It is also intended as a guide to best practice in systems that are free from problems. Column troubleshooting column protection guard column should be regarded as a costeffectivesacrifice to extend analytical column lifetime should contain identical packing material as the analytical column e. The columns can be regenerated rapidly using a few column volumes of organic solvent, preferentially the organic modifier in your mobile phase. Ideally the packing of the guard column should be the same as the analytical column so that the chromatography of the analytical column is not altered. With over 40 years as a leader in lc column technology, you can rely on the quality of thermo scientific hplc and uhplc columns. Comprehensive guide to successfully troubleshooting hplc columns. If system pressure is high loosen fittings beginning with the last connection in line and working backward to the pump 20 waters corporation 9. Introduction hplc lthough hplc method has been improved by advances in column technology and instrumentation, problems still arise. A troubleshooting guide successful hplc operation part of thermo fisher scienti. Fortis uhplc ultra high pressure liquid chromatography columns can be run up to bar. Mobile phase ph 7 likely column void due to silica dissolution unless specialty column used, zorbax extendc18 stable to ph 11 3.
Different types of hplc columns are used in analysis of different pharmaceutical compounds according to their nature and column separation capacity. Having an alternate column choice for a method reduces the risk of down time due to column problems such as a. Before hplc was available, lc analysis was carried by gravitational flow of the eluent the solvent used for lc analysis thus required several hours for the analysis to be completed. Contents page troubleshooting tables 2 peak shape 2 retention variation 3 ghost peaks 4 column backpressure 5 introduction 6 1. Comparison guide to c18 reversed phase hplc columns. When a problem occurs, it is advisable to perform a quick visual check of the instrument and column. Quickly and easily reversed hplc column develop methods based on compound type, usp methods, application, or desired column phase. We answer to the most frequently asked questions and common issues about hplc columns. Sometimes a similar packing may be used for the guard column. System pressure problems low pressure check if something has changed column, mobile phase, temperature, method if nothing has changed, check for leaks. The important segments of hplc system at the same, whether we use a modular system or a more sophisticated unit. Column instuments contribution extracolumn column contribution extracolumn band spreading instruments contribution 1. 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.
Although hplc method development has been improved by advances in column technology and instrumentation, problems still arise. If a cartridge system is being employed then an integral cartridge holder must also be used. In the late 1970s, new methods including reverse phase liquid chromatography allowed for improved separation between very similar compounds 6 by the. Retention and peak shape were as expected after cleaning. Experience new benchmarks the nexera series is a family of hplc systems that. Hplc and uhplc columns thermo fisher scientific in.
Mostly piston types of pumps, also called constant displacement pumps are widely used they have limitations of pressure i. Detector volume performance monitoring connectors 0. Sentry guard column symmetryshield rp18, 5m temperature. Use table 1 to determine which components may be causing the trouble. This is the hplc part which pumps the mobile phase at a constant pressure and flow rate through the column. Inject sample in mobile phase simultaneous elution of second component 1. Useful data is provided throughout the book, such as. Bill champion application engineer february 23, 2011 february 23, 2011 separation fundamentals agilent restricted. Hplc troubleshooting guide thermo fisher scientific. Hplc troubleshooting ebook page 7 hplc column maintenance prevention is better than cure guard columnsdisks a guard column is a short column typically 1020 mm in length that is packed with the same stationary phase material as the analytical column. The sample passes through the column with the mobile phase and separates in its. An alternative for people with smart phones is the thermo scientific hplc troubleshooting guide an app for iphone, android, and windows platforms. Alternatively add a small amount of sodium azide to the aqueous solvent to inhibit growth. Hplc troubleshooting hints and tips chromatography.
Ensure that an organic solvent is flushed through the hplc system and column at least once every 48 hours to kill unwanted microbial growth. Carryover, carryover, carry over, hplc, uhplc, injector, contamination, is a term used to describe a type of sample contamination which causes sample peaks to reappear in later runs which do not actually contain the sample e. Lc chromatography troubleshooting hplc columns in india. If the pressure is normal, there is a problem with the column. Because peak tailing is the most objectionable property associated.
Increasingly, chromatographers are seeking to identify alternate brands of hplc columns suitable for their assays. Introducing accucore hplc columns with coreenhanced technology. In addition to providing an introduction to hplc for pharmaceutical analysis it is intended that this book will be a useful resource. Hplc troubleshooting basic principles of hplc 176 the following table lists frequent problems in hplc and possible solutions for. Flush the column with 2030 column volumes of mobile phase. Being the most common principle hplcuhplc separation. If a cartridge system is being employed then an integral cartridge holder must also be. Accucore hplc and uhplc columns thermo fisher scientific. Carryover carryover contamination in hplc and lcms systems. Never allow a hplc column or system to stand with water or buffer in it for an extended period of time. Ipshita chattopadhyaya, ekta malhotra department of pharmacy, jaipur national university, jaipur, india.
