INTRODUCTION:

HPLC:

In order to separate, identify, and quantify active compounds, high-performance liquid chromatography-also referred to as high-pressure liquid chromatography - is a type of column chromatography that is often employed in biochemistry and analysis. It is a widely used analytical method for figuring out how to separate, identify, and measure each component of a mixture. One advanced method of column liquid chromatography is HPLC.

In the HPLC procedure, the solvent is forced through the column at pressures as high as 400 atmospheres, contrary to gravity's natural flow. This allows the sample to be divided into distinct components according to variations in relative affinities.

The typical components of HPLC system retention periods are a column packed with packing material (stationary phase), a pump that drives the mobile phase (s) through the column, and a detector that locates the molecule.

The interactions between the stationary phase, the molecules under study, and the solvent (s) used have an impact on the retention time. Little amounts of the samples to be examined are introduced to the stream of the mobile phase, where they are slowed down by certain chemical or physical reactions with the stationary phase. The kind of analyte and the makeup of the stationary and mobile phases both affect how much retardation occurs. The amount of time it takes for an analyte to elute is known as the retention time.

A common solvent is any miscible mixture of organic liquids or water. During the study, the mobile phase composition was altered using gradient elution. Analyte mixtures are separated by the gradient according to the analyte's affinity for the active mobile phase. The choice of gradients, additives, and solvents is influenced by the characteristics of the analyte and the stationary phase.¹

UHPLC:

Liquid chromatography with ultra-high performance (UHPLC) includes LC separations with columns that have particles smaller than the 2.5–5μm sizes often employed in HPLC. Greater efficiency per unit time is an advantage of utilising columns containing smaller particles, generally sub-2μm. Although UHPLC was first shown with custom equipment, the widespread use of this technology was made possible by the 2004 launch of the first UHPLC system on the market. In order to fully reap the benefits of sub-2-μm particle-containing columns, the equipment has to be optimised in tandem with the columns. The system had to have low extra-column dispersion and a column compartment that minimised radial temperature gradients in addition to being able to operate reliably at pressures of up to 1000 bar.²

Ultra Performance Liquid Chromatography, or UPLC, is a technique that is faster, more sensitive, and more resolved than HPLC. Its stationary phase employs sub 2μm particle technology to cut down on solvent use and shorten the total run time. The technology employed in Ultra Performance Liquid Chromatography allows peak capacity and speed during analysis to be enhanced and elevated to new heights with the aid of tiny particles, as per Van Demeter equation H = A + B/u + Cu. Analytes slowly diffuse into the stationary phase due to low diffusion coefficients in the liquid phase, which is why UPLC is less efficient than processes like gas chromatography and capillary electrophoresis. The Van Demeter Equation helps to explain the increased efficiency of UPLC, but it has a drawback in that it causes backpressure to grow during analysis. Short-length columns with sub-2μm particles are employed to address this problem. Utilising columns densely packed with ultra-small particles, UPLC makes full use of chromatographic principles 8 to perform the separations.

Pharmaceutical firms are now searching for ways to reduce the time and expense associated with medication development and analysis while upholding standards and quality. These are the advantages of quick analysis carried out using UPLC. UPLC may be used to transfer and optimise a standard test, resulting in a reduction in analysis time and an improvement in analysis sensitivity.

Principle:

Sub 2μm particle technology is employed by UPLC in its stationary phase9. The system operates on the premise of the Van Demeter equation, which essentially describes a connection between the plate height and flow rate. The following is the equation for the same:³

H = A + B/V + CV

Were,

A, B and C are constant.

A = Eddy diffusion,

H= HETP,

B = Longitudinal diffusion,

C = Equilibrium mass transfer,

V = flow rate.

Instrumentation:

Chromatographic separation can vary significantly across ultrahigh-pressure liquid chromatography (UHPLC) devices made by various manufacturers and with different settings. A more comprehensive assessment of the impact of instrument changes on the method is now necessary due to the increased complexity of the method development process brought about by the variance in instrument configuration. By measuring mobile-phase compositional accuracy, the investigations reported here identified the usual inter-instrument differences in dwell volume, additional column dispersion, and mixing efficiency.

In addition, the effects of individually and methodically varying the dwell volume and additional column dispersion on resolution for a small-molecule test combination during gradient elution were assessed. Translation and adjustment approaches for the dwell volume and wash-out volume method were assessed to take these inter-instrument variances into consideration.

Fig. UHPLC Instrument

Liquid chromatography separations utilising columns having particles smaller than the 2.5–5µm sizes often employed in HPLC are referred to as ultra-high-performance liquid chromatography (UHPLC). Greater efficiency per unit time is an advantage of utilising columns with smaller particles (usually sub-2µm).⁴

In order to fully benefit from the enhanced velocity, enhanced sensitivity, and improved resolution provided by tiny particles, instrument technology also needed to keep up. It was necessary to develop a whole new system with cutting edge technologies in the data system, auto sampler, detector, pump, and service diagnostics.⁵

Pumping Systems:

The pressure range that can be reached by today's HPLC technology is insufficient to achieve small particle, high peak capacity separations. Over a 15cm long column filled with 1.7µm particles, the estimated pressure drops at the flow rate that maximises efficiency is 15,000 psi. It is also necessary to have a pump that can provide solvent at these pressures smoothly and consistently, adjust for solvent compressibility, and function in both the gradient and isocratic separation modes. To provide a parallel binary gradient, the binary solvent manager makes use of two separate serial flow pumps. There are built-in valves for selecting from up to four different solvents. To fully use the sub-2µm particles, a pressure constraint of 15,000 psi, or roughly 1000 bar, is in place. ⁶

