Pharmaceutical Article

Easy Transfer of HPLC Methods to UHPLC

Using Fully Scalable Pinnacle® DB Columns

By Rick Lake, Pharmaceutical Innovations Chemist

Methods on Pinnacle® DB columns are easily transferred from 3 and 5µm to 1.9µm, allowing faster analysis without losing separation quality.
Pinnacle® DB columns are 100% Restek manufactured–from base silica to final packed column.
Restek offers the widest selection of stationary phases for UHPLC—more choices mean better selectivity for your analytes.

Ultra High Pressure Liquid Chromatography (UHPLC) is a rapidly growing technique that greatly increases sample throughput. UHPLC takes advantage of HPLC columns packed with <2µm particles, often used at elevated pressures, to significantly reduce analytical run times. By decreasing the particle size of the packing material, the analyst can maximize the number of theoretical plates, making shorter column lengths possible and increasing the range of usable flow rates. Ultimately, the analytical method is shortened without losing separation quality.

UHPLC is commonly used in two ways. First, to increase sample throughput, a conventional HPLC analysis can be scaled-down to UHPLC. Alternatively, to lessen time in method development, a preliminary separation can be developed in UHPLC and then scaled-up to conventional HPLC for routine analysis. In both cases, when transferring an analysis from conventional HPLC to UHPLC, or from UHPLC to HPLC, comparable method parameters must be used to maintain equivalent separations. First and foremost, to maintain selectivity while scaling an analysis, some column properties and operating conditions should remain consistent, while other parameters are optimized. For the analytical column, the pore size, carbon load, and support material must remain the same. Differences in selectivity can be seen when not using equivalent columns. However, when decreasing particle size and column dimensions, it is equally important that certain operating conditions be adjusted properly. To provide guidance, we will identify important method parameters that need to be adjusted and illustrate with an example of method transfer.

In this example, we will perform a scale-down method transfer for sulfonamides from a conventional HPLC analysis to a UHPLC analysis. The initial analysis was conducted on a standard analytical scale 150mm x 4.6mm ID x 5µm HPLC column using gradient elution. To speed the analysis, we decreased the internal diameter of the column to 2.1mm and reduced the particle size to 1.9µm.

The importance of selectivity in a chromatographic separation has been discussed in a previous article, Optimize Selectivity & Efficiency in UHPLC Separations. To ensure that we used the optimum stationary phase for this application, we first compared the selectivity of a Pinnacle® DB Biphenyl column to a C18 column. Sulfonamides are commonly assayed on alkyl phases; however, their fused ring structure (Figure 1) may show enhanced selectivity with a biphenyl phase that is capable of pi-pi interactions. When comparing C18 and biphenyl columns of identical dimensions, under identical conditions, the Pinnacle® DB Biphenyl showed better selectivity towards the early eluting sulfonamides (Figure 2). Therefore, for better selectivity and faster analysis times, we used a Pinnacle® DB Biphenyl stationary phase for this application.

When performing a scale-down procedure, a few simple calculations can be used to determine equivalent run conditions. Let’s go through them sequentially.

Adjusting Column Size

The first calculation determines the appropriate column length. Keeping the same column length while decreasing the particle size will increase the number of theoretical plates in that given column length. Therefore, column length can be shortened without losing resolution. By adjusting the column length properly, using Equation 1, we can maintain the same separation.

Adjusting Injection Volume

Once we have determined the proper column length, we can determine the appropriate injection volume. Decreasing the column internal diameter and length, decreases the overall column volume and sample capacity. Therefore, we must alter the injection volume as described in Equation 2. Please note that since overall column volume has decreased, it is important to match the sample solvent to the starting mobile phase composition. Mismatched sample solvents can cause irreproducible retention times, efficiencies, and even changes in selectivity.

Adjusting Flow Rate

Next, flow rate must be adjusted to maintain comparable linear velocity through a column with smaller internal diameter. Linear velocity is defined as the distance mobile phase travels over time, whereas flow rate is the volume of mobile phase that travels over time. To maintain the same linear velocity, which is important in maintaining efficiencies, flow rates must be decreased as column internal diameter decreases. Also, since smaller particle sizes give rise to higher optimal linear velocities, isocratic flow rates should be calculated with particle size taken into account. In this example a gradient elution was used and therefore particle size was not included in the equation. Equation 3 can be used to simply and quickly estimate the adjusted flow rate needed for equivalent chromatography. It is also important to note that <2µm particle sizes are less affected by higher flow rates, and therefore faster flow rates can be used in isocratic systems without detrimental effects on peak efficiency.

Adjusting Time Program

Lastly, after we have determined the proper column length, injection volume, and flow rate, we can find the equivalent time needed for gradient or step elutions. As an analytical method is scaled down, the time program needs to also be scaled down to keep the phase interactions the same. Time can be adjusted using Equation 4.

Conclusion

After determining the equivalent conditions for scaling-down the analysis of sulfonamides, we can see that the separations are equivalent, while the analysis time was greatly reduced (Figure 3). Under conventional HPLC the last compound eluted at 7.2 minutes and under UHPLC the last compound eluted at 2.6 minutes. By following the procedure described here to ensure that the columns are equivalent, scaling analytical procedures from HPLC to UHPLC, or vice versa, easily can be accomplished using Pinnacle® DB columns.