Being the most common principle hplc uhplc separation. Other extra column effects sampling rate time constant. Shodex hplc columns technical support for the americas. At the end of each chapter there is a list of references andor further reading which will help the reader to develop their expertise in the technique. Columns are the main component in hplc because the column is responsible for the separation of the sample components. Elution of organic bases with severe peak tailing is often associated with high silanol activity. Lc columns from different suppliers may have very different retention properties even if the bonding is the same. Even the improvements added in later time were able to shorten the analysis time slightly. Dr hayder obayes hashim if the calibration curve is a straight line, and it goes through zero, it is valid to. Hplc stands for high performance liquid chromatography. 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. Change selectivity by changing mobile phase or column phase column. Troubleshooting guide hplc before starting any troubleshooting, whether it is related to instruments or columns, it is essential that safe laboratory practices be observed. Therefore, it is important to monitor column back pressure regularly and solve the problem timely.
Abstract 1 the developments in column packing technology and suitable equipment paved the way for what is now called high performance or high pressure liquid chromatography hplc. Not enough solvent in bottle o replace the solvent bottle problem with check. How to set up your hplc column inline filter from injector guard cartridge holder to detector column coupler column table 1 selecting tubing id column id tubing id mm microns inches 12 100 0. There are five major of symptoms to help you quicklyidentify the source of the problems. Preventing column back pressure problems filter mobile phase. Hplc column troubleshooting what every hplc user should. Ultra high pressure liquid chromatography columns can be run up to bar.
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. Column instuments contribution extra column column contribution extra column band spreading instruments contribution 1. The peak shape was a secondary issue resolved by flushing the column. Hplc column troubleshooting what every hplc user should know. The contamination can last for several sequential runs, often decreasing in amount after each injection which is a key observation.
Continuous use of hplc columns under high pressure can cause deterioration and overload of the equipment. Hplc troubleshooting guide phenomenex uhplc, hplc, spe and gc. Column aging equilibration causes retentionselectivity changes 1 2 column 1 after cleaning with 1% h3po4 the primary analyte was sensitive to mobile phase aging of the column. With a c18 column in the system, a blank gradient is run and there are peaks in the chromatogram, there is contamination from somewhere. This guide is intended as an aid in troubleshooting problems that occur in the day to day running of your hplc system. So take off you lab coat, relax and let our web tool do the work.
If high back pressure still appears, then the blockage is in the system. Injection solvent stronger than mobile phase likely split and broad peaks, shape dependent on injection volume and k value. Aqueous gfc and nonaqueous gpc n amino acid analysis n sfc columns n hplc specialty columns for analysis of. A few of the most commonly used types are described below, classified by separation mechanism. 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. All other ham lc columns all other sho lc columns ax pak chi chiradex chiral cdph curosil taxol hypersil lichrosorb nucleodex nucleosil ab nucleosil hd nucleosil plgel sumichiral pdf link. Change selectivity by changing mobile phase or column phase column overloaded 1. Check if something has changed column, mobile phase, temperature if nothing has changed, remove the column and replace it with a union to check if the system pressure is the usual. This will pick up leaks, loose or disconnected tubing, changes. Column to column and batchtobatch reproducibility 5. Use table 1 page 5 to determine which components may be causing the trouble. The new technique provided much higher resolution, more. Lc column troubleshooting do you have an equivalent column for my lc column. No pressure 20 waters corporation 10 air in system o prime the pump methanol or ipa to remove air air in solvent lines.
Take this oneday course to explore the practical approach to hplc troubleshooting and maintenance. Page 2 troubleshooting in hplc 20 00 15 00 10 005 0 0 0 05 1 0 1 5 2 2 time 5 min ma u february 23, 2011. Its primary aims are to provide a logical process when troubleshooting, to maximize system. The chemical and physical properties of any solvents used should be known and the material safety data sheet msds for theses solvents should be readily available. Scientific hplc troubleshooting guide poster,4 where all troubleshooting suggestions are available at a glance and a useful suggestion can be found much faster. Hplc troubleshooting guide phenomenex uhplc, hplc, spe. In addition, they are carefully designed for broad applicability and easy operation. Pdf highperformance liquid chromatography is one of the most used analytical techniques in industry. The important segments of an hplc system are the same, whether you use a modular system or a more. In this guide we offer you a systematic means of isolating, identifying, and correcting many typical problems.
In an hplc system, problems can arise from many sources. Shimadzu hplc systems demonstrate high reliability, with outstanding performance, such as ultralow carryover and exceptional area reproducibility, and superior data quality. A process of elimination will usually enable you to pinpoint the specific cause and correct the problem. Although there are some reference tables for columns from different suppliers, the result may change under different chromatographic conditions. An ion exchange column can be either cationic or anionic. Hplc troubleshooting ebook page 7 hplc column maintenance prevention is better than cure guard columns disks a guard column is a short column typically 1020 mm in length that is packed with the same stationary phase material as the analytical column.
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