3. Troubleshooting for Increased Pressure
Contents
1. 3 Troubleshooting for Increased Pressure v a O Introduction S c Repeated analysis may increase back pressure Continuous use of HPLC columns under high pressure can cause deteri 3 oration and overload of the equipment Therefore it is important to monitor column back pressure regularly and solve the 2 problem timely _ Identification of the Clogging Site c The back pressure increase can be due to clogging of a column or clogging of the equipment First of all identify the a O clogging site a Q Schematic Diagram of HPLC 5 a UD 8 Q EE ca lt v oO g Waste Solution Recorder D Mobile Phase D Suction filter gt Pump gt Line Filter gt Column Pre filter lnjector gt Guard Column gt Analytical Column Detector i gt lt A Disconnect the system components one by one to identify the clogged component s Start by disconnecting the column D O from the system and measure the pressure of flowing mobile phase The pressure should be close to zero If the flow 2 v pressure without a column is normal then pressure increase is due to the clogged column The cause of clogging needs S to be determined and preventative measures implemented The column may need to be replaced 5 a Clogging of Equipment __ Identify the specific clogging site according to the method above lt S Case 1 High pressure caused by clogged tubing 8 Cause Salt dep2. in mobile phase Cause Samples have adsorbed to packing material or deposited in a column 2 Solution Wash the column for 30 minutes with half of the normal flow rate using a solvent that can dissolve the adsorbed 3 substances Here are washing procedures for each column type Q Reversed phase columns a When absorbed substances are not proteins wash with methanol or tetrahydrofuran 2 b When absorbed substances are proteins wash with 50 70 of acetonitrile water containing 0 1 of trifluoroacetic 2 acid However proteins may precipitate in high concentration of organic solvent gt COSMOSIL SL II Wash with methanol tetrahydrofuran or ethanol Fullerene columns Wash with o dichlorobenzene 1 2 4 trichlorobenzene COSMOSIL Sugar D NH2 HILIC columns Wash with 50 50 v v acetonitrile water for NH2 MS and 100 water for Sugar D and HILIC columns a Prevention a Choose appropriate pretreatment for each sample For more information please see page 193 Technical Oo Information 4 5 b We also recommend using guard columns please see page 199 Technical Information 6 2 lt Attention When wash columns do not connect column outflow end to the detector Let the solvent flow into waste gt Excessive washing may deteriorate the performance of columns Do not use strongly alkaline solution more than pH 7 5 or strongly acidic solution less than pH 1 5 for silica base packing material Store columns in manufacturer recommended s
3. osit in tubing Solution Disconnect the column and any other equipment before pumping water through the tubing Washing in a reversing a direction is also an effective way If the situation does not improve replace the tubing with a new one Case 2 High pressure caused by clogged pump Cause Line filter of pump is clogged Take apart the line filter and soak it in the solvent then clean in an ultrasonic cleaner If the situation does not improve replace the line filter with a new one Solution Case 3 High pressure caused by clogged manual injector ry gt 2 O a 2 o S 7 Cause Manual injector is clogged Inject with 20ml of contaminant dissolving solvent e g methanol by syringe Wash both lines in LOAD and INJECT position Cleaning the injector in an ultrasonic bath is also effective If solids caused the clogging wash the injector in a reversing direction If the situation does not improve replace the injector with a new one Solution x pul IIA 191 Technical Information v What should do when a clogged column caused pressure increase c E Case 1 Salt deposit in a column caused by pumping high organic solvent after using buffer solution z Cause Salt deposit in a column a Solution Wash columns for 30 minutes at half the normal flow rate using 10 organic solvent methanol or acetonitrile in water to dissolve salt deposit If the situation does no
4. t improve wash the column with 100 water under the same condition Prevention To switch to high organic solvent concentration after using a buffer first wash a column with a salt free mobile phase with the same concentration of organic solvent as the buffer then switch to the mobile phase of higher organic concentration E Example To change mobile phase from 10 90 v v acetonitrile 20mmol l phosphate buffer pH2 5 to 90 10 v v g acetonitrile water first wash the column for 15 minutes with 10 90 v v acetonitrile water and then switch o to 90 10 v v acetonitrile water a Case 2 The sample is not completely dissolved or unfiltered 2 T Cause Column frit is clogged by insoluble sample or impurities Solution Connect the column in the reverse direction and disconnect from the detector and then wash the column for 30 r minutes at half of the usual flow rate with the same mobile phase used for analysis If the situation does not improve g change the frit in the front end of the column We can replace end fittings with a paid service fee 2 Prevention We strongly recommend filtering sample and or mobile phase For more information please see page 193 Technical Z Information 4 Sample Pretreatment for HPLC 1 filtration S 2 Attention If the column is continually connecting in the reverse direction it may deteriorate S oo Case 3 Protein samples that adsorb easily to the column or samples that are slightly soluble
5. torage solvent after washing If the column performance does not improve after washing replace the column Case 4 Graduate pressure increase over time Casue 1 Contamination of column due to normal long term use Prevention Wash the column like Case 3 N oO e z T e c p o gt Casue 2 Column damage due to normal long term use Prevention Replace the columns No improvement in performance after washing When the column performance has not improved after washing we recommend replacing the column to lessen pressure burden on the instrument You could continue to use the column if peak shape is acceptable and the maximum pressure is less than 20 MPa VII Index 192
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