Gradient Optimization in Liquid Chromatography

When dealing with complex sample compositions, a single chromatographic system may struggle to achieve ideal separation—either requiring excessive time or yielding poor resolution. In such cases, gradient elution can significantly shorten analysis time and enhance separation efficiency.

In this article, we present a case study of gradient fine-tuning to demonstrate its remarkable impact on chromatographic performance.

1. Instrument Consistency: Develop the method on an instrument that will be available for long-term testing.
2. System Performance Awareness: Understand the instrument’s performance characteristics to prevent reproducibility issues after system changes.
3. Key System Parameters: Two fundamental system parameters to consider are gradient delay volume and proportional accuracy. These parameters can be determined using one specific experiment:
   • Use Ultrapure water as mobile phase A and 0.1% acetone in water as phase B;
   • Add a UV detector after phase B;
   • Set up a multi-step gradient where phase B increases from 0% to 100% in 5% increments, each increment for approximately 5 minutes, using a typical flow rate of 1 mL/min;
   • Run the gradient program through a T-union and record the chromatogram.
   • The difference between the programmed gradient time and the actual gradient transition time determines the gradient delay time. The plateau heights can be used to assess mobile phase composition accuracy—minor blending volume variations may cause slight blurring.
4. Method Design Based on System Characteristics: Once the system's performance is established, the method can be tailored accordingly. The biggest challenge in gradient method transfer is gradient delay volume differences between instruments:
   • If the new instrument has a larger delay volume than the original one on which the method was developed, add an isocratic hold at the beginning of the program to compensate.
   • If the new instrument has a smaller delay volume, add a gradient delay when transferring the method.
   • On rare cases where proportional inaccuracies arise mid-gradient, adjust the gradient program to eliminate the differences.
5. Initial Mobile Phase Equilibration: If the column has been incompletely equilibrated with the initial mobile phase, rapid gradient transitions will cause the first injection to differ from subsequent ones. Transferring methods between systems with different gradient delay volumes will cause different effects.