Zenix-C SEC Column User Manual - Sigma
Contents
1. Zenix C 150 Zenix C 300 00 20 40 460 80 100 120 140 Min Columns Zenix C 3 um 7 8x300 mm Mobile phase 150 mM Sodium Phosphate pH 7 0 Flow rate 1 0 mL min Detection UV214 nm Injection 10 ul 1 Thyroglobulin 670 kD 2 y Globulin 158 kD 3 Ovalbumin 44 kD 4 Myoglobulin 16 9 kD 5 Vitamin B12 1355 D Sample Column Characteristics Silica Spherical high purity lt 10 ppm metals Particle size 3 sum Pore sizes for protein separation 100 MW range 100 100 000 150 A MW range 500 150 000 300 A MW range 5 000 1 250 000 Safety Precaution The columns are normally operated under moderate pressure Loose connections will cause leaking of organic solvents and injected samples all of which should be considered as hazards In the case of leaking proper gloves should be worn while handling the columns When opening the columns proper protections should be used to avoid inhalation of the small silica particles Column Installation and Operation The column should always be capped at both ends when it is not in use When installing the column to the system first remove the end caps Unless a user has special purpose to reverse the flow direction for example removal of the inlet blockage follow the flow direction as marked on the column Column connections are an integral part of the chromatographic process If ferrules are over tightened not set properly or are not specific for the fitt2. Typically 10 15 column volumes of cleaning solution are sufficient Rinse well with 3 5 column volume of Nanopure water between each solution Cleaning solutions Low pH salt solutions help remove basic proteins Organics are useful when removing hydrophobic proteins Chaotropic agents help remove strongly adsorbed materials e g via hydrogen bonding Only use chaotropic agents when neutral salts or organics have not improved resolution Two cleaning solutions are recommended for general cleaning 1 Concentrated neutral salt e g 0 5 M Na SO at low pH e g pH 3 0 2 Water soluble organic MeOH ACN EtOH 10 20 in aqueous buffer e g 50 mM phosphate pH 7 0 Column Protection In addition to filtering the sample and the mobile phase the best way to protect the separation column is to install a guard column or a pre column filter in front of it In most cases a pre column filter helps to remove the residual particulates that are in the sample the mobile phase or leached from the HPLC system such as pump and injector seals However a guard column is highly recommended because it is more effective in trapping highly adsorptive sample components and residual particulates in the sample the mobile phase or from the HPLC system
3. 5 minutes for example 0 5 mL min and 1 25 mL min for 4 6x300 mm and 7 8x300 mm columns respectively pH For optimum performance and operation during the longest lifetime keep pH between 2 and 8 5 Pressure Even though the columns can operate at a pressure up to 3 500 psi the normal operating pressure is usually under 2 000 psi Continuous use at a high pressure may eventually damage the column Since the pressure is generated by the flow rate the maximum flow rate is limited by the backpressure It is expected that the backpressure might gradually increase with its service A sudden increase in backpressure suggests that the column inlet frit might be blocked In this case it is recommended that the column be flushed with reverse flow in an appropriate solvent Solvent compatibility Columns are compatible with aqueous buffers such as phosphate acetate Tris etc and water miscible organic solvents such as MeOH ethanol isopropanol acetonitrile THF etc When switching from an aqueous buffer to an organic solvent the column should be washed with nanopure T IM Zenix C water for at least 30 column volume then ethanol for 20 column volume When switching from an organic solvent to an aqueous buffer the column should be washed with ethanol for at least 30 column volume then nanopure water for 20 column volume and finally 20 column volume aqueous buffer After washing it is recommended that the column be stored in the aqueous buff
4. Sigma Aldrich Corporation is a worldwide distributor of HPLC columns manufactured by Sepax Technologies Inc For a complete listing of these and other Sepax columns please consult www sigmaaldrich com sepax Zenix C SEC Column User Manual Column Information Utilizing proprietary surface technologies and 3 um particle size Zenix C SEC phases are made of uniform hydrophilic and neutral nanometer thick films chemically bonded on the high purity and enhanced mechanical stability silica The proprietary surface technologies allow the chemistry of thin film formation to be well controlled which results in high column to column reproducibility The nature of the chemical bonding and the maximum bonding density of the thin film benefit Zenix C SEC phases with high stability The uniform surface coating enables high efficiency separation The narrowly dispersed spherical silica particles of the Zenix C packings for SEC 100 SEC 150 and SEC 300 have nominal pore sizes at 100 A 150 A and 300 A respectively With a small particle size of 3 um and specially designed large pore volume ca 1 35 mL g for Zenix C SEC 150 and 300 and ca 1 1 mL g for Zenix SEC 100 Zenix C phases have achieved unprecedented high separation efficiency and resolution Zenix C SEC columns are packed with a proprietary slurry technique to achieve uniform and stable packing bed density for maximum column efficiency Zenix C SEC phases are designed to ensure highes
