Page 1 of 3 PC HILIC 17.02.2014 http://hplc.shiseido.co.jp/e/column
Contents
1. polar compounds HPLC Conditions 3 Column 4 6 mm i d x 250 mm Sem HILIC mode provides another alternative to handle Mobile phase CHaCN H 0 80 20 extremely polar and hydrophilic compounds which are Flow rate 1 0 mL min unretainable in reversed phase e g a chromatogram of Column tamp 40 C allantoin shown below Sample Allamtain Detection UY 210mm Chromatogram of allantoin Amitriptyline a compound with a strong basicity is often used for discussing the quality of columns PC HILIC provides excellent peak shapes for basic compounds too http hplc shiseido co jp e column html ph_index htm 17 02 2014 PC HILIC Page 2 of 3 l HPLC Conditions Column 4 6 mim id x 250 mm Sem Mobile phase 10 mmol L HCOONH4 CHsCN H20 90 10 Flow rate 1 0 mL min Column tamp 30 C Sample Amitriptyline Detection UW 254 nm 10 min High sensitivity in LC MS Chromatogram of amitriptyline In LC MS of very hydrophilic or ionic compounds HILIC columns are often chosen to avoid extremely water rich or ion pair containing mobile phases used under reversed phase mode The preferable nature common to PC HILIC seems to be the use of mobile phases of higher organic contents which are advantageous in providing larger diffusion constants of analytes during their migration through the columns and also better ionization efficiency in electrospray ionization ESI Column Mobile phase MW 100 Flow rat2. PC HILIC Page 1 of 3 SHISEIDO Products PC HILIG List of Sales Technical Materials Representatives Overview User s Manual F Contact Shiseido Catalog List What is HILIC Hydrophilic interaction liquid chromatography HILIC is a relatively new LC technique that uses a hydrophilic stationary phase in most cases with organic dominant mobile phase The elution order of substances in HILIC mode is roughly the reverse of that in reversed phase mode HPLC Conditions 73 Column 4 6 mm id x 250 mm 5 em Mobile phase 10 mmol L HOCOONHas x36 CHaCN ag CHN Flow rate 1 0 mL min Column temp 40 C g0 CHaCN Detection UY 254 mm T0 CHaCN sample 1 Naphthalene 2 Thymine 3 Adenine 4 Cytosine 4090 CHEN 15 min What is PC Phosphorylcholine PC is a partial structure of phosphatidylcholine O lecitin one of the phospholipids forming cell membranes PC has a T N betaine structure and shows high hydrophilicity biocompatibility and R O P O Oe inhibitory effect of protein adhesion Its superhydrophilic character is suitable to the application as a HILIC phase O Features A silica based HILIC column with phosphorylcholine PC group Excellent retention and separation of very polar and hydrophilic compounds Allantain Large number of theoretical plates and outstanding peak profiles Also available in worldwide 2 4 E 10 min Strong retention of
3. any leaks 2 Inappropriate mobile phase e Secure adequate equilibration conditions time 3 Inadequate column equilibration time 4 Retention time too 1 Hydrolysis deterioration of a short bonded groups by strong acid or e Secure adequate equilibration base time 2 Inappropriate mobile phase conditions 3 Inadequate column equilibration time PC HILIC is shipped after a strict performance check However if you should find any defect please contact your dealer or Shiseido for replacement Note that Shiseido does not warrant the product against column life or deterioration caused by the failure to follow the above handling instructions Ten or more days after reception by the customer Shiseido will assume that the product was delivered in good condition and will not accept a later replacement request 2009 2 1 FAG l i F Our Chromatography Business Contact Shiseido Site Map MZ Analysentechnik GmbH _ Wohlerstralse 2 6 D 55120 Mainz Tel 49 6131 68 66 19 ANALYSENTECHNIK Fax 49 6131 68 66 20 e mail info mz at de www mz at de http hplc shiseido co jp e column html ph_manual htm 13 02 2014
4. are attributable to various causes that cannot all be listed up The table below describes some comparatively common problems related to the column Symptom Cause Measures 1 Column pressure Blocking with foreign matter e Sonicate the filter or replace it rise 1 Dust or insoluble matter in the e Filtrate the mobile phase and mobile phase or sample solution sample solution in advance using 2 Dirt in the tubing a membrane filter 3 Plunger seal fragment e Attach a line filter 4 Precipitation of sample Clean the tubing and replace the components plunger seal http hplc shiseido co jp e column html ph_manual htm 13 02 2014 PC HILIC User s Manual Page 3 of 3 e Prepare a sample solution with the mobile phase 2 Peak splitting 1 Void in the column head e Reconnect the tubing tailing and 2 Dead volume due to inappropriate Review the pH salt broadening connections concentration sample amount 3 Inappropriate mobile phase and other conditions conditions e Check the column performance e on suppression method using standard inspection Inadequate suppression Too solution much sample e Jon pair method Inadequate concentration of the ion pair agent Too much sample 4 Column deterioration Not repairable in the case of column deterioration or damage to the packing condition 3 Retention time too 1 Liquid leak Indicated on the e Check the pump and tubing for long or unstable pressure gage of the pump
