ 21.2 mm [11] For Precolumn Filter (0.5 µm) 5 units/pk [12] SS Refill Frits for 102020-21200, 5 units/pk