5. er for 48 hours to get well equilibrated for satisfactory performance Temperature The maximum operating temperature is 80 C The optimum operating temperature for the longest lifetime is 10 30 C Continuous use of the column at a higher temperature gt 80 C can damage the column especially under high pH gt 8 Flow rate Range Normal operating flow rate is 0 1 0 4 and 0 1 1 25 mL min for 4 6 mm and 7 8 mm I D columns respectively Storage When the column is not in use for an extended time the column should be stored in a 150 mM sodium phosphate buffer pH 7 0 Each column is shipped with two removable end plugs To prevent drying of the column bed seal both ends of the column with the end plugs provided Cleaning From time to time some samples could get adsorbed onto the inlet frit or the packing material When the adsorption accumulates to a certain level it is usually indicated by an increase in backpressure and a broader peak When this occurs it is time to clean your column The general procedure for column cleaning is as follows 1 Disconnect the column from the detector 2 Clean your column in the reverse flow direction 3 Run the column at less than 50 of the maximum recommended flow rate Monitor the backpressure If you see the pressure is much higher than the normal operating conditions you need to lower the flow rate or change the washing buffer as the cleaning solutions may be of different viscosities 4
6. ing leakage can occur Set the ferrules for column installation to the HPLC system as follows a Place the male nut and ferrule in order onto a 1 16 outer diameter piece of tubing Be certain that the wider end of the ferrule is against the nut b Press tubing firmly into the column end fitting Slide the nut and ferrule forward engage the threads and fingertighten the nut c Repeat this coupling procedure for the other end of the column Samples and Mobile Phases To avoid clogging the column all samples and solvents should be filtered through 0 45 um or 0 2 um filters before use Zenix C SEC columns are compatible with an aqueous mobile phase or a mixture of organic and water such as methanol or acetonitrile and water Always degas the mobile phase A simple way for degassing is to sonicate it for 5 minutes under water pumped vacuum Column Care Shipping Solvent New columns are shipped in 150 mM sodium phosphate buffer pH 7 0 During stocking and shipping the silica packing may become dried out It is recommended that 10 20 column volumes of 50 mM sodium phosphate buffer at pH 7 0 be purged to activate the column Flush the column with your mobile phase while gradually increasing the flow rate from 0 1 mL min to your operating condition until the baseline is stable If the column backpressure and baseline fluctuate this might be due to air bubbles trapped inside the column Flush the column with higher flow rate for 2
7. t resolution and maximum recovery for a very broad range of separation applications These applications cover large biological molecules such as proteins and nucleic acids small biological molecules such as peptides and oligonucleotides natural polymers such as polysaccharides synthetic polymers biological cells such as bacteria and virus and nanomaterials such as nanoparticles Typical applications for Zenix SEC columns are separation and detection in aqueous buffer mobile phases Column Stability and Performance Zenix C SEC columns use full coverage bonded silica packing which allows exceptionally high stability They are compatible with most aqueous buffers such as ammonium acetate phosphate tris etc When 150 mM phosphate buffer at pH 7 0 is used as the mobile phase to run Zenix C SEC columns 300 injections with protein standards of thyroglobulin BSA ribonuclease A and uracil or 1 month of usage has negligible deterioration for Zenix C SEC columns The neutral and hydrophilic Zenix C stationary phases have negligible nonspecific interactions with biological molecules such as proteins DNA RNA and peptides Combined with their high capacity Zenix C SEC columns enable high efficiency and high recovery separations Figure 1 shows an example of separating Biorad protein mixture using 7 8x300 mm Zenix C SEC columns Figure 1 Separation of Biorad protein mixture by Zenix C 100 150 and 300 columns Zenix C 100
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