Modern HPLC Method Development Tips (PART I):

This is the first of two articles which will provide suggestions on how to improve the HPLC (UHPLC) method development process. - PART I

Method Development, BEFORE YOU BEGIN

1. Start with a working system: Before you start to develop a method, make sure your chromatograph is operating in top condition! Passing an OQ test and regular maintenance are not enough. You need to have the proper training and experience which will allow you to identify signs of trouble all of the time. For example: Do you see any salt or buffer crystals on the outside of one or more parts (esp the fittings or the pump head)? If you do, then you have a leak or other problem which needs to be addressed. Do you know what the normal backpressure should be when the system is operating correctly at a fixed flow rate with a single pure solvent (or defined mixture) running through? You should, as this provides a quick check that the pump is functioning properly. These types of quick checks should be done each time you use the HPLC and, if applicable, should be part of a general SOP. Is the flow rate shown on the display correct? A small graduated cylinder and a stopwatch (watch, timer) to measure the mobile phase output from the detector can answer this question in a few minutes. Are the pressure and flow rate stable (ripple <1%)? Verify by recording the pressure readings over time and measuring the S/N of the baseline with your detector to estimate the flow rate stability (noise). If they are not stable, then do not start an analysis. Find and fix the cause of the instability before running samples or standards. Some common causes of flow instability include: a sticking check valve, failure to properly degas the mobile phase, a leak, poor mixing or a miscibility problem.

2. Mobile Phase Preparation: Have you prepared enough fresh mobile phase to use during the day? Mobile phase which is mostly aqueous and contains buffer salts often experiences significant growth of microbes in just one or two days (esp. at RT, so store at 4 C). Mobile phase must be clean, filtered and freshly prepared using an established standard method (this should be described in a SOP so it is made the same each time). This applies to all mobile phase additives used too (choose the highest grade possible. “HPLC Grade” or ultra-high purity and look for an analysis sheet). Water used should be freshly prepared, high resistance (~18 Mega ohm) HPLC grade water which has been filtered through a 0.22 u filter.

3. Degassing. Mobile phase solutions should be continuously degassed before being introduced into the HPLC pump(s). This is sometimes done via low pressure Helium gas sparging, but more commonly today, via in-line, electronic vacuum degassing modules. Placing bottles in an ultrasonic bath or using vacuum filtration systems alone to degas solutions before use does a very poor job of degassing and results in the gas slowly dissolving back into the solution the very moment you stop degassing them. Baseline instability, drift and flow rate variation are the result. These problems may contribute to poor quality integration and high %RSD. To insure that your electronic vacuum degasser provides the maximum benefit, it must be purged before use each day to remove the gas which has re-dissolved back inside them. To properly flush each chamber/channel, you will need to know its volume (provided by the manufacturer). *Individual vacuum chamber volumes vary from ~ 0.50 ml to as much as 50.00 ml EACH! At a flow rate of 1.000 ml/min, it could take from 0.5 to 50 minutes just to flush one vacuum chamber (and most systems have 4 chambers!). Luckily, most modern vacuum degassers have chambers with volumes of between 0.5 and 12 mls each and HPLC pumps which can be run at either 5 or 10 ml/min maximum to make this process go faster. Remember to purge these chambers with fresh solution at the start of each day. 

Best Practice: Each channel of your HPLC system should be flushed (purged) with freshly degassed mobile phase at the start of each day, before you run any samples.

 

Before starting ANY HPLC analysis, the HPLC pump must be running and operating with no problems, achieving a stable baseline, steady flow rate with as little pulsation as possible (~ 1% ripple or less). Accuracy depends on this.

Part II of this article discusses initial method development steps.