Over the years, analytical chemists have become very good at screening through organic solvents and reversed-phase columns with different properties to achieve targeted selectivity. Temperature and gradient slope are routinely adjusted to improve resolution.  In many cases, we take advantage of the stability of modern columns to screen both high and low pH mobile phases.

It's not as common to test small, incremental changes in pH as a routine tool in methods development. Small changes in pH dramatically change an elution order, even over a range of just 3/10ths of a pH unit. While it certainly can be valuable to use pH adjustment to develop a rugged method, chromatographers don’t frequently use this approach. 

Part of the challenge is that preparing mobile phases of different pH is a labor-intensive and time-consuming exercise: Do you spend Monday mornings making up buffer solutions to get ready for your analytical runs? How many measurements are involved? How many solvent bottles? Are the solutions exactly the same every time?

While the ACQUITY UPLC H-Class and ACQUITY UPLC H-Class Bio Systems are excellent quaternary-based chromatographic systems for developing and using high-resolution separations, one of the factors that contributes to method reproducibility is how consistently you make up your buffer solutions. That's why we've included Auto•Blend Plus Technology with each of our quaternary UPLC systems.

Automating mobile phase composition

Waters Auto•Blend Technology automates the formulation of mobile phases from reservoirs of pure solvents or concentrated stock solutions. Additionally, Auto•Blend Plus enables you to better manage the desired pH and ionic strength requirements for your selected mobile phase. Its software algorithm calculates how to deliver the required pH based on the definition of a buffer system. Our technique is based on the established principal of acid-base equilibria, where a buffer of desired pH can be created by mixing solutions of a weak acid and its corresponding salt or a base.

Auto•Blend Plus can be used to manage methods for both small and large molecules.

Select the Details tab for more information.

Since mobile phase combinations and gradients are automatically blended on-demand to meet the optimum retention and selectivity of the separation, Auto●Blend Plus technology is useful for routine assays, in complicated separations requiring multiple solvents, and in automatic method development or system flushing. It is included as a standard feature with the ACQUITY UPLC H-Class and H-Class Bio systems.

Makes method development more efficient:

• Explore all separation alternatives
• Test intermediate conditions
• Minimize errors in solution preparation

Streamlines routine analyses:

• Minimizes errors in solution preparation
• Conserves costly solvent use (and disposal)
• Readily switch between multiple methods

Programming with AutoBlend Plus

1. Use a pre-defined buffer system or create your own from any components that are suitable for your sample.
2. Directly program a gradient table in pH and salt concentration.
3. Program pH and salt concentration independently, using linear, concave, or convex curves for either or both. 

AutoBlend Plus also provides a graphic display of the programmed gradient, below.

Principles of Auto●Blend Plus Technology

Auto●Blend Plus Technology is the use of specific algorithms to program a quaternary solvent manager to deliver buffers of a specified pH and salt concentration. 

The function is derived from the  practice of preparing buffer solutions by blending acidic and basic  stock solutions.  In the quaternary solvent manager, the percentage of flow drawn from each of the four reservoirs provides the same blending as manual buffer preparation. 

A typical system configuration is shown in Figure 1. The software calculates the proportions of reservoirs A, B, C, and D based on the definitions of the buffer system.  Proportions are calculated from the known pKa of the buffer or from an empirical calibration table.  The required proportions from each reservoir are calculated at each pump stroke to deliver the pH and salt concentration specified by the separation method.  The system and software have been validated for accurate pH delivery, both at constant pH and with simple pH gradients.

Figure 1. Principle of Auto•Blend Plus Technology .  The four mobile phase reservoirs of the ACQUITY UPLC^® H-Class System are filled with concentrated stocks and the Auto•Blend Plus Technology algorithm is used to calculate the proportions required from each reservoir for a specified pH and salt concentration.

 For details, see the HPLC 2011 poster: Efficient Use of pH Gradients in the Ion Exchange Analysis of Proteins