Last time, the author briefly introduced the differences between high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC). It is believed that everyone
now has a more specific understanding of the two. With the development of small particle chromatographic columns, more and more industry professionals expect to use sub-2μm particle size chromatographic columns in UHPLC to broaden selectivity, improve work efficiency, reduce energy consumption, and reduce pollution.
Today, we will guide everyone on how to convert an HPLC method to a UHPLC method.
The premise for ensuring a successful conversion is:
● Same selectivity: Fillers made with the same brand and the same bonding process are only different in particle size.
● Same or larger column length-to-particle size ratio: This ensures equivalent or higher theoretical plate numbers.
How to Convert the Method
● Study the differences between the two and conduct conversion research: The chromatographic column filler particle sizes are different, the system volume is different, and the inner diameter of the instrument delivery flow system is different, which affects the flow rate, injection volume, and gradient elution program.
● Adjust parameters: flow rate, injection volume, and gradient elution program.
- Flow rate is proportional to the square of the column radius.
HPLC column: 4.6*150mm, 5μm Flow rate: 1.5mL/min
UHPLC column: 2.1*100mm, 1.8μm The flow rate would be:
- The sample concentration remains unchanged, and the injection volume is proportional to the column volume.
HPLC column: 4.6*150mm, 5μm Injection volume: 20μL
UHPLC column: 2.1100mm The injection volume would be: [the column volume][proportion of injection volume to column volume]
- Conversion of gradient elution program
For isocratic elution, only the flow rate and injection volume need to be adjusted. However, for gradient elution, the gradient elution program needs to be adjusted. The basis for adjustment is to calculate the time required to deliver the same multiple of column volume of solvent to the target column under the flow rate of geometric scaling.
3. Optimization of method parameters
- To improve the separation, adjusting the gradient elution program and increasing the column length are convenient and feasible measures.
2. If the impurity conditions detected in the destruction test are inconsistent, and the converted impurities are more and the material balance is better than the original method, and there is no significant difference in the measurement results between the two, the conversion is successful.
3. If the results are significantly inconsistent or involve unqualified situations, the relevant responsible person should be reported to decide whether to re-evaluate the original method or abandon the method conversion.
4. When is a conversion considered failed
- Principle: The system suitability test does not meet the requirements.
2. The separation cannot reach the required level even after adjusting the parameters.
3. The material balance is significantly worse than the HPLC method in the destruction test, and the optimization of the method cannot solve the issue.
4. If the test results are inconsistent with HPLC, or even unqualified, the original method should be evaluated or the method conversion should be abandoned.
5. Items that need to be validated for UHPLC conversion method
- Principle: The system suitability test meets the requirements.
2. The peak purity meets the requirements.
3. The separation meets the requirements.
4. The sensitivity meets the requirements.
5. For items without system suitability requirements, destructive tests should be performed on 2-3 sample conditions, and impurity separation and material balance should be equivalent.
In daily work, when an HPLC method is successfully converted to a UHPLC method, not only is the selectivity broadened and work efficiency improved, but also energy consumption is reduced and pollution is decreased.