5. e 200 gcLmin Column temp Detection sample Ferchloric acid PG HILIC 0 mm id x 150 mm 5am 0 1 Formic acid in He CHaCN 40 680 40 C ESI Negative mode Perchlioric acid Separation of perchloric acid PC HILIC shows large numbers of theoretical plates compared to conventional HILIC columns http hplc shiseido co jp e column html ph_index htm 17 02 2014 PC HILIC Page 3 of 3 HPLC Conditions Column 4 6 mim id x 250 mm Sem Mobile phase 10mmal L HOOONH4 PH3 5 CHCH 10 90 Flow rate 1 0 mL min Column tamp 40 C Detection UY 254 nm Sample 1 Naphthalene 2 Thymine N 271200 3 Adenina 4 Cytosine PC HILIC 15 min Chromatorgam of nucleobase PAGE TOP Our Chromatography Business Contact Shiseido site Map Copyright CC 1999 2008 Shiseido Co Ltd All rights reserved http hplc shiseido co jp e column html ph_index htm 17 02 2014 PC HILIC User s Manual Page 1 of 3 SHL EIDO Products PC HILIC Users Manual List of Sales Technical Materials Overview User s Manual IF Contact Shiseido J Catalog List PC HILIC User s Manual 1 Handling the Column 2 Attaching the Column 3 Analysis 4 Storing the Column 5 End Fittings 6 Replacement Parts and Repair Items 7 Troubleshooting la Le la be Lo oe A PC HILIC is made of totally porous spherical silica bonded with phosphorylcholine PC groups 1 Handling the Column 1 Handle the c
6. eport 5 End Fittings 1 An analytical column of up to 6 mm ID uses a filter embedded end fitting as shown in Fig 1 The filter cannot be changed alone If the filter is clogged or the column pressure is high replace the end fitting See Table 3 for the replacement parts and repair items 2 See Fig 1 for the column connection If the tubing is inappropriate especially if a tube for a different type of column is used the length after the ferrule tip V in Fig 1 1s often different from the end fitting length L and a problem may occur If L is greater than V dead volume may be generated and cause peak broadening or tailing or deterioration of separation performance If L is smaller than V liquid may leak because of inadequate ferrule adhesion Therefore we recommend replacing the ferrule together with the column If the column is replaced frequently the male nut may crush the ferrule and liquid may leak Since tightening the nut too much may cause its head to come off replace the ferrule at an early Stage End fitting Ferrule 1 16 K Wale nut EE Tube 11 bi Filter Fig 1 Column connection PAGE TO 6 Replacement Parts and Repair Items Table 3 Replacement parts and repair items Part No Part Name Description 21105 End fitting 4 6 mm 2 pieces 21107 End fitting 6 mm 2 pieces 21110 Ferrule 1 16 Ferrules 1 16 10pieces sili E TQ 7 Troubleshooting Problems in high performance liquid chromatography
7. n the test for shipment When using a mobile phase containing inorganic salts cares should be taken in arranging a liquid replacement procedure to avoid a sudden pressure increase by salt precipitation 5 To prevent column deterioration avoid the following Frequent mobile phase change or changing directly to a mobile phase of low compatibility eRapid pressure change in column inlet eLarge column pressure under the use of a high viscosity mobile phase eLeaving 100 water flowing through the column for more than one hour 3 2 Preparing a Sample Solution 1 IF possible prepare the sample with the mobile phase or something closer to it 2 Using a solvent with high content of water may lower the separation efficiency Use a solvent with more than 50 content of organic solvent 3 Sample solutions should be aqueous Remove insoluble matters if any by using a filter of 0 45 um or smaller 4 The pH of the sample solution should be set in acceptable pH range for packing material http hplc shiseido co jp e column html ph_manual htm 13 02 2014 PC HILIC User s Manual Page 2 of 3 4 Storing the Column 1 Seal the column with the accessory plug and store it in a cold place where there is little temperature fluctuation 2 For storage replace the mobile phase with a solution of organic solvent and water having the same composition as the mobile phase and then fill it with the solvent used at the time of shipment Refer to the column r
8. olumn with great care A strong shock may cause damage 2 Attach or detach the column when the pressure gage indicates zero 3 The maximum column operating pressure is 20 MPa 2 Attaching the Column 1 The column joint is of the male nut type for tubing of 1 16 inch OD Check that the tubing joints of the system fit correctly and that the ferrule tips are deeply inserted into the joints See Fig 1 2 Before attaching the column replace the liquid in the system with the mobile phase to be used Note the replacement procedure to avoid salting out The shipment solvent is described in the column report enclosed with the column 3 Attach the column according to the direction of the arrow 3 Analysis 3 1 Mobile Phase 1 A mobile phase should be a mixture of acetonitrile and water or aqueous buffer Mobile phase has to contain more than 70 of acetonitrile and minimum 3 of water or aqueous buffer for the HILIC mode to function Upon preparing a buffer solution ammonium formate or ammonium acetate are recommended to avoid precipitation and accidental pressure rise 2 The acceptable pH range for PC HILIC is 3 to 7 5 3 After complete degassing filtrate the mobile phase using a membrane filter 0 45 um or smaller to remove dust A 2 um filter is used at the column inlet To prevent foreign matter from clogging the column inlet filter the use of a line filter is recommended 4 A brand new column contains the mobile phase used i
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