Quality of mobile phase and buffers:

It is crucial to remember that the frits and particles found in UHPLC columns are far smaller than those found in conventional HPLC packings. For instance, an HPLC column's input frit pore size is typically equivalent to 2 µm, however in a UHPLC column, it could only be equal to 0.2µm (this number greatly depends on the column supplier). Consequently, in the UHPLC setup, minute particles that may be present in the mobile phase but do not impact the HPLC materials might become crucial. As a result, it's critical to ensure that there are no insoluble particles in the solvents. To do this, there are a few crucial guidelines that must be followed while preparing the mobile phase in UHPLC:

· Use only premium organic solvents (preferably filtered via a 0.22µm membrane); acetonitrile of UHPLC quality may be obtained from a number of sources, including Biosolve®, JT Baker®, Fisher Scientific®, and others.

· Be cautious with the microbiological growth (especially when using phosphate buffer): always use freshly prepared mobile phases.

· The water should be ultra-pure and filtered through a 0.22µm membrane (Milli-Q® system or similar high-quality water is recommended).

· Don't top off the bottles and clean glasses with caution.⁷

UHPLC Columns:

For the purpose of separating sample components, a bonded phase that offers selectivity and retention is necessary. UHPLC columns have a smaller diameter because bigger diameter columns require greater flow rates, which in turn demand larger quantities of mobile phase, to obtain the necessary linear velocity.⁸

The most advanced UHPLC column technology available today comes in 2.1mm column configurations that are jam-packed with sub-2micron particle technology.

For many years, 4.6mm columns were considered the industry standard; however, in order to mitigate the effects of viscous heating during ultra-high-pressure operations, 2.1mm column forms had to be adopted. A notable temperature rise is seen near the column outlet when driving the flow through the interstitial spaces in the column packing, which subsequently impacts the retention qualities. 25A radial temperature gradient is created as heat is dispersed through the column wall, and the ensuing parabolic flow profile will negatively affect chromatographic efficiency.

The impact of frictional heating on the chromatographic separation is significantly less than what would be expected for 4.6 mm. columns running at similar linear velocities when the column's cross section is reduced to 2.1mm. and an optimised column-oven configuration is used. This results in a nearly five-fold increase in flow rate.

Regarding particle size and type, significant advancements in column technology have also been achieved. 12 Because the resistance to mass transfers (C-term) reduces according to the square of the particle diameter and the eddy-dispersion contribution (A-term) is proportional to particle diameter, decreased particle diameter results in lower plate height values.⁹

Advantages:

1. Use of a unique separation material with extremely tiny particle size that allows for faster analysis

2. Lower operating expenses.

3. Reduced use of solvents

4. Shortens process cycle times, allowing for the production of more goods with the same number of resources.

5. Shortens the runtime and boosts sensitivity.¹⁰

Disadvantages:

1. Higher back pressures shorten the columns' lifespan as compared to traditional HPLC.

2. Because the majority of particles smaller than 2μm are not regenerable, their applications are limited.¹¹

CONCLUSIONS:

UHPLC technology, which is characterised by columns filled with sub-2µm particles and operated at extremely high pressure, has shown to be a potent means of enhancing chromatographic analysis in terms of resolving power and throughput. Its inherent performance is particularly favourable when compared to other available methods, such high temperature liquid chromatography or monoliths, for increasing chromatographic efficiencies in the 1,000–80,000 plate range.

The less environmentally friendly analytical techniques may eventually give way to ultra-high-performance liquid chromatography, if the procedures utilising this type of chromatography have been adequately verified. The goal of UHPLC technique modifications is most likely to eliminate friction heating by exploring novel approaches to the manufacture of stationary and mobile phases.

REFERENCES:

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2. Thomas H. Walter, Richard W. Andrews, Recent innovations in UHPLC columns and instrumentation, Trends in Analytical Chemistry. 2014. doi: 10.1016/j.trac.2014.07.016

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7. Davy Guillarme, Jean-Luc Veuthey. Guidelines for the use of UHPLC Instruments. Davy Guillarme, Jean-Luc Veuthey Lcap, University of Geneva.

8. Patel Niti Bharatbhai, Jasmina Surati et al., A Review on UHPLC Instrumentation with Advancements. Journal of Advances in Pharmaceutical Sciences. 2023; 1(1): 33-44 2023.

9. De Vos J, Stoll D, Buckenmaier S, Eeltink S, Grinias JP. Advances in ultra-high-pressure and multi-dimensional liquid chromatography instrumentation and workflows. Anal Sci Adv. 2021; 2: 171–192. https://doi.org/10.1002/ansa.202100007

10. Samatha et al. Ultra Performance Liquid Chromatography (UPLC). World Journal of Pharmacy and Pharmaceutical Sciences. 2015; 4(8); 356-367.

11. Swetha Sri. R, Bhavya Sri. K, Mounika. Ch. A Review on Comparative study of HPLC and UPLC. Research J. Pharm. and Tech. 2020; 13(3): 1570-1574. doi: 10.5958/0974-360X.2020. 00284.Χ

Received on 05.03.2024 Modified on 30.03.2024

Res. J. Pharmacology and Pharmacodynamics. 2024;16(2):123-126.

DOI: 10.52711/2321-5836.2024.00022