Sample prep
Matrix effects
Method steps
Automated methods
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Sample preparation is central to successful HPLC and UHPLC analyses. Examples of sample preparation include:

  • Converting solid samples into a liquid form
  • Simplifying complex mixtures
  • Removing interfering matrix
  • Concentrating or diluting

There are many  sample preparation techniques established, and each method has a particular benefit or specific application.

Preparation method

Analytical principle

Application(s)

Dilution

Decrease analyte, solvent, or matrix concentration in sample

Preventing column/detector overloading, reducing sample solvent elution strength

Centrifugation

Sedimentation based on density

Removing large cellular components from solution

Filtration

Remove particulates from sample

Extending column lifetime, preventing clogging of fluidics

Protein precipitation

Desolubilize proteins by adding salt, solvent, or altering pH

Removal of protein from solution

Liquid-liquid extraction

Isolate sample components based on solubility differences in two miscible solvents

Purifying compounds based on polarity/charge

Solid phase extraction

Selective separation/purification of target analytes using a sorbent stationary phase

Isolating small molecules from biological matrices, desalting large biomolecules

Immunoaffinity capture

Selective purification of analyte using an antibody

Isolating  small molecules from biological/environmental/food and beverage matrices

Protein digestion

Enzymatic cleavage of protein into peptides

Generating peptides for bottom-up proteomics/peptide mapping

Derivatization

Chemical reaction to alter the physicochemical properties of analyte

Improving analyte retention, stability, or detectability

Size exclusion

Separate samples components based on size

Protein buffer exchange/desalting, macromolecule removal

Homogenizing

Break down solid sample structure using mechanical force

Disruption of biological tissue, environmental samples for improved extraction

QuEChERS

Multi-step process including homogenizing, liquid-liquid extraction, centrifugation, solid phase extraction

Extraction of pesticides from food and environmental samples

What is the sample matrix?

You can think of the sample matrix as anything in a sample except the analytes of interest, which includes everything from salts to other compounds and solvents. The matrix-type can dictate the sample preparation, the mode of chromatography, and the detection method. Understanding the sample matrix is a fundamental consideration in method development.

What are matrix effects?

Matrix effect is a broad term describing the tendency of specific analyte matrices to alter the detection or quantification of an analyte. This effect usually manifests itself as a bias and results in under or overestimating the solution's existing analyte concentration.

Matrix effects can appear in nearly any stage within an analysis, including sample preparation, separation on the column, and detection. Here are a few general examples:

  • Co-elution of a UV absorbing compound with the analyte of interest
  • Sample pH altering the retention factor of ionizable analytes
  • Ion suppression of the analyte in electrospray ionization

How to mitigate interfering matrix

There are a few common ways to mitigate matrix effects. The correct choice depends on the specifics of the analysis.

If analyte sensitivity is adequate, the most straightforward approach is to dilute the sample in a proper injection solvent. A more dilute sample gives a more negligible matrix effect.

Other solutions include an extraction before analysis, which improves the separation by eliminating possible sources of sample contamination. Using a 2D-LC or switching to a more selective detection method can also circumvent matrix effects.

Lastly, you can perform standard addition without changing the method. But this technique is generally avoided due to the increased number of injections per sample. 

 

Method development steps

For situations where no established method is available, careful planning and execution are necessary to develop a robust procedure. Aside from sample preparation, there are four main steps to know when creating an HPLC or UHPLC method:

  1. Method scouting. Involves screening various column and eluent conditions. The purpose of this phase is to select the best combinations for a successful HPLC separation. In practice, method scouting requires significant manual work for column and mobile phase switching and instrument method creation. By understanding the target analyte properties, scouting can be initially limited to several of the most promising column candidates.

  2. Method optimization. Includes iterative testing of various separation conditions of the HPLC method and is performed to achieve the best possible resolution, speed, and reproducibility. This step is the most time-consuming part of method development and often requires expert knowledge to perfect.

  3. Robustness testing. Done to determine the impact of changing parameters of the separation method. Optimizing robustness is important for many method development and validation processes. 

  4. Method validation. The industry-specific process for determining whether a developed analytical method fits the desired application.

HPLC Method Development Step by Step

Developing a robust, reproducible, and reliable HPLC or UHPLC method can be cumbersome even for an experienced liquid chromatographer. This video teaches you all the steps required to properly develop an LC method.

 

Method development considerations

For method development, three parameters play a role which are—with increasing significance—the compound retention (k), efficiency (N), and selectivity (a). A common way to adjust the selectivity is to change the column chemistry and eluents. If this change is manual, such work is often time-consuming. Fortunately, modern technology allows the automation of such processes.

Method development resources

Developing a robust, reproducible, and reliable HPLC or UHPLC method can be cumbersome even for an experienced liquid chromatographer. 

In some cases, you can entirely avoid method development by searching the Thermo Scientific AppsLab Library of Analytical Applications. This online library contains a searchable repository of thousands of applications with detailed method information and prepopulated eWorkflow™ procedures.

Another source that can be helpful to define starting method parameters are monographs from established pharmacopoeias such as the US Pharmacopeia (USP) and the European Pharmacopoeia (Ph. Eur.).

Automated method development

Developing an LC method is still a bottleneck in many laboratories, but automated method development is a significant time and resource-saving process. Various hardware and software tools are available to accelerate the method development process, enhance final method quality, and reduce development time from weeks or even months to days.

Method development hardware

The two key hardware capabilities required for automated method development are:

  1. Automated solvent switching. This technology provides the ability to switch mobile phases during a sequence without manually exchanging bottles and purging the system. The Thermo Scientific Vanquish Method Development Systems include a solvent extension kit with an external selection valve for automated scouting of up to 10 solvents per channel.

  2. Automated column switching. Used for early-stage method development and generally includes scouting several stationary phase chemistries. Automatic column switching saves both time and user effort by eliminating pausing sequences to switch fittings between columns manually. The Thermo Scientific Viper Method Scouting Kit includes all fluidic connections and capillaries required to scout four-column chemistries.

    3.  

Method development software

A fully automated method development process requires specialized software to guide the process from method scouting through validation. Several software packages include features from predicting analyte retention behavior to sequence generation.

ChromSwordAuto Chromeleon Connect, for instance, utilizes an artificial intelligence-driven approach for method optimization. ChromSword AutoRobust Chromeleon Connect uses a multivariate approach for streamlining automated method robustness and system stability evaluation. Both options are fully integrated into Chromeleon for a streamlined user experience.

Another prominent software package compatible with Thermo Scientific HPLC systems is S-Matrix® Fusion QbD®, which is based heavily on the quality-by-design approach to method development.

 
 
 
FAQ

What are the steps in HPLC method development?

Developing an HPLC method requires four different steps: method scouting, method optimization, robustness testing and method validation.

What is HPLC method validation?

Method validation is a formal and systematic process of performing investigational procedures with the aim of verifying that the HPLC method is appropriate and fit for the purpose to provide satisfactory and consistent results within the limits being described for that method.

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