The Quest for Maximum LC Performance: A Well-Packed Column Bed

As chromatographers seek maximum separation performance on their liquid chromatography (LC) systems, a key factor is the use of LC columns with an optimally packed column bed density. Solid-core columns manufactured with strict quality control for their packed bed density enable top efficiency, mechanical stability, and performance - no matter how tough your method conditions.

The evolution of packed columns

The introduction of the ACQUITY UPLC System in 2004 showcased the advantages in speed, sensitivity, and resolution that could be obtained by using columns packed with smaller, sub 2 µm particles, on a low dispersion instrument.

Many LC column vendors now offer columns packed with smaller particle sizes to improve separation performance. Columns packed with 2.x µm particle sizes have become popular for a lot of different workflows. This particle size provides increased column efficiency over traditional columns packed with >3 µm particles while generating backpressures that do not exceed the pressure limits of most HPLC/UHPLC systems.

The introduction of 2.x µm columns packed with solid-core particles further advanced particle technology in providing even more column efficiency. Solid-core particles increase column efficiency by reducing the longitudinal diffusion within the analytical column which decreases peak widths and ultimately results in increased efficiency. Solid-core columns also have lower backpressures when compared to equivalent columns packed with fully porous particles of similar size; this is due to the ability to pack these particles 'less densely' or to a higher interstitial porosity.

Warning: Not all packed columns are created equal

The goal of every column vendor is to manufacture reproducible columns, that have the highest column efficiencies, with the lowest column backpressures, and rock-solid mechanical stability. However, differences in LC column production methods can significantly impact the performance of the column. Figure 1, an illustration of two simulated packed columns, highlights one column that has been optimally packed and one column that has been poorly packed. The key difference between the columns is in the formation of the packed bed; variations in the packed bed density have a significant impact on the column's performance for both column efficiency and column lifetime.

Figure 1. Images of a well-packed and a poorly-packed LC column bed
(Top) Optimally packed bed displays highest column efficiency and delivers long column lifetimes
(Bottom) Poorly packed bed displays lowest column efficiency and delivers short column lifetimes

Waters: Experts in packed column manufacturing

The true measure of excellence for Waters is consistently providing LC columns that have an optimal packed bed density and deliver the highest column efficiency while maintaining superior mechanical stability.

Table 1 shows a comparison of column efficiency (USP Plates), tailing factor, and backpressure for Waters CORTECS 2.7 µm C18 columns vs Competitor A: C18, 2.7 µm solid-core columns. Two different column configurations, 2.1 x 50 mm and 4.6 x 50 mm were compared in this study.

Table 1. Efficiency, tailing, and pressure values for competitor C18, 2.7 µm solid-core columns and Waters CORTECS C18, 2.7 µm columns. Test conditions 75/25 Acetonitrile/Water, 30ºC, 254 nm UV detection, reporting Octanophenone (kPrime ~3.6). Flow rates: 2.1mm, 0.30mL/min, 4.6mm, 1.44 mL/min

The results in table 1 show on average ~24% higher column efficiency for the CORTECS C18 columns vs the Competitor A: C18 2.7 µm solid-core columns for both the 2.1 x 50 mm and the 4.6 x 50 mm columns. Tailing factors are equivalent between the four columns; backpressure is equivalent for the 2.1 x 50 mm column, but the competitor A: C18, 2.7 µm 4.6 x 50 mm column did show ~20% more backpressure vs the CORTECS 4.6 x 50 mm column. Though efficiency alone is not an indicator of a well-packed column, packed bed stability is.

Packed column bed stability testing

Advancements in LC systems require that columns operate at the elevated pressures that can be generated by modern UHPLC systems. Sudden changes in pressure across the column due to the LC system's injection cycle can cause the packed bed of a column to shift slightly and to fail prematurely.

To test the packed bed stability of Waters LC columns, an accelerated packed bed stability test is used to ensure mechanical stability, by simulating the injection cycles of the system on a column during the columns lifetime. For this testing, columns are subjected to repeated pressure pulses that simulate the extremes of an LC system's injection cycles. Column efficiency and peak shape are measured intermittently throughout the test to track any changes and to assess the quality of the packed bed.

Figure 2 shows the packed bed stability test results for Competitor A: C18, 2.7 µm 2.1 x 50 mm and 4.6 x 50 mm solid-core columns compared to CORTECS C18, 2.7 µm columns of equivalent dimensions. As you can see from the results, there is a significant loss in efficiency over injection cycles on the competitor C18, 2.7 µm solid-core columns, indicating a poorly packed bed.

• Lower initial column efficiencies for both the Competitor A: C18 2.7 µm solid-core columns
• Complete Column failure at < 200 pressure cycles for both the Competitor A: C18 2.7 µm solid-core columns
• 50% loss in column efficiency at < 100 pressure cycles for both the Competitor A: C18 2.7 µm solid-core columns

Figure 2. Results from the accelerated bed stability testing. 2.1 mm 8,000 psi max pressure, 4.6 mm 4000 psi max pressure

Following the packed bed stability testing, the column inlets of both 2.1 x 50 mm columns were opened and inspected, Figure 3 shows a significant void at the inlet of the packed bed for the competitor A: C18 2.7 µm solid-core column, clearly showing an unstable packed bed. The CORTECS C18, 2.7 µm column showed no voiding.

Figure 3. 2.1 x 50 mm column inlets after accelerated stability testing
(Left) Competitor A: C18, 2.7 µm 2.1 x 50 mm solid-core column
(Right) CORTECS C18, 2.7 µm 2.1 x 50 mm column

Ensure maximum performance and best method results with an optimally packed column

The quality of your column can have serious repercussions on your method results and LC performance. When it comes to choosing an LC column vendor, not all vendors are the same, and not all vendors build the same high-performance columns. Spending a little extra on a higher quality column can pay dividends in the long run, resulting in more analysis/column, consistent performance/less troubleshooting, and the ability to push your LC system to its operation limits, maximizing your laboratory's productivity.

Waters expertise in column packing, specifically in producing high-efficiency columns with optimal bed densities, ensures that columns are engineered to last, and won't crumble under the pressure that a UHPLC/HPLC system can put onto it. CORTECS 2.7 µm columns are designed specifically to increase efficiency, provide lower column backpressures, and are mechanically stable to withstand your toughest method conditions.

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