Showing results for 
Search instead for 
Did you mean: 
Team TFS
Team TFS

092221 Image 1 Blog.jpg


HPLC method development is a bit like trying to solve a maze. The goal – the cheese, if you will – is to develop a method that provides adequate resolution, sensitivity, robustness and throughput.


Sounds simple enough, right? But when you begin to consider all relevant parameters, things can get a little overwhelming. Experimental variables include stationary phase, mobile phase (identity, modifiers, pH), column dimensions, temperature, gradient program. The list goes on.


As if this “maze” isn’t complicated enough, each parameter is intimately connected to the other. This interconnectivity means even a small change in one parameter can shift the optimal range for others, ultimately affecting the separation efficiency.


If at this point you aren’t starting to feel like the mouse, then you are probably the one who should be writing this blog post.


Before we dive into the top ways to simplify and accelerate your HPLC method development, let’s take a moment to consider the process for developing a typical method. While developing a sample preparation protocol is an essential part of this process, for the purposes of this blog post, we will focus on steps directly related to the method parameters.


At the risk of oversimplifying things, HPLC method development consists of roughly four key steps:


Step 1: Method scouting


After settling on your target parameters for method critical quality attributes (e.g., resolution > 1.5) and selecting a separation mode, the first step is to screen column and mobile phase (MP) conditions for a promising starting point.


For experienced chromatographers, this step entails selecting a few promising column and mobile phase candidates and comparing results using an isocratic or simple gradient method. Because downstream method development and the overall success of the method strongly depend on method scouting decisions, method development experience often saves time and effort.


Novice chromatographers are forced to cast a much wider net, testing more column types, MP compositions, and MP additives. Without thorough initial scouting, methods run the risk of not meeting targets, particularly concerning throughput. On top of that, method scouting requires a significant amount of effort for manual MP preparation plus column and MP switching.


Step 2: Method optimization


Ok, so you’ve settled on the most promising mobile phase and column combination – great job! Sadly, you aren’t out of the woods yet. Odds are you have some unresolved critical pairs, or at least a method long enough to allow you to eat lunch and run some errands.


Either way, method optimization is required to speed up the separation and parse out analytes with similar retention factors. Probably the most time-consuming step, this stage involves iterative testing of various combinations of separation parameters until target critical quality attributes such as resolution or peak symmetry are achieved. At this point you should explore various gradient programs to maximize resolution between critical peak pairs.


Overall, this phase requires operators to run large numbers of samples and repeatedly make decisions based on each set of results. While user experience and knowledge are invaluable, the process requires critical analysis of numerous data sets, making HPLC method optimization laborious even for the most seasoned veterans.


Step 3: Robustness testing


Within the pharmaceutical industry, “the robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage.” 1


Rather than repeated analysis of a sample under the same conditions, robustness testing requires an exploration of how predetermined changes in method parameters impact critical quality attributes. Testing is performed either by varying a single parameter or multiple parameters at a time, with respective trade-offs in experiment time and data complexity. The result is an understanding of the range of each parameter within which a method delivers consistent results. This mechanistic understanding is required for confidence in long-term routine analysis as well as transferring HPLC methods successfully between instruments and labs.


While the other stages of method development always proceed in the same order (scouting → optimization → validation), robustness testing tends to be less standardized across industries and even between labs and can be performed during any stage within method development, including scouting, optimization and validation.


In the pharmaceutical industry, best practice is to perform robustness testing using quality by design principles during method development. If performed during method development, robustness testing is not required during future method validation.1


Step 4: Method validation


Following development, methods are validated to ensure suitability for a given application based on a set of industry performance standards. Such standards may include, but are not limit to, method characteristics such as accuracy, precision, detection/quantitation limits, linearity, and robustness (if not performed during development).


While the quality by design approach requires a rigorous method development process, it ultimately helps streamline method validation. Successful method validation often necessitates following Good Laboratory Practice (GLP), which includes maintaining proper documentation throughout the entire process.2 Once validated, methods are deemed fit for usage within regulated environments.


Accelerate your HPLC method development


Hopefully, with this high-level overview, you now have a slightly better grasp on the steps and requirements for developing HPLC methods in regulated industries. The one universal ingredient in all method development processes: time.


While developing methods may take shorter or longer depending on the sample complexity and performance requirements, it is almost always time-consuming and labor-intensive. Even the most seasoned veterans take weeks to generate methods.


And it isn’t going to get any easier, due to increasing demands on analytical methods stemming from ever-tightening government restrictions on the food and pharmaceutical industries.


“But isn’t there some way to improve my lab’s method development process?”


I’m glad you asked.


Yes, there are a variety of tools available for increasing the rate of your method development – and even enhancing final method quality. Thermo Scientific™ Vanquish™ Method Development Systems accelerate method development through a comprehensive suite of automated hardware and software tools. With this new system, you can screen up to six columns and 13 eluents with the Thermo Scientific™ Viper™ Method Scouting Solutions Kit and Solvent Extension Kit.


Integration of ChromSwordAuto with Thermo Scientific™ Chromeleon™ CDS automates experimental planning and execution during all stages, from method scouting through robustness testing and validation. To ensure digital regulatory compliance, all your data is stored securely within the Chromeleon Data vault. Chromeleon CDS is also compatible with the S-Matrix™ Fusion QbD™ method development software package.


Learn more

Vanquish Method Development HPLC and UHPLC systems

Product Spotlight: Vanquish Method Development Systems




  1. International Conference on Harmonization (ICH) Tripartite Guideline, Topic Q2 (R1): Validation of Analytical Procedures: Text and Methodology, 2005.
  2. Food and Drug Administration, 81 FR 58341: Laboratory Practice for Nonclinical Laboratory Studies, 2016.