Hplc Troubleshooting
Páginas: 6 (1485 palabras)
Publicado: 7 de septiembre de 2011
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Bulletin 826E
HPLC Troubleshooting Guide
How to identify, isolate, and correct the most common HPLC problems
Although HPLC method development has been improved by advances in column technology and instrumentation, problems still arise. In thisguide we offer you a systematic means of isolating, identifying, and correcting many typical problems. The important segments of an HPLC system are the same, whether you use a modular system or a more sophisticated unit. Problems affecting overall system performance can arise in each component. Some common problems are discussed here. Solutions to these problems are presented in easy-to-use tables.E000648
CONTENTS:
Isolating HPLC Problems ................................................... 2 How to Prevent Mobile Phase Problems ............................ 2 Isolating Pump Problems .................................................. 3 Injector and Injection Solvents .......................................... 3 Column Protection ...........................................................3 Getting the Most from Your Analytical Column ................ 3 Solving Detector Problems ................................................ 4 Column Heater, Recorder ................................................. 4 Keeping Accurate Records ................................................ 4 Problem Index .................................................................. 4 HPLC Problems, Causesand Remedies ........................ 5-13 Restoring Your Column's Performance ...................... 14-15 Preventing and Solving Common Fitting Problems .......... 16 A Selection of Column Protection Products ............... 17-20
sigma-aldrich.com
Isolating HPLC Problems
In an HPLC system, problems can arise from many sources. First define the problem, then isolate the source. UseTable 1 (page 5) to determine which component(s) may be causing the trouble. A process of elimination will usually enable you to pinpoint the specific cause and correct the problem.
To prevent bubbles in the system, degas the mobile phase. Generally an in-line degasser is a first choice, but sparging with helium can be an alternative if the mobile phase does not contain any volatile components. Useion pair reagents carefully. The optimum chain length and concentration of the reagent must be determined for each analysis. Concentrations can be as low as 0.2 mM, or as high as 150 mM, or more. In general, increasing the concentration or chain length increases retention times. High concentrations (>50%) of acetonitrile or some other organic solvents can precipitate ion pair reagents. Also, somesalts of ion pair reagents are insoluble in water and will precipitate. Avoid this by using sodium-containing buffers in the presence of long chain sulfonic acids (e.g., sodium dodecyl sulfate), instead of potassium-containing buffers. Volatile basic and acidic modifiers, such as triethylamine (TEA) and trifluoracetic acid (TFA), are useful when you wish to recover a compound for furtheranalysis. These modifiers also let you avoid problems associated with ion pair reagents. They can be added to the buffer at concentrations of 0.1 to 1.0% TEA or 0.01 to 0.15% TFA. Increasing the concentration may improve peak shape for certain compounds, but can alter retention times. Recycling the mobile phase used for isocratic separations has become more popular in recent years as a means of reducingthe cost of solvents, their disposal, and mobile phase preparation time. An apparatus such as the Supelco SRS-3000 or SRS-1000 Solvent Recovery System uses a microprocessor controlled switching valve to direct the solvent stream to waste when a peak is detected. When the baseline falls under the selected threshold, uncontaminated solvent is directed back to the solvent reservoir.
How to...
Bulletin 826E
HPLC Troubleshooting Guide
How to identify, isolate, and correct the most common HPLC problems
Although HPLC method development has been improved by advances in column technology and instrumentation, problems still arise. In thisguide we offer you a systematic means of isolating, identifying, and correcting many typical problems. The important segments of an HPLC system are the same, whether you use a modular system or a more sophisticated unit. Problems affecting overall system performance can arise in each component. Some common problems are discussed here. Solutions to these problems are presented in easy-to-use tables.E000648
CONTENTS:
Isolating HPLC Problems ................................................... 2 How to Prevent Mobile Phase Problems ............................ 2 Isolating Pump Problems .................................................. 3 Injector and Injection Solvents .......................................... 3 Column Protection ...........................................................3 Getting the Most from Your Analytical Column ................ 3 Solving Detector Problems ................................................ 4 Column Heater, Recorder ................................................. 4 Keeping Accurate Records ................................................ 4 Problem Index .................................................................. 4 HPLC Problems, Causesand Remedies ........................ 5-13 Restoring Your Column's Performance ...................... 14-15 Preventing and Solving Common Fitting Problems .......... 16 A Selection of Column Protection Products ............... 17-20
sigma-aldrich.com
Isolating HPLC Problems
In an HPLC system, problems can arise from many sources. First define the problem, then isolate the source. UseTable 1 (page 5) to determine which component(s) may be causing the trouble. A process of elimination will usually enable you to pinpoint the specific cause and correct the problem.
To prevent bubbles in the system, degas the mobile phase. Generally an in-line degasser is a first choice, but sparging with helium can be an alternative if the mobile phase does not contain any volatile components. Useion pair reagents carefully. The optimum chain length and concentration of the reagent must be determined for each analysis. Concentrations can be as low as 0.2 mM, or as high as 150 mM, or more. In general, increasing the concentration or chain length increases retention times. High concentrations (>50%) of acetonitrile or some other organic solvents can precipitate ion pair reagents. Also, somesalts of ion pair reagents are insoluble in water and will precipitate. Avoid this by using sodium-containing buffers in the presence of long chain sulfonic acids (e.g., sodium dodecyl sulfate), instead of potassium-containing buffers. Volatile basic and acidic modifiers, such as triethylamine (TEA) and trifluoracetic acid (TFA), are useful when you wish to recover a compound for furtheranalysis. These modifiers also let you avoid problems associated with ion pair reagents. They can be added to the buffer at concentrations of 0.1 to 1.0% TEA or 0.01 to 0.15% TFA. Increasing the concentration may improve peak shape for certain compounds, but can alter retention times. Recycling the mobile phase used for isocratic separations has become more popular in recent years as a means of reducingthe cost of solvents, their disposal, and mobile phase preparation time. An apparatus such as the Supelco SRS-3000 or SRS-1000 Solvent Recovery System uses a microprocessor controlled switching valve to direct the solvent stream to waste when a peak is detected. When the baseline falls under the selected threshold, uncontaminated solvent is directed back to the solvent reservoir.
How to...
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