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. Shodex hplc columns technical support for the americas. Detector volume performance monitoring connectors 0. Dr hayder obayes hashim if the calibration curve is a straight line, and it goes through zero, it is valid to. Injection solvent stronger than mobile phase likely split and broad peaks, shape dependent on injection volume and k value. Inject sample in mobile phase simultaneous elution of second component 1. Troubleshooting guide hplc before starting any troubleshooting, whether it is related to instruments or columns, it is essential that safe laboratory practices be observed. We answer to the most frequently asked questions and common issues about hplc columns. If the pressure is normal, there is a problem with 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. Hplc and uhplc columns thermo fisher scientific in. 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. Having an alternate column choice for a method reduces the risk of down time due to column problems such as a. Because peak tailing is the most objectionable property associated.
Even the improvements added in later time were able to shorten the analysis time slightly. Useful data is provided throughout the book, such as. Fortis uhplc ultra high pressure liquid chromatography columns can be run up to bar. The contamination can last for several sequential runs, often decreasing in amount after each injection which is a key observation. Troubleshooting for increased pressure introduction repeated analysis may increase back pressure.
Continuous use of hplc columns under high pressure can cause deterioration and overload of the equipment. In addition to providing an introduction to hplc for pharmaceutical analysis it is intended that this book will be a useful resource. System pressure problems low pressure check if something has changed column, mobile phase, temperature, method if nothing has changed, check for leaks. This guide is intended as an aid in troubleshooting problems that occur in the day to day running of your hplc system. The new technique provided much higher resolution, more. Contents page troubleshooting tables 2 peak shape 2 retention variation 3 ghost peaks 4 column backpressure 5 introduction 6 1. 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. Increasingly, chromatographers are seeking to identify alternate brands of hplc columns suitable for their assays. Elution of organic bases with severe peak tailing is often associated with high silanol activity. 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.
In an hplc system, problems can arise from many sources. Quickly and easily reversed hplc column develop methods based on compound type, usp methods, application, or desired column phase. If a cartridge system is being employed then an integral cartridge holder must also be used. 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. Take this oneday course to explore the practical approach to hplc troubleshooting and maintenance. Ultra high pressure liquid chromatography columns can be run up to bar.
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. If a cartridge system is being employed then an integral cartridge holder must also be. 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. Use table 1 page 5 to determine which components may be causing the trouble. 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. Alternatively add a small amount of sodium azide to the aqueous solvent to inhibit growth. Change selectivity by changing mobile phase or column phase column overloaded 1. Accucore hplc and uhplc columns thermo fisher scientific. Hplc troubleshooting basic principles of hplc 176 the following table lists frequent problems in hplc and possible solutions for. Lc column troubleshooting do you have an equivalent column for my lc column. The peak shape was a secondary issue resolved by flushing the column. Although there are some reference tables for columns from different suppliers, the result may change under different chromatographic conditions. Column instuments contribution extra column column contribution extra column band spreading instruments contribution 1.
Pdf highperformance liquid chromatography is one of the most used analytical techniques in industry. Sentry guard column symmetryshield rp18, 5m temperature. 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. This will pick up leaks, loose or disconnected tubing, changes. Ensure that an organic solvent is flushed through the hplc system and column at least once every 48 hours to kill unwanted microbial growth. Retention and peak shape were as expected after cleaning. There are five major of symptoms to help you quicklyidentify the source of the problems. The important segments of hplc system at the same, whether we use a modular system or a more sophisticated unit. Hplc column troubleshooting what every hplc user should. A process of elimination will usually enable you to pinpoint the specific cause and correct the problem. When a problem occurs, it is advisable to perform a quick visual check of the instrument and column. Shimadzu hplc systems demonstrate high reliability, with outstanding performance, such as ultralow carryover and exceptional area reproducibility, and superior data quality.
Ipshita chattopadhyaya, ekta malhotra department of pharmacy, jaipur national university, jaipur, india. In the late 1970s, new methods including reverse phase liquid chromatography allowed for improved separation between very similar compounds 6 by the. Column instuments contribution extracolumn column contribution extracolumn band spreading instruments contribution 1. The important segments of an hplc system are the same, whether you use a modular system or a more. It is also intended as a guide to best practice in systems that are free from problems.
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. If system pressure is high loosen fittings beginning with the last connection in line and working backward to the pump 20 waters corporation 9. 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. Scientific hplc troubleshooting guide poster,4 where all troubleshooting suggestions are available at a glance and a useful suggestion can be found much faster. Different types of hplc columns are used in analysis of different pharmaceutical compounds according to their nature and column separation capacity. Being the most common principle hplcuhplc separation. In addition, they are carefully designed for broad applicability and easy operation. The sample passes through the column with the mobile phase and separates in its. So take off you lab coat, relax and let our web tool do the work.
Comparison guide to c18 reversed phase hplc columns. The columns can be regenerated rapidly using a few column volumes of organic solvent, preferentially the organic modifier in your mobile phase. Change selectivity by changing mobile phase or column phase column. An ion exchange column can be either cationic or anionic. Although hplc method development has been improved by advances in column technology and instrumentation, problems still arise. Use table 1 to determine which components may be causing the trouble. A few of the most commonly used types are described below, classified by separation mechanism. With over 40 years as a leader in lc column technology, you can rely on the quality of thermo scientific hplc and uhplc columns. Hplc column troubleshooting what every hplc user should know. In this guide we offer you a systematic means of isolating, identifying, and correcting many typical problems. 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.
Experience new benchmarks the nexera series is a family of hplc systems that. 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. An alternative for people with smart phones is the thermo scientific hplc troubleshooting guide an app for iphone, android, and windows platforms. A wide variety of hplc column types are now available for various analytical applications. Introduction hplc lthough hplc method has been improved by advances in column technology and instrumentation, problems still arise. 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. 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. Hplc questions and answers pdf free download in biochemistry mcqs,interview questions,objective questions,multiple choice.
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. Columns are the main component in hplc because the column is responsible for the separation of the sample components. This is the hplc part which pumps the mobile phase at a constant pressure and flow rate through the column. Column to column and batchtobatch reproducibility 5. Hplc troubleshooting guide thermo fisher scientific. 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. Never allow a hplc column or system to stand with water or buffer in it for an extended period of time. A troubleshooting guide successful hplc operation part of thermo fisher scienti. Hplc troubleshooting guide phenomenex uhplc, hplc, spe. You will be logged off in seconds due to inactivity. Therefore, it is important to monitor column back pressure regularly and solve the problem timely. Preventing column back pressure problems filter mobile phase. Introducing accucore hplc columns with coreenhanced technology. Comprehensive guide to successfully troubleshooting hplc columns.
Lc chromatography troubleshooting hplc columns in india. Flush the column with 2030 column volumes of mobile phase. If high back pressure still appears, then the blockage is in the system. Not enough solvent in bottle o replace the solvent bottle problem with check. Carryover carryover contamination in hplc and lcms systems. Aqueous gfc and nonaqueous gpc n amino acid analysis n sfc columns n hplc specialty columns for analysis of. Other extra column effects sampling rate time constant. Mobile phase ph 7 likely column void due to silica dissolution unless specialty column used, zorbax extendc18 stable to ph 11 3. 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. Hplc troubleshooting guide phenomenex uhplc, hplc, spe and gc. Hplc troubleshooting hints and tips chromatography. Sometimes a similar packing may be used for the guard column. Being the most common principle hplc uhplc separation.