Ultra Performance Liquid Chromatography (Uplc): A New Trend in Analysis

Chromatography is a technique used to separate the components of a mixture, known as solutes, by examining how well each solute distributes itself between a stationary phase and a mobile phase, which is a flowing fluid. The stationary phase may be either solid or liquid, while the mobile phase can be a liquid or gas. Separation is influenced by factors such as the molecular properties related to adsorption, affinity, and partitioning, as well as differences in molecular weights. Consequently, some mixture components move quickly through the mobile phase and exit the chromatography system, while others move more slowly through the stationary phase and remain longer. This method consists of three key components: Chromatography is a technique employed to separate mixtures into their individual components. The term comes from Greek, with "chromo"meaning "color"and"graphic" meaning "writing." All types of chromatography operate on the same fundamental principle, utilizing both a stationary phase (which can be a solid or a liquid supported on a solid) and a mobile phase (either a liquid or a gas). The process involves the transfer of mass between these two phases. Stationary phase: This consists of either a solid phase or a layer of liquid that is absorbed on a solid support Separated molecules. Mobile phase: This typically involves a liquid or gas component Many HPLC methods can be carried out on UPLC for its less time consumption and high sensitivity. These methods could be optimized to get better results. In this respect, an HPLC method for quality control (QC) was optimized for UPLC.

Table 1: Comparison of Original HPLC and Optimized UPLC Parameters

While the underlying principles of UPLC and HPLC are the same, their performance differs significantly. UPLC utilizes column materials with particle sizes of less than 2 μm, which enhances its efficiency. To fully leverage the benefits of these columns, UPLC offers apowerful,robust, and reliable solution. The familiar design of the UPLC H-Class's Quaternary Solvent Manager (QSM) and Sample Manager (SMFTN), featuring a flow-through needle design, provides the flexibility and usability similar to traditional HPLC, while achieving the superior separations unique to UPLC

Principle of UPLC:

 UPLC operates on the principle of using stationary phases made up of particles smaller than 2 μm. This advancement is influenced by the Van Deemter equation, an empirical formula that outlines the relationship between linear flow rate and plate height (HETP or column efficiency).                                 The equation is represented as follows:

 H=A+B/V+CV

Where:

A, B, and C are constants                                           

 H = Height Equivalent to a Theoretical Plate (HETP)

A = Eddy diffusion

B = Longitudinal diffusion

C = Equilibrium mass transfer

V = flow rate

Eddy diffusion:

refers to the movement of the mobile phase through a column filled with stationary phase, where solute molecules follow various random paths. This randomness leads to band broadening.

Longitudinal diffusion:

occurs because the concentration of analytes is lower at the edges of the band compared to the center. As a result, analytes diffuse from the center toward the edges, contributing to band broadening. Eddy diffusion (A) is at its lowest when the particles in a packed column are small and uniform. the B term, which reflects longitudinal diffusion or the natural tendency of molecules to diffuse, decreases at higher flow rates, so it’s divided by B. The C term represents equilibrium mass transfer, which is affected by kinetic resistance during the separation process. This resistance is the delay experienced by molecules moving from the gas phase to the stationary phase and back. As gas flow increases, molecules in the packing tend to lag behind those in the mobile phase

Chemistry of Small Particles:

The design and development of sub-2 µm particles is a significant challenge, and researchers have been very active in this area to capitalise on their advantages [2, 3]. Although high efficiency nonporous 1.5 µm particles are commercially available, they suffer from low surface area, leading to poor loading capacity and retention. To maintain retention and capacity similar to HPLC, UPLC must use a novel porous particle that can withstand high pressures. Silica based particles have good mechanical strength, but suffer from a number of disadvantages. These include tailing of basic analytes and a limited pH range. Another alternative, polymeric columns, can overcome pH limitations, but they have their own issues, including low efficiencies and limited capacities. In 2000, Waters introduced a first generation hybrid chemistry, called XTerra®, which combines the advantageous properties of both silica and polymeric columns - they are mechanically strong, with high efficiency, and operate over an extended pH range. XTerra columns are produced using a classical solgel synthesis that incorporates carbon in the form of methyl groups. However, in order to provide the kind of enhanced mechanical stability UPLC requires, a second generation hybrid technology was developed [4], called ACQUITY UPLCTM. ACQUITY UPLC 1.7 µm particles bridge the methyl groups in the silica matrix, as shown in Figure 3, which enhances their mechanical stability. Packing a 1.7µm particle in reproducible and rugged columns was also a challenge that needed to be overcome. The column hardware required a smoother interior surface and the end frits were re-designed to retain the small particles and resist clogging. Packed bed uniformity is also critical, especially if shorter columns are to maintain resolution while accomplishing the goal of faster separations. All ACQUITY UPLC columns also include the eCordTM microchip technology that captures the manufacturing information for each column, including the quality control tests and certificates of analysis. When used in the Waters ACQUITY UPLC system, the eCord database can also be updated with real time method information, such as the number of injections, or pressure information, to maintain a complete column history pler, detector, data system, and service diagnostics was required. The ACQUITY UPLC system has been holistically designed for low system and dwell volume to take full advantage of low dispersion and small particle technology. Achieving small particle, high peak capacity separations requires a greater pressure range than that achievable by today's HPLC instrumentation. The calculated pressure drop at the optimum flow rate for maximum efficiency across a 15 cm long column packed with 1.7 µm particles is about 15,000 psi. Therefore a pump capable of delivering solvent smoothly and reproducibly at these pressures, which can compensate for solvent compressibility and operate in both the gradient and isocratic separation modes, is required. Sample introduction is also critical. Conventional injection valves, either automated or manual, are not designed and hardened to work at extreme pressure. To protect the column from experiencing extreme pressure fluctuations, the injection process must be relatively pulsefree. The swept volume of the device also needs to be minimal to reduce potential band spreading. A fast injection cycle time is needed to fully capitalise on the speed afforded by UPLC, which in turn requires a high sample capacity. Low volume injections with minimal carryover are also required to realise the increased sensitivity benefits. With 1.7 µm particles, half-height peak widths of less than one second are obtained, posing significant challenges for the detector. In order to accurately and reproducibly integrate an analyte peak, the detector sampling rate must be high enough to capture enough data points across the peak. In addition, the detector cell must have minimal dispersion (volume) to preserve separation efficiency. Conceptually, the sensitivity increase for UPLC detection should be 2-3 times higher than HPLC separations, depending on the detection technique. MS detection is significantly enhanced by UPLC; increased peak concentrations with reduced chromatographic dispersion at lower flow rates (no flow splitting) promotes increased source ionisation efficiencies.

 Instrumentation

The fundamental principles and instrumentation of a UPLC system are similar to those of HPLC, with enhancements in the equipment and hardware. The UPLC system includes a binary solvent system, a sample manager, a column manager, and a detector. The solvent manager features two flow pumps that create a parallel binary gradient under high pressure. A degassing system removes dissolved gases from the mobile phase, which can be selected from up to four solvents via a valve. UPLC systems can handle pressures of approximately 15,000 psi (around 1000 bar), allowing them to utilize sub-2-mm particles effectively. Additionally, the sample manager employs advanced technology, enabling sample temperatures to be lowered to 0°C, while the column manager can regulate temperatures up to 90°C. This high-temperature capability in ulta performance liquid chromatography (UPLC) significantly reduces analysis time without compromising efficiency

Components of instrumentation

1. Pumping system

2. Sample introduction device

3. UPLC columns

4. Detection instruments