Maximize Sample Throughput with Ascentis Express Fused-Core Technology

By: Wayne K. Way, Reporter US Vol 27.1


Wayne K. Way
wayne.way@sial.com

Because most analytical laboratories will at some point experience the need to do more and do it faster, product development in the HPLC arena has focused largely on approaches that improve resolution or reduce analysis time. Equally important, however, are the economics, for any innovation that is too costly will not be widely and readily adopted. Ascentis Express with Fused-Core technology is the rare innovation: it accomplishes both high resolution and high speed. Moreover, because it can be run on any HPLC instrument, it is an economical alternative to technologies that require capital investment for special instruments, like sub-2 μm particles designed for ultra high pressure HPLC (UHPLC).

Throughout a series of Reporter articles, we have systematically described the various features of Ascentis Express, and how analysts can put these benefits into practice to achieve high resolution, high throughput HPLC separations. In this article, we will focus on the speed component.

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Methods to Improve the Speed of an HPLC Separation

There are both physical (kinetic) and chemical (thermodynamic) techniques to reduce analysis time. Chemically, one can change the stationary phase to one that is less retentive, using a C8 instead of a C18, for example, or increase the strength of the mobile phase. However, these approaches may adversely affect resolution by altering the selectivity. Temperature also can be increased to decrease retention, but this requires special instrumentation and has limits in practice for many reasons. Physical methods to increase speed include increasing the flow rate, decreasing the column length and decreasing the particle size. These also have practical limits.

Flow rate is the easiest approach to reduce analysis time. However, the maximum flow rate is limited by the most pressure-sensitive component of the system. Also, conventional HPLC particles have a distinct flow rate where maximum performance is obtained. Both below and above this flow rate, efficiency is lost due to various kinetic parameters described in the van Deemter relationship shown in Figure 1.

Figure 1. van Deemter Relationship Between Particle Size, Flow Rate and Separation Efficiency


Reducing column length also will increase analysis speed, both by reducing retention and permitting higher flow rates. However, for a typical 100,000 plate/m HPLC column, every cm in column length reduction also sacrifices 1,000 theoretical plates.

Particle size provides an interesting lever to reduce analysis time. The van Deemter relationship in Figure 1 shows that smaller particles provide higher efficiency at any practical flow rate. Note the different shapes of the curves in Figure 1. The smaller the particle size, the faster it can be run without loss of efficiency. Pressure is the tradeoff, however, because it varies with the inverse square of the average particle diameter. This relationship is shown in Figure 2.

Figure 2. Influence of Flow Rate on Pressure for Various Particle Sizes

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Speed Benefits of Ascentis Express Compared to

So the challenge is this: How can one combine the efficiency benefits of small particles, with speed benefits of higher flow rates? Even further: Can this be achieved on conventional HPLC systems? That’s where the innovative Ascentis Express comes in.

Compared to conventional porous particles, including the 3 μm HPLC and sub-2 μm UHPLC particles, Ascentis Express has two important physical attributes that permit high-speed operation without loss of efficiency: Fused- Core technology and extremely narrow particle size distribution. Both of these properties work on the kinetics of the separation by positively influencing the A and C terms of the van Deemter equation shown in Figure 1.

  • Compared to 3 μm HPLC particles, the speed benefit of Ascentis Express is derived from the flat van Deemter relationship shown in Figure 1. Ascentis Express with 2.7 μm particles has comparable pressure per unit column length as 3 μm particles, but it permits faster analysis because it can be run at higher flow rates without loss of efficiency. It also has nearly twice the efficiency as 3 μm particles at any flow rate.
  • Compared to sub-2 μm particles for UHPLC, the speed benefit of Ascentis Express is derived from the pressure relationship shown in Figure 2. Ascentis Express with 2.7 μm particles has comparable efficiency per unit column length as sub-2 μm particles, but it permits faster analysis because it generates less than half the pressure. If using a UHPLC system, Ascentis Express can provide even higher efficiency than sub-2 μm particles because longer columns can be used.

The example in Figure 3 typifies the speed improvements that are possible with Ascentis Express over conventional HPLC particles, in this case over the workhorse HPLC particle, C18-bonded 5 μm porous silica. In this example we took a typical 4 minute isocratic separation on a 5 μm C18 (panel A) and adjusted the flow rate and mobile phase composition to obtain equal N and k’ on a shorter Ascentis Express column (panel B). Injection volume was also reduced in proportion to the column length. Note that these simple changes gave nearly 5-fold increase in throughput. The flow rate was then increased on the Ascentis Express column to 1.2 mL/min. (panel C), which reduced the analysis time to 23 seconds – a nearly 10-fold improvement in throughput on a conventional HPLC instrument. If the analyst has available a mid-pressure system, the retention time could be further reduced by increasing flow rate, as in panel D. Here, we show an analysis time of only 14 seconds at 6400 psi, a 15-fold increase in throughput over the original 4 minute separation.It is important to note that these dramatic improvements in throughput did not come at the expense of efficiency. The resolution of a critical pair remained virtually constant throughout the experiment because of the flat van Deemter plot.

Figure 3. Throughput Improvements on Ascentis Express (53811-U)

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Rugged, High-Speed Separations

The narrow particle size distribution around the average of 2.7 μm means Ascentis Express columns can achieve this remarkable performance using 2 μm porosity frits. The presence of fines in sub-2 μm particles and conventional 3 μm particles forces the use of much finer porosity frits, which can foul more easily and reduce the lifetime of the columns. Sub-2 μm particles typically require 0.2 μm frits, while 3 μm typically need 0.5 μm frits. Figure 4 compares the particle size distribution of Ascentis Express to typical porous HPLC particles. Rugged Ascentis Express columns last longer, like 5 μm columns, a valuable attribute for both method development and routine sample assay.

Figure 4. Comparison of Particle Size Distribution of Ascentis Express and Conventional 3 μm HPLC Particles


Ascentis Express columns are currently available in four phases, C18, C8, RP-Amide and HILIC, and from 2.1 to 4.6 mm I.D. New capillary dimensions of Ascentis Express range from 75 to 500 μm I.D. For technical and ordering information, please visit sigma-aldrich.com/express.

The following table presents a partial listing of Ascentis Express columns.

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Materials

     
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