The sample passes through the column with the mobile phase and separates in its. Additional checks of the system are now included in the checklist below. This sop shall be applicable for use of new column and, maintenance and retrieval of old hplc gc columns. We answer to the most frequently asked questions and common issues about hplc columns.
Connect the column taking care to match the arrow to the direction of flow slowly ramp the flow rate to that required, ensuring no pressure surges occur. Fortis uhplc ultra high pressure liquid chromatography columns can be run up to bar. The attached manual describes the xselect csh hplc columns and includes user recommendations. Overdue instrument maintenance can sometimes cause peak shape problems. Back pressure column back pressure should not exceed 400 psi. Highlight the key consumables that need to be replaced by the lc user on a regular basis on any hplc to maintain performance. Connect the column to the pump, but not the detector. View enhanced pdf access article on wiley online library html view download pdf for offline viewing. Page 1 care, maintenance, and troubleshooting of hplc columns columns and consumables edward kim applications engineer january 17, 2008. Use the correct guard column or guard cartridge system securityguard to help remove particulates before they foul your column. Analytical column flow rate direction as indicated on the column label typical flow rate 0. In addition, they are carefully designed for broad applicability and easy operation. Mobile phase ph 7 likely column void due to silica dissolution py,unless specialty column used, zorbax extendc18 stable to ph 11 3. Maintaining your agilent 1100 series hplc system ensuring optimal performance and results.
Use hplc grade solvents and filter all solutions before use. Hplc troubleshooting and maintenance crawford scientific. Columns are the main component in hplc because the column is responsible for the separation of the sample components. When using any hplc uhplc column, be sure to only use hplc grade solvents and materials while also avoiding immiscible solventbuffer combinations. Care, maintenance, and troubleshooting oftroubleshooting of. No matter how good the hplc system performance and the sample preparation are, successful separations may. Flush the column with 2030 column volumes of mobile. Hplc preventative maintenance an annual preventative maintenance visit is required. Components of an hplc system column and integrated guard column solvent return line isolating hplc problems in an hplc system, problems can arise from many sources. Hplc column care guide phenomenex uhplc, hplc, spe and gc. High pressure liquid chromatography 20 chem 4 introduction chromatography can be described as a mass transfer process involving adsorption using a nonpolar stationary phase and a mobile polar phase titrating through the column. Diol reason for column degradation partially blocked plugged frit or column bed. Care, maintenance, and troubleshooting oftroubleshooting.
Use table 1 page 5 to determine which components may be causing the trouble. Protocols, applications and pricing for hplc columns, micro dialysis membranes and kits, 96 well dialysis or equilibrium dialysis plates, and micro sample spe. Maintaining your hplc system publication 59884269 title. It describes common maintenance procedures that can be performed by the user. An ineffective hplc column can affect your analysis, resulting in inaccurate outcomes. The simplest method would be washing with high polar solvent like water first and then go for a slow gradient of organic solvent like acn or methanol until 90 % concentration in low flow of 0. Elution of organic bases with severe peak tailing is often associated with high silanol activity. This sop shall be applicable for use of new column and, maintenance and retrieval of old hplcgc columns. This gives rise to the following principles for a secure analysis cycle. Agilent maintaining your agilent 1100 series hplc system. Completely scalable hplc column solutions from uhplc to.
Setup peaks for integration setup will prevent integrating every wiggle ii. Frequently encountered troubleshooting problems are classified into four categories pressure, baseline, peak, and data. The following general procedures are recommended for regeneration of column performance. Injection solvent stronger than mobile phase likely split and broad peaks, shape dependent on. These guidelines depend on the one hand on the nature of the chromatographic support silica. Alternatively add a small amount of sodium azide to the aqueous solvent to inhibit growth. Hplc, or high performance liquid chromatography, is an analytical tool used in laboratories to detect individual solutes within a complex mixture.
Care, maintenance, and troubleshooting of hplc columns columns and consumables edward kim applications engineer january 17, 2008. Manual injection valves 5 sample loops 7 section 2. Always use a pre column to protect the hplc column. Troubleshooting in hplc preventing column back pressure problems 2. Hplc troubleshooting and maintenance a logical approach to hplc troubleshooting and maintenance is fully explored within this oneday course.
Column pressures will usually increase as the column ages, though sudden increases in pressure are usually a result of a blocked frit see column maintenance. Introduce the major considerations in 12001100 lc user maintenance. Chromatography columns and supplies hichrom limited 1 the markham centre, station road theale, reading, berks, rg7 4pe, uk tel. Cleaning your hplc column might be simple, but it is exceptionally important to the efficiency, lifetime, and productivity of the instrument. Column guide usage and maintenance of hplc columns. Introduce the most commonly observed column related. Manual injection valves 7 replacement rotor seals and stators 9 sample loops 11 section 2.
