Ascentis Express UHPLC Columns with 2.0 μm Fused-Core Particles

By: Richard A. Henry, Gaurang Parmar, Reporter US, Volume 32.2

  • Proven column performance and ruggedness using the latest Fused-Core particles
  • Lower operating pressure than typical sub-2 µm particle columns

Column Performance

The new Supelco Ascentis® Express column line has been designed specifically for UHPLC instruments with 2.0 µm Fused-Core® silica, as diagrammed in Figure 1. Ascentis Express 2.0 µm UHPLC columns are optimized to preserve the efficiency advantages of using sub-2 µm particles while minimizing pressure disadvantages. As shown in Figure 2, the plot of reduced plate height (h) vs flow velocity for Fused-Core 2.0 µm particle columns matches a Fused-Core 2.7 µm particle column by delivering h values that are consistently below 2 (typically ~ 1.7). This is significantly lower than typical commercial sub-2 µm UHPLC columns that employ either porous or core-type particles.

Ascentis Express Fused-Core Silicas for Small Molecule Separations

Figure 1. Ascentis Express Fused-Core Silicas for Small Molecule Separations

 

Reduced Plate Height van Deemter Plots Showing Performance of Ascentis Express Columns

Figure 2. Reduced Plate Height van Deemter Plots Showing Performance of Ascentis Express Columns with 2.0 µm and 2.7 µm Fused-Core Particles Compared to Columns with sub-2 µm Particles at Various Flow Velocities
CONDITIONS: columns: as listed, 5 cm x 2.1 mm I.D.; sample: naphthalene; mobile phase: 50:50 acetonitrile:water; instrument: Shimadzu Nexera

 

Columns with lower reduced plate height values demonstrate higher efficiency for a given particle size. Ascentis Express columns with 2.0 µm particles show comparable efficiency at significantly lower pressure than competitor columns that use smaller particles. Achieving high performance at lower pressures can extend useful column life and minimize selectivity changes created by frictional heating that creates temperature variation inside the column bed. Ascentis Express 2.0 µm columns will show highest performance when used in Waters ACQUITY, Agilent Infinity, Thermo Scientific™ Dionex UltiMate™ and Vanquish™, Shimadzu® Nexera, and other modern UHPLC instruments. Figure 3 plots pressure drop vs flow rate for the columns compared in Figure 2. Figure 4 shows a QC chromatogram for Ascentis Express 10 cm x 2.1 mm, 2.0 µm column, and Table 2 compares test results to other Supelco and competitor columns. The bar chart in Figure 5 illustrates column performance in plates per pressure unit. Note that Supelco Ascentis Express columns easily outperform typical commercial C18 columns when ranked by a plates/pressure measurement. The only other column that compares favorably to Ascentis Express C18, 2.0 µm in plates/pressure is the new Supelco Titan C18, 1.9 µm column which features a similar, narrow particle distribution.

Table 2. Comparison of Theoretical Plates and Pressure

CONDITIONS: columns: as listed, 10 cm x 2.1 mm; mobile phase: (A) water, (B) acetonitrile, (50:50, A:B); flow rate: 0.4 mL/min; pressure and performance: as listed in Table 2; system background pressure: 370 psi; column temp.: 35 °C; detector: UV, 250 nm; injection: 0.5 µL; sample: dissolved in 75:25 water:acetonitrile; instrument: Dionex UltiMate 3000

 

Pressure Drop Comparison of Express 2.0 µm and 2.7 µm Fused-Core Particles and other sub-2 µm Columns

Figure 3. Pressure Drop (flow resistance) for Ascentis Express 5 cm x 2.1 mm Columns with 2.0 µm and 2.7 µm Fused-Core Particles Compared to sub-2 µm Columns at Various Flow Rates
CONDITIONS: columns: as listed, 5 cm x 2.1 mm I.D.; sample: naphthalene; mobile phase: 50:50 acetonitrile:water; instrument: Shimadzu Nexera

 

Suitability Test Chromatogram

Figure 4. Suitability Test Chromatogram
CONDITIONS: columns: Ascentis Express C18, 10 cm x 2.1 mm I.D., 2.0 µm, (Product No. 50813- U); mobile phase: (A) water, (B) acetonitrile, (50:50, A:B); flow rate: 0.4 mL/min; pressure and performance: see Table 2; system background pressure: 370 psi; column temp.: 35 °C; detector: UV, 250 nm; injection: 0.5 µL; sample: dissolved in 75:25 water:acetonitrile; instrument: Dionex UltiMate 3000

 

Performance Ranking for Modern UHPLC Columns in Plates per Pressure Unit

Figure 5. Performance Ranking for Modern UHPLC Columns in Plates per Pressure Unit
CONDITIONS: columns: as listed, 10 cm x 2.1 mm I.D.; mobile phase: (A) water, (B) acetonitrile, (50:50, A:B); flow rate: 0.4 mL/min; pressure and performance: see Table 2; system background pressure: 370 psi; column temp.: 35 °C; detector: UV, 250 nm; injection: 0.5 µL; sample: dissolved in 75:25 water:acetonitrile; instrument: Dionex UltiMate 3000

Column Selectivity

Ascentis Express C18 and F5 (pentafluoropropyl or PFP) columns are currently available in the 2.0 µm particle size, in a range of phases. C18 phases with flexible alkyl chains are extremely versatile because solutes are retained by a simple partition mechanism where dispersive interactions and solubility in organic solvent controls retention and selectivity. The Ascentis Express F5 phase behaves differently because sample components can interact not only by dispersive forces but also with the aromatic ring that becomes highly polarized by five fluorine substituent groups. C18 and PFP phases typically show very large selectivity differences so they are complementary choices for method development.

