Troubleshooting, HPLC systems column

Set the flow rate of the HPLC system to 1 ml/min across a 5-p.m x 250-mm x 4.6-mm C18 polymeric support reversed-phase column or equivalent at ambient temperature. Set the detector at 520 nm. Inject 50 ul sample into the HPLC system and start a gradient similar to that outlined in Table FI. 3.2. Analyze data as described (see Data Anaylsis in Critical Parameters and Troubleshooting). 

It is imperative that the HPLC instrument, including the detector, is working correctly. The easiest way to check this is by first running a blank. If there is no response, one can move onto injecting the standards. If there is a response to the blank, the column may have been overloaded prior to this run. Refer to a troubleshooting guide for the specific HPLC system. The internet is also an invaluable source for troubleshooting (e.g., see Internet Resources). Keep in mind that the source of the problem may not be the system but may in fact be the column. 

Records also help prevent mistakes, such as introducing water into a silica column, or precipitating buffer in the system by adding too much organic solvent. Many analysts occasionally modify their HPLC systems for a variety of reasons. Reliable records are the best way to ensure that a modification does not introduce problems. For problems relating to pumps, detectors, automatic samplers, and data systems, instrument manuals provide suitable troubleshooting guides. 

Part II shows you how to make the best use of the common columns and how to keep them up and running. (Chapter 6 on column healing should pay for the book in itself) It discusses the various pieces of HPLC equipment, how they go together to form systems, and how to systematically troubleshoot system problems. We will take a look at the newest innovations and improvements in column technology and how to put these to work in your research. New detectors are emerging to make possible analysis of compounds and quantities that previously were not detectable. 

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