Glycomix-SAX - Sepax Technologies, Inc.
Contents
1. Sepax Technologies Ine Delaware Technology Park 5 100 Innovation Way Newark DE 19711 USA Phone 302 366 1101 Fax 302 366 1151 Toll Free 1 877 SEPAX US www sepax tech com USER MANUAL Glycomix SAX lon Exchange Phase Column Information Glycomix strong anion exchange phase is made of narrow dispersed and hydrophilic polymer particles It is a strong anion exchanger with quaternary ammonium functional groups that are chemically bonded to the hydrophilic surface of the polymer particles Glycomix aes SMe 1 S AX l i f 7 F M A Figure 1 Chemical compositions of Glycomix SAX Column Stability and Performance With well controlled surface chemistry and resin production Glycomix resins exhibit high reproducibility The Glycomix SAX columns are specially designed for highly charged polysaccharides separation such as heparin sodium Figure 2 shows a representative separation profile of heparin sodium dermatan sulfate DS and oversulfated chondroitin sulfate OSCS Figure 2 Separation of heparin dermatan sulfate and oversulfated chondroitin sulfate Heparin OSCS 5 er 5 0 25 3 0 35 Column Glycomix SAX 4 6 x 250 mm Guard column 4 6 x 50 mm Mobile phase A 0 04 NaH PO pH 3 0 B 0 04 NaH PO 14 NaClO pH 3 0 Flow rate 0 22 mL min Gradient 20 90 in 60 minutes Wavelength 202 nm Column temp 40 C Injection volume 10 uL Samp2. 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 in the sample the mobile phase or leached from HPLC system such as pump and injector seals However a guard column is highly recommended because it will more effectively trap highly adsorptive sample components and residual particulates in the sample the mobile phase or from the HPLC system Glycomix Products ID x Length mm Part number Glycomix SAX 4 6 x 250 mm 901665 4625 COMI ORS Jaen 901665 4605 guard column Glycomix Kit Column Guard 901665 KIT Heparin Chondroitin Oversulfated Chondroitin 5 mg OSCS 5 Sulfate Giycomix SAX
3. e replace the filter or the guard column The washing solutions are 150 mM potassium nitrate in 75 acetonitrile at pH 2 adjusted by HCl for Glycomix strong anion exchange columns 2 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 the increasing backpressure and the broadening peaks When this occurs it is time to clean your column The general guidelines for column cleaning are 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 Typically 10 15 column volumes of cleaning solution are sufficient Some general guidelines are recommended for choosing cleaning solutions e A low pH salt solution will help to remove basic proteins e A high pH salt solution will help to remove acidic proteins e Organics will help to remove hydrophobic proteins e One cleaning solution is recommended for general cleaning 150 mM potassium nitrate in 75 acetonitrile at pH 2 adjusted pH by HCl Column Protection In addition to filtering the sample and the mobile phase the
4. le 20 mg mL Heparin Sodium mg mL Dermatan Sulfate DS and 1 mg mL Oversulfated chondroitin sulfate OSCS Table 1 Gradient table for column running and re equilibration Time Flow A B min mL min OoOo w o 0 22 mL min 10 9 w o o o 74 1 w o 0 22 mL min so A 0 04 NaH POx pH 3 0 B 0 04 NaH PO 14 NaClO pH 3 0 Table 2 Glycomix SAX Technical Specifications Typical backpressure for 4 6x250 10 bar mm 0 22 mL min Maximum temperature C 0 Heparin highly charged polysaccharides Mobile phase compatibility Applications Safety Precaution Glycomix SAX ion exchange columns are normally operated under low pressure Loose connections will cause leaking of buffers and injected samples all of which should be considered as hazards In the case of leaking proper gloves should be worn for handling the leaked columns When opening the columns proper protections should be used to avoid inhalation of the small polymer particles Column installation and Operation When column is shipped or not in use it should always be capped at both ends When installing the column to the system first remove the end caps Make the flow direction as marked on the column unless a user has special purpose to reverse the flow direction such as the removal of the inlet blockage Column connections are an integral part of the chromatographic process If ferrules are over tightened not set prope
5. m can be operated up to 1 mL min flow rate and a back pressure of 100 bars can be tolerated 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 plugged In this case it is recommended that the column be flushed with reverse flow in an appropriate solvent Temperature The maximum operating temperature is 80 C The optimum Operation temperature for longest lifetime is 10 50 C Continuous use of the column at higher temperature gt 80 C can damage the column especially under extreme pH gt 12 or lt 2 0 Flow rate Range Normal operation is 0 22 mL min for 4 6 mm I D columns for heparin sodium separation using the recommended USP running conditions Storage When not in use for extended time store the Glycomix SAX columns in deionized water with 20 ethanol Flush the column with water and then 20 ethanol for at least 15 column volumes for each step Seal both ends with the removable end plugs provided with the column to prevent the drying of the column bed Column clean up 1 If a pre column filter or a guard column is used before the separation column clean the pre column filter or the guard column first by flushing the filter or the guard column in reverse flow direction using washing solutions for 15 30 min If washing does not improve the column performanc
6. rly or are not specific for the fitting 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 O D 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 finger tighten the nut c While continuing to press the tube firmly into the end fitting use a 1 4 wrench to further tighten d Repeat this coupling procedure for the other end of the column Column Care Glycomix SAX columns are shipped with 20 ethanol First time use During stocking and shipping the packing could be dried out Flush the column with HPLC water while gradually increasing the flow rate from 0 1 mL min to your operation condition flush the column with water for 10 20 column volumes at 1ml min Then equilibrate the column with the running mobile phase for another 10 20 column volumes or until the baseline is stable If the column backpressure and baseline fluctuate there might be air bubbles trapped inside the column Flush the column with higher flow rate for 2 5 minutes for example 1 0 mL min for a 4 6 x 250 mm column If the mobile phase or pH is quite different from the storage buffer in the column it is recommended that the column is washed first with new mobile phases for 10 column volume Pressure Glycomix SAX 4 6 x 250 m
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