Ultra High Performance Liquid Chromatography UPLC Beyond HPLC - The New Height of Chromatography Lab

Over the past 30 years, high performance liquid chromatography (HPLC) has become one of the most widely used technologies in laboratories around the world. This is because HPLC is a powerful multi-purpose technology. Its power is reflected in its wide range of applications, from capillary to preparative, and it is a versatile technology that detects a wide range of compounds within the relevant detection technology. Fundamentally, at present, there are three main directions in the development of HPLC technology: chemistry, detectors and systems/data management. Advances in chromatography in these areas have led to the widespread acceptance of this technology. Despite this, there is still room for further development in chromatographic techniques. So, what is the future of HPLC? What follow-up developments can expand and extend the usefulness of this recognized technology?

Definition of UPLCTM <br>The cutting-edge technology of liquid chromatography is under development. Dr. J. Jorgensen of the University of Northern California and Dr. M. Lee of Brigham Young University have recently published insights on the future scope of the chromatography laboratory. Their research portrays a new picture for separation science. The separation process reached a new end point by using much smaller particles than before. The basic principle of the method is embodied in the Fandamt equation. The Fandamt equation is an empirical formula that describes the relationship between line speed and plate height (column efficiency).

Since particle size is one of the variables, it can be used to describe the theoretical properties of various particle sizes (Figure 1). As can be seen from Figure 1, once the particle size of the filler is less than 2 μm, the chromatographic technique can reach a new level: not only is the column efficiency higher, but the column efficiency is not reduced as the flow rate is increased.


Figure 1: These van dermatt curves show the reduction in particle size over the past 30 years. A reduction in the height of the theoretical plate means an increase in efficiency. But even for 2.5 μm particles, the efficiency will start to drop at higher flow rates, and the system back pressure will increase accordingly. If UPLCTM technology and particles below 2 μm are used, efficiency can be maintained even at higher line speeds. The use of shorter columns and/or higher flow rates speeds up analysis and increases resolution and sensitivity, making it now possible to take full advantage of chromatographic principles for separation.

This presents a scenario where both peak capacity and separation speed are increased to new limits. In short, peak capacity is defined as the number of peaks that can be resolved per unit time by chromatography. Chromatography with improved performance such as peak capacity and separation speed is called ultra-high performance liquid chromatography, or UPLCTM. Figure 2 illustrates the effect of increased peak capacity on chromatographic separation performance as the particle size is reduced from 5 μm to 1.7 μm.


Figure 2: Comparison of 5 μm particles with 1.7 μm particles. Peak capacity and resolution have increased significantly.

Speed, Sensitivity, and Resolution <br>The additional efficiency stemming from smaller particles brings more significant benefits. Shorter columns or higher flow rates increase speed while small particles also increase resolution. For any given separation, the best combination of speed and resolution can be achieved by adjusting these variables. Figure 3 shows the results of UPLC and HPLC speed comparison. This experiment compared the separation speeds of 3 μm particles and 1.7 μm particles at the same resolution; the results showed that the separation speed of the smaller particles was faster. The increase in efficiency also brings further benefits: increased sensitivity. As the zone becomes narrower during the separation process, the analyte is more concentrated on the test point. This will be discussed in more detail when considering the instrument design requirements of UPLC.

It should be noted that 3 μm is the limit of the particle size of a 15 cm long normal column. This is the minimum particle size that can be used by the operating system when the column reaches the optimum flow rate without exceeding the pressure limits of commercially available instruments (using any ratio of methanol-water mixed solvent). (Note: The smallest particle used for HPLC to date is 0.67 μm [3].)

As mentioned above, the Van dermatt equation (and its curves) indicates the need to use particles below 2 μm to achieve an increase in column efficiency. Therefore, there is a need for technological innovations that enable the UPLC concept to be applied to chromatography laboratories2.


Figure 3: The top chromatogram is a 5-component mixed sample overlay obtained using normal (3 μm) HPLC and 1.7 μm UPLC, respectively. The bottom chromatogram is the first 6 minutes of the separation process, indicating that UPLC increases the separation speed while leaving the resolution constant. The amount of solvent used in UPLC is also greatly reduced.

Smaller particles, larger peak capacity
Developing and filling particles below 2 μm into a reproducible and durable column is a challenge in itself. Figure 4 shows the apparent difference between the current 5 μm particles commonly used in laboratories and the smaller 1.7 μm particles recommended for UPLC columns. Currently, non-porous 1.5 μm particles are available. Although such particles are more efficient, they have the disadvantage of having a smaller surface area. A small surface area results in a small amount of sample loading and a short retention time. In order to maintain a similar retention time and load capacity with HPLC, UPLC must use porous particles. UPLC requires a novel high pressure resistant porous particle. The uniformity of the packed bed is also critical; especially when shorter columns are used to maintain resolution stability while at the same time achieving the goal of speeding up separation. Another requirement is that the inner surface of the column must be smooth enough to fill smaller particles. The sieve plates at both ends of the column should be redesigned to retain both small particles and blockages.

Figure 4: To illustrate the significant difference between 5 μm particles and 1.7 μm particles, the scanning electron micrograph compares two particles to 60 μm human hair. When the measurement distance is the same, the hair has a diameter approximately equal to 12 particles of 5 μm and 33 particles of 1.7 μm.