Different particle sizes in the Ascentis Express 90 A Fused-Core column family are designed to have the same retention and selectivity for easy method development or method transfer. Log k values for a 60-component sample containing acidic, basic and neutral pharmaceuticals are compared in Figure 6 between the new Ascentis Express C18, 2.0 µm column and the popular Ascentis Express C18, 2.7 µm column that has become well-established in many HPLC methods; acetonitrile and methanol conditions were investigated at pH 4.0 and 7.0. Note correlation is essentially 100% for all four conditions, indicating that a C18 phase bonded to either the 2.0 µm or 2.7 µm Fused-Core particle will produce the same selectivity factor (α = k2/k1) for a wide range of different chemical structures. Note also the slopes for each log k plot are essentially unity, indicating that two C18 columns will also match one another in retention factor (k).

This identical selectivity and retention performance is demonstrated in Figures 7A and 7B for Ascentis Express C18, 2.0 µm and 2.7 µm columns using a mixture of sulfa drugs. Performance is also compared to a commercial coretype C18, 1.7 µm column (Figure 7C) to demonstrate that Ascentis Express columns have similar retention and selectivity to other C18 columns regardless of particle size or design. Note that the Ascentis Express C18 2.0 µm column matches the sub-2 µm C18 core-type in efficiency, but operates at lower pressure.


Figure 6. Performance Ranking for Modern UHPLC Columns in Plates per Pressure Unit
CONDITIONS: columns: Ascentis Express C18, 10 cm x 2.1 mm I.D., 2.7 µm (Product No. 53823-U), Ascentis Express C18, 10 cm x 2.1 mm I.D., 2.0 µm (Product No. 50813-U)
mobile phase 1 (M1): 10 mM ammonium formate (70:30 water:acetonitrile), pH 7.0
mobile phase 2 (M2): 10 mM ammonium formate (70:30 water:acetonitrile), pH 4.0
mobile phase 3 (M3): 10 mM ammonium formate (50:50 water:methanol), pH 7.0
mobile phase 4 (M4): 10 mM ammonium formate (50:50 water:methanol), pH 4.0
flow rate: 0.3 mL/min; column temp: 35° C; injection: 2 µL; sample: 60-component mix containing acidic, basic and neutral pharmaceuticals;  instrument: Agilent 1290 Infinity with 6210 TOF MS in ESI+ mode, 100-1000m/z

 

Sulfa Drug Comparison

Figure 7. Sulfa Drug Comparison
CONDITIONS: column: as listed, 10 cm x 2.1 mm I.D.; mobile phase: (A) water with 0.1% acetic acid; (B) acetonitrile with 0.1%; acetic acid, (90:1 A:B); flow rate: 0.8 mL/min; pressure: see above; column temp.: 45 °C; detector: UV, 250 nm; injection: 0.5 µL; sample: 25 µg/mL in 90:10 water:methanol; instrument: Agilent 1200

 

Figure 8 illustrates the selectivity difference between Ascentis Express C18 and F5 columns using a steroid example. Hydrocortisone and prednisolone differ by only one double bond and have nearly identical solubility. Since the main difference between these rigid solutes is shape rather than solubility, C18 columns typically show poor selectivity because alkyl chains can readily change conformation to provide nearly equivalent amounts of solute interaction. For rigid isomers, the PFP column can provide additional selectivity because one isomer may approach the planar aromatic ring differently and interact more strongly. Aromatic phases, such as PFP, often show different, enhanced selectivity over C18 between closely-related, rigid compounds in any sample.

Steroid Comparison on Ascentis Express F5 vs C18 Phases

Figure 8. Steroid Comparison on Ascentis Express F5 vs C18 Phases
CONDITIONS: column: as indicated, 10 cm x 2.1 mm I.D., 2.0 µm; mobile phase: (A) water, (B) methanol (50:50, A:B); flow rate: 0.5 mL/min; pressure: 11430 psi (788 bar); system background pressure: 420 psi; column temp.: 35 ° C; detector: UV, 240 nm; injection: 0.5 µL; sample: 50 µg/mL in 85:15, water:methanol; instrument: Dionex UltiMate 3000

Conclusion

  • Fused-Core particles have revolutionized U/HPLC performance.
    Ascentis Express 2.0 µm Fused-Core particles are designed to provide UHPLC performance for practical small molecule analysis.
  • Ascentis Express column line now features three 90 Å Fused-Core particle sizes for high speed separation of small molecules.
  • Ascentis Express 2.0, 2.7 and 5 µm Fused-Core columns represent the best selection of phases and geometries for every analytical application and instrument.

Trademarks

Ascentis is a registered trademark of Sigma-Aldrich Co. LLC
Titan is a trademark of Sigma-Aldrich Co. LLC
Fused-Core is a registered trademark of Advanced Materials Technology, Inc.