Converting a method from HPLC to UHPLC has a number of benefits, as outlined in the first 2 articles in our series, including faster analytical separations, better peak resolution and lower long-term costs. Once the decision has been made to convert a method from HPLC to UHPLC, a number of factors and best practices should be considered to ensure consistency of data quality and efficiency of the new method.
Choosing the Right Analysis
Before making the decision to transfer a method from HPLC to UHPLC, you should first consider whether the method itself is a good choice for transfer, as not all analytical methods are inherently good candidates. Some methods may not yield sufficient financial or efficiency gains to support the inherent costs associated with implementing new methods. Good candidates for migration to UHPLC are methods with longer run times (≥20 minutes), those that use reverse phase columns, as well as methods or labs with heavy sample loads.
Choosing the Right Column
After evaluating the method itself, the next step in the method transfer process is to evaluate the column utilized in both the currently HPLC method, and the one that will be used in the new UHPLC method. Ideally, the same type of column should be utilized in both methods to maintain the analyte retention order and ensure consistency over time. The dimensions of the column, however, will differ between the methods. The optimal inner diameter of a UHPLC column is around 2.1 mm, as this column size reduces mobile phase consumption and frictional heating, which can lead to column degradation. Taking time to choose the right UHPLC column will simplify the migration from the HPLC method. As a general “rule of thumb,” the following conversions can be used when transferring from a 4.6 mm internal diameter, 5 µm particle HPLC column:
Figure 1 Column Dimension Transfer Guidelines
Calculating Method Parameters
Once the appropriate column has been selected, several method parameters must be evaluated and converted for the UHPLC method1.
- Injection Volume: The injection volume (Vinj)must be changed when transferring a method from HPLC to UHPLC to avoid column overload, as well as maintain sensitivity and reduce extra-column band broadening. As a best practice, the injected volume onto the column should only represent 1-5% of the total column volume. A smaller column volume requires a smaller injection volume. The UHPLC injection volume (V_inj2) can be calculated utilizing the column internal diameter (dc) and the length (L):
- Mobile Phase Flow Rate: The flow rate (F) for the UHPLC method must be adjusted to maintain a mobile phase linear velocity (u) similar to what was used in the HPLC method. The linear velocity within a column is directly proportional to the column diameter (dc), but also depends on the particle size (dp) of the stationary phase. Therefore u*dp must be maintained at a constant value to account for changes in column diameter and particle size.
- Isocratic Step Time: The ratio between the isocratic step time (tiso) and the column dead time (which depends on the mobile phase flow rate (F), column diameter (dc) and length (L)) must be maintained between the HPLC and UHPLC conditions.
- Gradient Slope and Step: As a best practice, the initial and final mobile phase compositions in any HPLC gradient step should be maintained in the UHPLC method. The slope and time of the gradient step in the UHPLC method must be adjusted such that the product of the gradient slope and the dead time remain constant between the HPLC method and the UHPLC method. The UHPLC gradient slope (slope2) can be calculated using the following formula:
For slope segments, it is essential that the initial and final gradient composition (%B) remain constant. The UHPLC gradient step time (tgrad2) can be calculated utilizing the following formula:
In the fourth, and final, article in our series, we will utilize and apply these best practices and look at potential efficiency and production gains associated with the conversion of a method to UHPLC.
- Guillarme, D., & Veuthey, P. (2009). UHPLC Instrumentation and Method Transfer Guidelines [Scholarly project]. In PerkinElmer White Paper.