Design Requirements for UPLC Systems <br> In order to achieve a small particle size and large peak capacity separation, a wider pressure range than the currently used HPLC instrument is required. At the optimum flow rate for maximum efficiency, the calculated pressure drop is ~15,000 psi for a 10 cm long column with 1.7 μm particles. Therefore, there is a need for a pump that can deliver solvent at this pressure. Another consideration is that under these conditions, the compressibility of the solvent will become significant, especially under multi-solvent and gradient separation conditions. UPLC's solvent delivery system should not only have high-pressure pump suction, but also use various solvents in isocratic and gradient separation modes. The solvent delivery system must offset a wide range of potential pressure fluctuations caused by solvent compressibility for smoothing. Reproducible fluid delivery.

Injection is also critical. Ordinary injection valves, whether manual or automatic, do not have the durability of extreme pressure. Typical performance criteria for reproducibility and linearity of the injection are required for analytical applications, but other features are also necessary. To protect the column from extreme pressure fluctuations, the injection process should eliminate pulse interference as much as possible. The instrument's system size is small to reduce the possible zone broadening of the sample being tested. In the fast injection cycle, UPLC can work at maximum speed, large sample throughput, and can be used without manual inspection for a long time. At the same time, the low volume injection volume and the minimum cross-contamination match the sensitivity improvement.

In theory, a UPLC system with 1.7 μm particles produces a detection peak with a half-width of less than 1 second. This poses a challenge for UPLC inspection. First, the detector must have a high sampling rate to capture enough data points as the peak passes to accurately and reproducibly identify (and integrate) the detected peaks of the analyte. Second, the detector must have a minimum diffusion volume to ensure that the separation efficiency is not reduced. The optical components of the detector must also have performance metrics that reflect the sensitivity of the UPLC. Conceptually, for different detection techniques, the sensitivity of UPLC detection should be 2-3 times the sensitivity of HPLC separation (Figure 5). For example, mass spectrometry can greatly benefit from the performance characteristics of UPLC. An increase in peak concentration and a reduced chromatographic diffusion at a lower flow rate (without shunting) will increase the efficiency of the ion source, thereby increasing the sensitivity of the mass spectrometer connected to the UPLC by at least a factor of three.

Figure 5: Decreasing the chromatographic broadening of an analyte during chromatographic analysis of peak capacity may help to increase sensitivity.

A comprehensive system approach <br>After thinking about the chromatographic process and how to apply these principles to UPLC, it is clear that in addition to the chemistry, pumps, injectors and detectors, the system needs to be considered. The size of the system is critical. If the high performance of UPLC separation is to be maintained during chromatographic analysis, the total system volume of UPLC must be significantly lower than current HPLC systems. It is also important to note that not only must the performance specifications of the system components mentioned above be carefully considered, but also the connecting tubes and fittings need to be carefully considered. In order for UPLC's good performance to be widely used, it is necessary to develop a comprehensive system approach that includes both the required performance specifications and control of accessory variables.

In the chromatography laboratory, the advantages of the UPLC system can be fully utilized, bringing more benefits to the laboratory. High-throughput library screening for characterization can be done at a faster rate or with better resolution, both of which can translate into increased efficiency. Metabolite identification and bioanalysis will benefit from increased speed, resolution and sensitivity. The peptide map time can be significantly shortened to speed up the characterization process. For peptide studies, the UPLC system yields a larger peak capacity than the currently used HPLC. The overall development of the stability indication method and method will result in more information per injection and will also reduce separation time. The most common use of chromatographic separation techniques - quantitative analysis can achieve faster speeds, better baseline separation, which will make quantitative integration easier and more reproducible, increasing laboratory productivity and credibility.

UPLCTM: Redefining Chromatography Labs <br>Many scientists have encountered separation barriers and are currently working to break the limits of common HPLC; ultra-high performance liquid chromatography offers the potential to expand and extend this widely used separation technique (Figure 6). UPLC technology promises to increase LC speed, resolution and sensitivity, providing more information for each analysis; therefore, the technology will be widely recognized in the laboratory.

Figure 6: UPLC presents a new speed, resolution and sensitivity that will redefine the chromatography lab.

references
[1] Anton D. Jerkovich, J. Scott Mellors, and James W. Jorgenson, "The Use of Micrometer-Sized Particles in Ultrahigh Pressure Liquid Chromatography," LCGC North America Volume 21, Number 7 (2003).
[2] N. Wu, JA Lippert, and ML Lee, "Practical Aspects of Ultrahigh Pressure Capillary Liquid Chromatography," J. Chromatogr. 911, 1 (2001).
N. Wu, DC Collins, JA Lippert, Y. Xiang, and ML Lee, "Ultrahigh Pressure Liquid Chromatography/Time-of-Flight Mass Spectrometry for Fast Separations," J. Microcol. Sep. 12, 462 (2000).
[3] JM Cintron, LA Colon, "Organo-silica Nanoparticles Used in Ultrahigh- Pressure Liquid Chromatography," The Analyst, 127 (2002) 701 - 704.

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