Agilent ZORBAX HILIC Plus HPLC Columns handbook
Contents
1. best to remove salts from the instrument and column by purging the column with the same mobile phase without the buffer for example using 90 10 ACN H 20 to remove a 90 10 ACN 0 01 M formate buffered mobile phase Re equilibration is faster with the original mobile phase when using this approach but several 3 to 6 injections should be made to verify column equilibration Agilent Ordering Information For more information on our products and services visit our Web site at www agilent com chem For technical support in the U S and Canada call 1 800 227 9770 or call your local Agilent sales office 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 2009 Printed in the USA March 18 2009 Part No 820114 001 yi a Agilent Technologies2. General Description Agilent ZORBAX HILIC Plus HPLC columns are non bonded silica columns made with ZORBAX Eclipse Plus silica high purity Type B silica specially treated for good peak shape and optimized for hydrophilic interaction chromatography HILIC separations HILIC is typically used for the retention and separation of small polar ana lytes The ZORBAX HILIC Plus columns ship containing acetonitrile water and are ready to use for HILIC separa tion HILIC columns require more equilibration than reversed phase columns and more details on this are pro vided in the method development section of this data sheet Operating conditions Column Agilent ZORBAX HILIC Plus OC Chromatogram 4 6 mm x 100 mm 3 5 pm Mobile phase 85 acetonitrile 15 water Flow rate 1 mL min Temperature Ambient 23 C Detection UV 254 nm Sample volume 2uL Sample Caffeine 100 pg mL mAU 604 504 404 304 204 104 04 i T T T ae ee ee i T T re tet 0 2 04 0 6 0 8 1 1 2 1 4 1 6 1 8 min Figure 1 ZORBAX HILIC Plus QC chromatogram Agilent ZORBAX HILIC Plus HPLC Columns Data Sheet Column Characteristics A typical quality control QC chromatogram for a 4 6 mm x 100 mm 3 5 um ZORBAX HILIC Plus column is shown in Figure 1 The QC test with the performance of your column is shown on the Column Performance Report enclosed with your column Safety Considerations e All points of connection in a liquid chromatographic system are potential s
3. aphy tech niques Some typical analytes may include melamine and other polar analytes including such compounds as acrylamide nucleosides metformin and other compounds in the U S EPA 1694 Method Column Care Samples that contain particulate matter may plug the column inlet frit and should be filtered before injection into the column If solvent flow appears to be restricted unusually high column back pressure check first to see that solvent flow is unobstructed up to the column inlet If the column has the restriction there may be particulate matter on the inlet frit An attempt should be made to remove any inlet debris by backflushing 25 to 30 mL of mobile phase through the column If this fails to return the column to near its original back pressure the column should be replaced To remove strongly retained materials from the column flush the column with a strong solvent which is water in the HILIC mode Storage Recommendations Acetonitrile water 95 5 is recommended as the long term storage solvent for the HILIC Plus column It may be necessary to flush the column with 60 acetonitrile 40 water to remove strongly retained compounds prior to switching to the storage solvent Before storing the column the end fittings should be tightly capped with the end plugs to prevent the packing from drying out Columns may be safely stored for short periods in most HILIC mobile phases However to protect equipment it is
4. best to equilibrate the column with 30 to 40 water in acetonitrile before use The column must be equi librated with 20 to 50 column volumes before use Several injections should be done to verify that the column is properly equilibrated A typical mobile phase for the HILIC Plus column will be acetonitrile water with an acetate or formate buffer This will most commonly be ammonium acetate or formate to achieve compatibility with an MS detector To optimize retention for HILIC methods increase the percent acetoni trile in the mobile phase and decrease the aqueous buffer to increase retention In addition it is critical to optimize pH and buffer strength for the best results A recom mended starting buffer concentration is 5 to 10 mM and increase up to 20 mM for improved peak shape and reten tion A typical pH range for HILIC separations will be pH 2 to 7 using formate and acetate buffers Applications ZORBAX HILIC Plus columns can be used for typical HILIC applications The most common HILIC applications are for the retention of small polar and or basic analytes HILIC is considered a good alternative to ion pair chro matography and the use of bonded phases with polar groups in the bonded phase These applications typically require additives and high aqueous conditions that are less compatible with MS detectors therefore HILIC may be a preferred column choice HILIC can also be a pre ferred alternative to normal phase chromatogr
5. ources of leaks Users of liquid chromatographic instruments should be aware of the potential toxicity or flammability of their mobile phases Because of the small particle size dry ZORBAX pack ings are respirable Columns should only be opened in a well ventilated area Operational Guidelines e The direction of flow is marked on the column e While generally not harmful to the column reversing flow should be avoided except to attempt to remove contamination from the inlet frit e ZORBAX HILIC Plus columns are shipped containing acetonitrile and water e ZORBAX HILIC Plus columns are compatible with water and all common organic solvents e Maximum operating pressure is 400 bar e Maximum operating temperature is 40 C e The operating pH range of this column is pH 1 to 8 Note HILIC Plus columns are silica based columns with no bonded phase All silica has some solubility in pH gt 6 aqueous mobile phases Solubility of the silica is also increased at elevated temperatures Therefore using the HILIC Plus column above pH 6 and 40 C will reduce the column lifetime S o Agilent Technologies Method Development with HILIC Plus The ZORBAX HILIC Plus column is best used for separa tions of polar analytes inadequately retained on typical reversed phase columns For the HILIC mechanism to work effectively the column must be equilibrated with water to create a water layer on the silica sorbent There fore it is
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