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Agilent Bio SEC-5 Columns handbook

1. 1 0 4 mL min for 4 6 mm id columns lt Agilent Technologies Buffers and Samples Prior to use filter and degas all buffers and if possible sam ples through a 0 2 um or 0 45 um filter This will prevent col umn clogging and air bubbles in the buffers The use of a 0 5 um frit filter or an Agilent Bio SEC 5 guard can further protect the column Agilent Bio SEC 5 columns are compati ble with commonly used aqueous buffers Water and organic mixtures such as acetonitrile and methanol can also be used The use of an inline degassor is recommended or at minimum the buffers should be degassed by filtration or sonicated under water pumped vacuum Column Equilibration Agilent Bio SEC 5 columns are shipped in 0 05 azide in 0 1 M sodium phosphate buffer pH 7 0 Prior to the first sample injection purge the column with 20 column volumes of 150 mM sodium phosphate buffer pH 7 0 at 0 1 mL min Gradually increase the flow rate until you reach your intend ed operating conditions and allow the baseline to flatten If the baseline or column back pressure fluctuates increase the flow for 3 5 minutes keeping in mind the maximum par ticle pressure of 240 bar 3 500 psi Once equilibrated and the baseline is flat the column is ready for a sample injec tion Keep in mind that equilibration time is needed after each run pH Stability Agilent Bio SEC 5 columns can be used in the range pH 2 8 5 Using the column within this range will opt
2. About Agilent Bio SEC 5 Columns Agilent Bio SEC 5 columns are packed with 5 um silica par ticles coated with a proprietary neutral hydrophilic layer The thin polymeric layer is chemically bonded to highly pure and mechanically stable silica in an extremely controlled process This uniform surface layer provides a highly effi cient and stable size exclusion particle Agilent Bio SEC 5 columns are available in 5 um particles with 100 A 150 A 300 A 500 A 1000 A and 2000 A nominal pore sizes The highly spherical and narrowly dispersed 5 um particles are packed using a proprietary technique ensuring a uniform and stable packed bed The Agilent Bio SEC 5 columns offer superior column lifetime and reproducibility Agilent Bio SEC 5 columns are designed for a broad range of size based bio molecule separations including antibodies proteins peptides oligonucleotides polysaccharides polymers bio logical cells bacteria viral particles and other large bio mol ecules Agilent Bio SEC 5 columns are best used with most aqueous buffers and have excellent stability in high and low salt conditions Basic Characteristics Data Sheet Agilent Bio SEC 5 Columns Safety Precautions Agilent Bio SEC 5 columns are designed for use with high pressure liquid chromatography HPLC systems Loose fit tings and connections can cause buffer leaks and sample loss Many samples and buffers are considered hazardous and should be treated as such Alway
3. gilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2010 Printed in the USA February 18 2010 5973 1743 Agilent Technologies
4. imize performance and lifetime Column Hardware and Particle Pressure The recommended operating pressure for both the 7 8 mm and 4 6 mm id columns is 137 bar 2 000 psi Column life time is increased when operating at or below this pressure Do not exceed the maximum particle pressure of 240 bar 8 500 psi Long term use at high flow rates may damage or decrease the lifetime of the column Column back pressure commonly increases over the lifetime of the column If there is a sudden increase in column back pressure it may be due to a clogged inlet frit Reversing the flow to flush the column may clear the clogged inlet frit Temperature Column lifetime is optimized when used between 10 30 C The maximum column operating temperature is 80 C Long term use at 80 C or higher will damage the column especially when being used at above pH 8 Flow Rate Range The working flow rate is 0 1 1 25 mL min for the 7 8 mm id columns and 0 1 0 4 mL min for 4 6 mm id columns Guard and Column Cleanup An increase in guard or column back pressure is likely to occur over time Absorption of protein to the packing mater ial or on the inlet frit will cause this increase in pressure and will decrease column performance Cleaning the guard or column may decrease the back pressure and improve per formance When using a guard column or precolumn filter remove the main column and flush the guard filter in the reverse flow direction with cleaning buffe
5. r for at least 15 minutes or replace the guard filter during your next col umn use To clean the main column flush the column in the reverse direction with cleaning buffer for at least 15 column volumes at no more than 50 of the maximum particle pres sure limit Rinse well with 3 5 column volumes of Milli Q or Nanopure water after each cleaning solution and prior to reconditioning the column for use Two cleaning buffers are recommended 1 Concentrated neutral salts such as 0 5 M Na SO pH 3 0 2 Aqueous buffer with water soluble organic such as 50 mM phosphate pH 7 0 with 10 to 20 methanol acetonitrile or ethanol Extended Storage For extended column storage flush the column in a 0 05 NaN sodium azide or 20 ethanol containing buffer at pH 7 0 for at least 10 column volumes and tightly seal both ends with the removable end plugs provided with the column The column can be refrigerated or stored at room temperature 4 35 C Column Protection Guard columns can be used for added column protection and are recommended Guards and filters often capture par ticulates coming from the samples the buffers or from the HPLC system being used Agilent Ordering Information For more information on our products visit our Agilent Technologies home page on the World Wide Web at http www agilent com chem supplies For Technical support in the US and Canada call 1 800 227 9770 or call your local Agilent sales office www a
6. s wear gloves and safe ty glasses when using columns in an HPLC system Agilent does not recommend opening HPLC columns for any reason Installing the Column Before installing the column remove both endcaps and ensure that your flow direction matches the arrow on the column Use the recommended flow direction unless reverse flow is being used to remove material blocking the inlet Prior to applying flow over the column make tight ferrule connections The recommended tubing is 1 16 od PEEK or stainless tubing with standard HPLC PEEK or stainless fer ules and nuts Column Phase Size Exclusion Packing Spherical high purity porous silica with a hydrophilic polymeric coating Particle size 5 um Pore structure 100A 150A 300A 500A 1000A 2000A Column exclusion limits in Daltons 100 A MW range 100 100 000 150 A MW range 500 150 000 300 A MW range 5 000 1 250 000 500 A MW range 15 000 5 000 000 1000 A MW range 50 000 7 500 000 2000 A MW range gt 10 000 000 pH stability 2 8 5 Operating temperature limit Recommended range 10 30 C maximum 80 C Operating pressure limit Recommended operating pressure 137 bar 2 000 psi Maximum pressure 240 bar 3 500 psi Mobile phase compatibility Recommended 150 mM phosphate buffer pH 7 0 other aqueous buffers with high and low salt can be used Working flow rate 0 1 1 25 mL min for 7 8 mm id columns 0

Agilent Bio SEC-5 Columns handbook

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