Take this oneday course to explore the practical approach to hplc troubleshooting and maintenance. Additionally, use of solvent filtration is highly recommended to remove trace impurities from your mobile phase of choice. Shimadzu hplc systems demonstrate high reliability, with outstanding performance, such as ultralow carryover and exceptional area reproducibility, and superior data quality. Maintaining your agilent 1100 series hplc system optimize performance and maximize efficiency in your lab. Be sure to heat the column before turning on the hplc pump, otherwise the column bed will be crushed. Dolan, lc resources, present session 4 of the hplc troubleshooting and preventive maintenance. Hydrocarbon phases, such as c18, are relatively chemically inert. Hplc and column maintenance lcms wiley online library. No matter how good the hplc system performance and the. Types of column used in the laboratory and their storage solvent reversed phase. Retention and peak shape were as expected after cleaning. Mobile phase ph 7 likely column void due to silica dissolution unless specialty column used, zorbax extendc18 stable to ph 11 3.
Hplc column technical guide introduction specifications shipping solvent mobile phase sample diluentinjection solvent column cleaning and storage tips to increase hplc column lifetime benefits of scaling down column internal diameter size tips on maximizing efficiency on uhplc columns benefits of metalfree peek columns. Because peak tailing is the most objectionable property associated. Ensure that an organic solvent is flushed through the hplc system and column at least once every 48 hours to kill unwanted microbial growth. This chapter gives an overview of hplc system maintenance and summarizes strategies and guidelines for hplc troubleshooting.
Care, maintenance, and troubleshooting oftroubleshooting of hplc columns columns and consumables edward kim apppp glications engineer january 17, 2008. The cleaning and regeneration of reversedphase hplc columns. Column care maintenance analytical hplc applications o n h o h3c ch3 oh o n n ch3 ch 3 n h n n n ncbzvaline methaqualone isoamarine mianserin. Clean up samples before injection either filtering to remove particulates or solid phase extraction techniques. The column is the heart of the high performance liquid chromatographic system.
Silica, cn, nh2, diol, alumina isopropanol or hexane ion exchange. Column agingequilibration causes retentionselectivity changes 1 2 column 1 after cleaning with 1% h3po4 the primary analyte was sensitive to mobile phase aging of the column. Experience new benchmarks the nexera series is a family of hplc systems that. Our extensive family of products offers a variety of particle sizes and column designs to meet all separation needs, including improved resolution, enhanced sensitivity, faster analysis and consistent performance. In this guide we offer you a systematic means of isolating, identifying, and correcting many typical problems. Different types of hplc columns are used in analysis of different pharmaceutical compounds according to their nature and column separation capacity. Jun 26, 2019 column care is exceptionally important in a lab, however, if not done properly can affect your columns health. Hplc column troubleshooting what every hplc user should know. Never allow a hplc column or system to stand with water or buffer in it for an extended period of time. Column care is exceptionally important in a lab, however, if not done properly can affect your columns health.
Each restek liquid chromatography lc column is individually packed and tested to. The important segments of an hplc system are the same, whether you use a modular system or a more. With over 40 years as a leader in lc column technology, you can rely on the quality of thermo scientific hplc and uhplc columns. Cover the primary preventative maintenance tasks that the lc user needs to be concerned with and plan for. Hplc and uhplc columns thermo fisher scientific us.
Agilent technologies hplc systems all stackable modules for small bench space single system control module front access for customer maintenance ergonomic tubing organization for lowest delay volume and bandspreading single can cable connection all modules have rs. Column maintenance to maximize the life of this ultradurable column, we recommend the following. These tasks are critical to maintain the performance, reliability and accuracy of the instrument. Care, maintenance, and troubleshooting of hplc columns. All consumables are to be checked by looking at the ims counters page for each module of the system. Flow restrictions voidingcrushing of the column bed and loss of performance will occur if. Guidelines for use and care of aminex resinbased columns. Hplc column manufacturer and supplier of the worlds largest range of hplc columns. Although hplc method development has been improved by advances in column technology and instrumentation, problems still arise. Waters offers a large selection of analytical columns and cartridges packed with. When changing to a different mobile phase, make sure the solvents andor buffers are miscible see. For example watermethanol mixtures will generally give higher back.
Injjgpection solvent stronger than mobile phase likely split and broad peaks, shape dependent on injection volume and k value. The active component of the column, the sorbent or the stationary phase, is typically a granular. Autosampler maintenance 8 injection valve maintenance 9 exchanging the needleneedle seat 12 autosampler parts and supplies 15 section 3. Autosampler maintenance 12 injection valve 14 needleneedle seat 17 autosampler parts and supplies 19 section 3. Follow the appropriate flushing procedure for the type of column see page 373, using 1020 column volumes of each solvent see table below. When possible, use eluent filters and guard columns between the hplc pump and the injection valve. The peak shape was a secondary issue resolved by flushing the column. Trace impurities can dramatically degrade hplc columns.
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