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June 27

What is Your HPLC Doing?

The quiet workhorse of most laboratories is usually a HPLC (in other words, HPLC is the most common technique used in analytical laboratories). There are SOPs, test methods, training, non-conformance, and verification to ensure analysts maintain consistent working practices, but how do you ensure that your HPLC maintains consistency? It is vital that the equipment is giving accurate and reproducible data.

System Suitability Tests (SST) are great to run within your data to ensure that the HPLC, mobile phase and column are working correctly as a whole system throughout your sequence; However, these can fail or even show that they are irregular for a routine test method. 

Good Routine/Preventative Maintenance is key to understanding the possibilities of what could go wrong with the system, and how these could be solved or prevented. Planned Maintenance can be simple tasks such as ensuring there are no early signs of leaks around the many connections.  It is also important to clean or replace consumable parts which would be exposed to general wear and tear. More complex tasks would be to ensure the system is working sufficiently by performing predetermined tests.  These tests ensures the HPLC is performing accurately within qualification, especially when performing tests against required specifications.

Most leaks in a system can be easily identified during routine use with pressure changes or shifting retention time. Pump based leaks, if small yet consistent, would reduce the pressure without seeing a wandering retention time of peaks; However, a leak that occurs after the detector, possibly with waste tubing connections, would not change the pressure scale or affect the chromatography during the analysis performed. If a leak is left unfixed, it can build up and cause greater issues with the detector or result in other possible electrical issues.

Critical issues that go undetected and could cause routine running to generate unusable data would most commonly be from the pump or the detector. Autosampler issues can usually be seen through RSD (Relative Standard Deviation) SSTs of a sequence – these are vital to ensuring confidence in the LC’s data throughout the run, but an understanding of what is expected from your test method is required due to expected peak size.

A HPLC pump is the module most affected by the specific mobile phase used in the analysis, which can have many different characteristics. At Broughton, we run many different test methods which means that it is important to ensure the pumps are thoroughly cleaned. This can prevent any buffer crash out in the pump which subsequently could cause damage or blockages throughout the system if not dealt with. This can all occur without producing unusable data. The pump also has the most moving parts of a HPLC and therefore, will suffer from general wear and tear. Other parts and consumables that will suffer from general wear and tear issues throughout normal use are seals around the pistons. These need to be tight enough to form a seal to ensure that the flow path keeps flowing freely and liquid is under control.

Detectors could be classed as the most important module within the HPLC system as this produces the data, and UV is the most commonly used type of detector. The quality of the chromatogram produced will be dependent on noise and peak shape which both can be affected by the UV lamp, flow cells, and the wavelength accuracy of the detector set up. A signal to noise ratio, baseline noise and lamp intensity all are observation points to understand how well a detector is working.

Having a good preventative maintenance schedule that consists of a regular inspection and performance review of the system in full would ensure that the HPLC keeps on going under any pressure.  Routine qualification is required to ensure that the system is working correctly, alongside the routine SSTs that will be performed throughout routine analysis, giving the consistent confidence that your system is working well.

Techniques and capabilities

 

About the Author

Michael joined Broughton Laboratories as an Analytical Chemist in 2012, before specialising in chromatography techniques and supporting the growth in technology capabilities within the business. He is the Scientific Technology Systems Specialist here at Broughton Laboratories. He supports the laboratory with equipment, technical and troubleshooting queries. He also carries out routine maintenance, performance qualification, training, and is an expert in Chromeleon CDS.