Agilent VariTide RPC Columns Manual
Contents
1. VariTide RPC columns are physically and chemically stable across the complete pH range This guide is intended to help maximize column performance and lifetime The column is optimized as a universal solution for the analysis and purification of synthetic peptides Installation Analytical and preparative columns A 1 16 in stainless steel tubing is recommended for column con nections 0 010 in id for analytical work and 0 020 in id for prepar ative work using the 21 2 id column Connecting tubing lengths should be minimized to avoid excessive dead volume which will diminish system performance Column connections should be made using Parker compatible 1 16 in nuts and ferrules with spe cial reference to compatibility of column connectors as illustrated in Figure 1 Connect the HPLC column in the flow direction indi cated The nut and ferrule should be tightened 1 4 of a turn past finger tight by applying the wrenches as show in Figure 2 0 090 0 210 p E Figure 1 Compatible connectors The distance x for the standard HPLC column end fitting is 0 090 in and a minimum male nut length of 0 210 in is required Some fittings from other manufacturers may not be compatible for example Waters and Rheodyne If unsure please contact Agilent Technologies for advice Flats l en rs ee Figure 2 Don t use wrenches on the flats To avoid loosening the endfittings and causing leaks wrenches must be used on the en2. dfitting adjacent to the connecting nut and NOT on the column barrel or the opposite endfitting 3 p Agilent Technologies Data Sheet Agilent VariTide RPC Columns Shipping Eluent VariTide RPC columns are supplied containing 7 1 w w acetoni trile water Columns are securely sealed with end caps which must ALWAYS be replaced when the column is disconnected from the HPLC system to prevent the column from drying out Column Conditioning It will be necessary to wash out the shipping eluent and condition the column with the required mobile phase before use Ensure the mobile phase components are soluble whenever changing eluent and never use 100 aqueous eluents Mobile Phases All eluents should be HPLC grade they should be filtered lt 0 5 um filter and thoroughly degassed before use The physical and chemical stability of VariTide RPC adsorbents allows buffered mobile phases across the complete pH range to be used The maximum buffer salt concentration is determined by solubility Reversed Phase Chromatography It is strongly recommended that a minimum of 1 organic modifier is maintained in the mobile phase Column performance may be reduced when reintroducing organic modifier after use in 100 aqueous eluents VariTide RPC absorbents can be used with all common organic modifiers in both isocratic and gradient elution modes Flow Rate Column Pressure The columns are packed to enable operating pressures up to 3000 ps
3. i 207 bar to be used This pressure should not be exceeded Optimum column performance will usually be found at flow rates of less than 360 cm h equivalent to 1 0 mL min ona 4 6 mm id column If column pressures are unusually high due to eluent viscosity increasing the operating temperature will be ben eficial The increased temperature will reduce the eluent viscosi ty which will result in a lower operating pressure and improved mass transfer Sample Preparation Samples should be filtered lt 0 5 um to remove particulate matter which may otherwise foul the column inlet In line filters can be used to provide additional protection Samples should be prepared in the starting eluent or in a solvent of lower strength whenever possible for maximum solute interaction Column Efficiency Testing Each column is provided with its own individual test certificate It is recommended that the column be re checked from time to time to monitor its performance 7 1 w w acetonitrile water is used with acetone as the unretained test probe System fac tors such as dead volume flow rate temperature etc can sig nificantly affect the results obtained It is important that these factors are taken into account when comparing the results with the test certificate data Column Clean Up If the column begins to exhibit signs of deterioration then the fol lowing clean up procedures may be beneficial Particulate matter blocking the inlet frit a
4. nd causing excessive back pressure may sometimes be removed by gentle flushing in the reverse direction The use of a higher strength organic modifier such as acetonitrile or tetrahydrofuran may remove hydrophobically bound contami nants Peptide contamination may sometimes be removed by an aqueous acetonitrile gradient containing 1 v v TFA Strong acids and bases including sodium hydroxide can be used for cleaning in place and depyrogenation All cleaning eluents must contain a minimum of 1 organic modifier Storage On removing the column from the system the end plugs must be replaced to prevent the column from drying as this would disrupt the packed bed The end plugs need only be applied finger tight Long term storage in buffer should be avoided to prevent the risk of crystallization of buffer salts A high organic content is recommended to inhibit bacterial growth Agilent Ordering Information For more information on our products visit our web site at www agilent com chem columns www agilent 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 2011 Printed in the USA February 2011 PK134 3 iE Agilent Technologies
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