Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, December 8, 2018

Part 1, Overlooked HPLC Chromatography Standard Operating Procedures (SOP's); Mobile Phase Preparation

As a scientific consultant, I often review company overall laboratory operations and make recommendations regarding documentation and procedures which may improve their accuracy and results. Some of these recommendations come in the form of SOP's.

Here is the first example (of three installment posts) of a 'must have' SOP' which should be in place for any laboratory performing HPLC analysis.

Part 1 :  
Procedure(s) For the Preparation of Mobile Phase:

Proper documentation of HPLC methods should always include all of the information that someone would need to reliably reproduce the method in another laboratory. This includes the instrument brand, model, module numbers, configuration, details of the column type with the dimensions and particle size, flow rate, mobile phase composition, all detection parameters including flow cell dimensions, path length/volume, wavelength(s) and bandwidth (if applicable), sampling rate, injection solution, injection volume, sample concentration and other critical information. 
  • An area which is often overlooked is HOW the mobile phase solutions are prepared. In addition to stating the chemical grades used, pH measurement checks/adjustments and if any filtering is required, mobile phase preparation often include weighing, dispensing and mixing steps, each of which needs to be described in detail if they are to be reproduced. Without clear directions, the composition of the mobile phase may be different. For example, do you weigh or measure out all liquids? What type of glassware are used to measure volumes? Volumetric flasks, beakers, graduated cylinders (and if so, what tolerance grade or class are they?) When mixing two solutions, do you measure and prepare them separately in two containers (if so, which containers?), then mix them (how do you mix them)? Do you fill one container with one liquid, then fill to the desired level with the second one? Do you need to check the pH of the solution (as well as how to adjust the pH of the solution? With what?)? When and how? Do you have a SOP for the pH meter and how/when to calibrate it? Number of standards used (usually 3 are used)? Is the final solution filtered, and if so, by what method (be specific)? I have seen people use different methods each time they prepare a solution. As you can see, each procedure results in a final composition which will be different. Different mobile phase compositions usually lead to different results. The important message here is to use the SAME method to prepare solutions and to document it in a SOP for the method (and for all methods). Additionally, be sure there is formal training to insure that everyone prepares solutions in the same manner. Most labs will need multiple SOP's for mobile phase preparation, but as a general guideline, you should have one master SOP for the preparation of mobile phase solutions. This will set the standard from which the other SOP's can be based on.
  • Be sure to prepare FRESH, filtered mobile phase solution for use each day. Prepare only enough solution for one or two days use (refrigerate the solution to get 2 days), then dispose of any remaining solution and start with fresh solution.Do not store aqueous solutions at room temperature or for extended periods.
Notes on SOP creation and editing: Make sure you have several people review the draft SOP's before approving. Sometimes what appears clear to you may in fact have a different meaning to someone else. Clear procedures should contain enough detail that people with different backgrounds will each carry out the procedure in the same manner. Often, these types of documents will go through many drafts and even after approved, should also be open to future suggestions to make them even better.