What causes high pressure in HPLC?
What causes high pressure in HPLC?
High back pressure in LC instruments is usually caused by foreign material blocking the flow of mobile phase. Although crimped PEEK or stainless steel tubing will occasionally be the culprit, particulates clogging the system are most often the cause.
What are the reasons for getting negative peaks in HPLC?
Negative peaks are most often caused by difference in refractive index between the sample solvent, sample and mobile phase. They are also caused after routine maintenance when the system has not been reconfigured correctly. Qualitative assays do not measure exact quantities of an analyte in solution.
Why sometimes retention problem arises in HPLC?
Increasing back-pressure may indicate a contamination of the column, but even a clogged frit can affect retention times. The pressure needed to push the mobile phase through the frit warms up the mobile phase by friction, and this increase in temperature can affect the retention times.
How do you reduce negative peaks in HPLC?
Solution: Adjust or change sample solvent. Dilute sample in mobile phase whenever possible. d) Mobile phase more absorptive than sample components to UV wavelength (vacancy peaks). Solution: Change UV wavelength or use mobile phase that does not adsorb chosen wavelength.
What affects retention time HPLC?
Retention time depends not only on the structure of the specific molecule, but also on factors such as the nature of the mobile and stationary phases, the flow rate of the mobile phase, and dimensions of the chromatographic column. Retention time is usually characteristic for a specific compound in a given separation.
Why do peaks shift in HPLC?
If all of the peaks are shifted by about the same time interval, the variations are likely caused by variation in flow rate in the instrument. This can be a shift in either direction, but is often toward longer retention times/slower flow rate.
How do you flush flow cell HPLC?
Reverse the flow cell inlet and outlet lines. Start the flow at a very low rate, ensuring that the back pressure remains below the maximum rating (1000 psi for UV, 100 psi for RI) for the flow cell. Flush at a low flow for at least one hour. Flush with HPLC grade water until the effluent returns to neutral.
What are some of the problems with HPLC?
HPLC Problems Waters Corp. © 2002 Potential Sources of Chromatographic Problems z Mobile Phase z Injector z In-Line Filter z Column z Detector z Sample z Pump z Guard Column z Connecting Tubing and Fittings z Integrator/Recorder Software Scientist/Analyst — need for logical approach to save time
How to troubleshoot a Sigma Aldrich HPLC column?
HPLC Troubleshooting Guide 1 Further Recommendations. We also suggest referring to the maintenance and troubleshooting sections of your instrument manual. 2 Restoring Your Column’s Performance. 3 Preventing and Solving Common Hardware Problems. 4 A Selection of Column Protection Products.
What should I do if my HPLC detector is clogged?
A clogged column frit is another common HPLC problem. To minimize this problem from the start, use a precolumn filter and guard column. To clean the inlet, first disconnect and reverse the column. Connect it to the pump (but not to the detector), and pump solvent through at twice the standard flow rate.
Why does retention time increase in HPLC chromatography?
As a result of using a longer tubing before the column, an increase in the retention time to the right occurs, as shown above. This increase in retention time is more obvious in the later peaks (from bottom to the top chromatograms). The reason is illustrated in the next graph.