A Simple Yet Effective Trick for Isomer Separation
Introduction
When developing methods for isomer separation, have you ever encountered this situation? A shallow gradient or isocratic elution provides better separation than a standard gradient, but when the organic phase ratio is slightly increased, the isomers fail to achieve complete separation. On the other hand, reducing the organic phase ratio beyond a certain point no longer improves resolution. In some cases, although the peaks appear fully separated, broad peak shapes result in inadequate resolution after integration.
If adjusting the organic phase ratio alone cannot balance peak shape and resolution, does that mean we are out of options? In this article, we introduce a simple yet effective trick to solve this issue through two case studies.
Application 1
Method 1
| Column | Ultisil® Phenyl-Ether (4.6×150 mm, 3 μm) | ||
| Elution Program | Time (min) | Buffer Salt (%) | Acetonitrile (%) |
| 0 | 55 | 45 | |
| 45 | 40 | 60 | |
| 45.01 | 55 | 45 | |
| 60 | 55 | 45 | |
When separating the target component using Ultisil® Phenyl-Ether (4.6×150 mm, 3 μm) under gradient conditions, the resolution between peaks was poor, leading to peak coalescence. To address this, we attempted to slow down the gradient.
Method 2
| Column | Ultisil® Phenyl-Ether (4.6×150 mm, 3 μm) | ||
| Elution Program | Time (min) | Buffer Salt (%) | Acetonitrile (%) |
| 0 | 60 | 40 | |
| 40 | 47 | 53 | |
| 40.01 | 60 | 40 | |
| 50 | 60 | 40 | |
After slowing the gradient, the resolution between the main peak and adjacent impurities are 1.55 and 1.79, respectively. However, further gradient adjustments failed to yield additional improvements.
In this case, we introduced a 20-minute isocratic hold at 40% organic phase prior to the gradient, allowing the sample to migrate more slowly within the column and enhancing the separation between components. The subsequent gradient elution then facilitated their elution.
Method 3
| Column | Ultisil® Phenyl-Ether (4.6×150 mm, 3 μm) | ||
| Elution Program | Time (min) | Buffer Salt (%) | Acetonitrile (%) |
| 0 | 60 | 40 | |
| 20 | 60 | 40 | |
| 60 | 47 | 53 | |
| 60.01 | 60 | 40 | |
| 70 | 60 | 40 | |
Under these conditions, the resolution between the main peak and adjacent impurities were improved to 1.75 and 1.84, respectively.
This demonstrates that by incorporating a low organic phase isocratic segment before the gradient (20 minutes in this example), the sample moves more slowly through the column, enhancing component separation before applying the gradient for elution. This strategy effectively improves resolution.
Application 2
During method development on Ultisil® Cellu-JR (4.6×250 mm, 5 μm), two peaks exhibited a tendency for separation, with isocratic conditions yielding better resolution than gradient elution. However, as the organic phase ratio decreased, the peaks remained incompletely separated, and the peak shapes deteriorated.
Method 1
| Column | Ultisil® Cellu-JR (4.6×250 mm, 5 μm) |
| Mobile Phase | 0.1% Diethylamine / Methanol = 15/85 |
Under isocratic conditions, the resolution between the two peaks was 1.4.
To improve separation, we introduced a 20-minute isocratic hold at 70% methanol, allowing the chiral isomers to migrate more slowly within the column for better resolution. A subsequent 5-minute gradient transition to 80% methanol was applied to accelerate elution and refine peak shape.
Method 2
| Column | Ultisil® Cellu-JR (4.6×250 mm, 5 μm) | ||
| Flow Rate | 0.35 mL/min | ||
| Elution Program | Time (min) | 0.1% Diethylamine (%) | Methanol (%) |
| 0 | 30 | 70 | |
| 20 | 30 | 70 | |
| 25 | 20 | 80 | |
| 40 | 20 | 80 | |
| 40.01 | 30 | 70 | |
| 60 | 30 | 70 | |
Under these modified conditions, the resolution between the two components increased to 1.9.
This case further illustrates that by adding a low organic phase isocratic segment before the gradient (20 minutes in this example), the chiral isomers migrate more slowly within the column, enhancing separation. The subsequent gradient transition restores elution speed and peak shape. This approach proves to be an effective way to improve resolution.