INTRODUCTION:

Ultra performance liquid chromatography systems take advantage of technological pace in particle chemistry performance, system optimization, detector design and data processing. When taken together, these achievements have created an improvement in chromatographic performance. UPLC retains the practicality and principles of HPLC and along with that increases the overall interrelated attributes of speed, sensitivity and resolution.

Speed allows a greater number of analyses to be performed in a shorter amount of time thereby increasing sample throughput and lab productivity. Faster analysis and hence called as ultra performance liquid chromatography, achieves both higher sample analysis throughput and better assay sensitivity. Analysis of operation cost and sample throughput UPLC cost advantageous over HPLC.¹

The factor responsible for the development of UPLC technique was evolution of packing materials used to effect the separation. The principles behind this evolution are governed by the van Dee meter equation that describes the relationship between linear velocity and plate height.

According to the van Dee meter equation, decrease in particle size increases the efficiency of separations while on other hand efficiency diminishes and peak capacity can be extended to new limits, termed ultra performance liquid chromatography (UPLC).

This technology takes full advantage of chromatographic principles to run separations using columns packed with smaller particles and/or higher flow rates for increased speed, with superior resolution and sensitivity.¹ The use of non-porous particles, however, has been limited in the pharmaceutical industry due to their low sample loading capacity. The Milford, Massachusetts based company Waters Corporation introduced ACQUITY UPLC, the commercially available system that addresses the challenge of using elevated pressure and 2 mm particles, which makes it a particularly attractive and promising tool for fast Liquid Chromatographic method development. Engineering challenges of operating at high pressures and the high performance expected from such columns necessitates new developed pumps, redesigned injector, reduced system volumes, an increased detector sampling rate, and other improvements. To be suitable for the analysis of pharmaceutical development samples under GMPs, the UPLC instrument and columns must not only deliver on its promises for fast, high resolution separations but do so reproducibly and with the required sensitivity.² In addition to the speed at which the data can be obtained, the quality of the data is also improved. It is clear that the quality of the UPLC-MS spectra is better than that of the Capillary LC-MS spectra with much improved signal-to-noise ratio.³ This new category of analytical separation science retains the practicality and principles of HPLC while increasing the overall interrelated attributes of speed, sensitivity and resolution. Today’s pharmaceutical industries are looking for new ways to cut cost and shorten time for development of drugs while at the same time improving the quality of their products and analytical laboratories are not exception in this trend. These are the benefits of faster analysis and hence the ultra performance liquid chromatography. UPLC presents the possibility to extent and expand the utility of conventional HPLC.

THEORETICAL PRINCIPLES OF UPLC:^{4, 5}

A good chromatographic method is characterized by improved efficiency, sensitivity, resolution and time of the analysis. Reduction of stationary phase particle size from 5 to 1.7 µm resulted in development of new separation technology UPLC.

EFFICIENCY:

Efficiency (N) is inversely proportional to particle size (dp).

As the particle size is lowered from 5µm (HPLC) to 1.7 µm (UPLC), N is increased by a factor of three.

SENSITIVITY:

If N increases then peak width decreases to its square the decrease in peak width produces taller peaks, as peak width is inversely proportional to peak height (h).

Increase in sensitivity is obtained as a result of narrow and tall peaks.

RESOLUTION:

Resolution between two chromatographic peaks is determined by measuring the distance between two peaks relative to their respective peak widths. The resolution equation provides quantitative model for the three parameters that controls resolution (efficiency, selectivity and retentivity).

(Efficiency) (Selectivity) (Retentivity)

Developing a chromatographic method is based upon the systematic manipulation of these parameters. Most method development strategies focus on retention and sensitivity because they are easy and economical to manipulate.

Resolution is improved by increasing the retention (k) of all peaks. Increasing retention, however, increases peak width, resulting in lower sensitivity and reduces sample throughput.

Selectivity (elution sequence) can be manipulated by several parameters including mobile phase pH, organic modifier and bonded phase.

Efficiency is less often used to improve a separation because it is difficult to change experimentally and any improvements only contribute to resolution as the square root. Efficiency however, can be significantly improved by reducing the diameter of the particle. A column packed with 1.7 µm particles would offer a 1.7 fold improvement in resolution compared to a column packed with 5 µm materials.

SPEED:

Efficiency is proportional to the column length and inversely proportional to the particle size.

The column can be shortened by the same factor as the particle size without loss of resolution. Using a flow rate three times higher due to the small particles and shortening the column by one third, the separation is completed in one-ninth time while maintaining the resolution.

INSTRUMENTATION:

To truly take advantage of the increased speed, superior resolution and sensitivity afforded by small particles, instrument technology also had to keep pace. A completely new system design with advanced technology in the pump, auto sampler, detector, data system, and service diagnostics was required. The ACQUITY UPLC system has been designed for low system and dwell volume to take full without loss of resolution. The application of UPLC resulted in the detection of additional drug metabolites, superior separation and improved spectral ^6-7.The lines of attack for fast LC method development are varied. Method development simulation software’s such as ACD™ ⁸, Dry Lab ™⁹ or Chromsword ™ ¹⁰ are valuable tools for optimizing and streamlining methods. Such softwares allow to increase the information obtained from a limited number of runs and to predict the best possible separation conditions.

PUMPING SYSTEMS:

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. The binary solvent manager uses two individual serial flow pumps to deliver a parallel binary gradient. There are built-in solvent select valves to choose from up to four solvents. There is a 15,000-psi pressure limit (about 1000 bar) to take full advantage of the sub-2μm particles.

SAMPLE INJECTION:

In UPLC, sample introduction is critical. Conventional injection valves, either automated or manual, are not designed and hardened to work at extreme pressure. To protect the column from extreme pressure fluctuations, the injection process must be relatively pulse-free and 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 capitalize on the speed afforded by UPLC, which in turn requires a high sample capacity. Low volume injections with minimal carryover are also required to increase sensitivity¹¹. There are also direct injection approaches for biological samples.^12-13

SAMPLE MANAGER:

The sample manager also incorporates several technology advancements. Using pressure assisted sample introduction, low dispersion is maintained through the injection n process, and a series of pressures required. The binary solvent manager uses two individual serial flow pumps to deliver a Parallel binary gradient. There are built-in solvent select valves to choose from up to four solvents. There is a 15,000-psi pressure limit (about 1000 bar) to take full advantage of the sub-2μm particles. Transducers facilitate self-monitoring and diagnostics. It uses needle-in-needle sampling for improved ruggedness and needle calibration sensor increases accuracy. Injection cycle time is 25 seconds without a wash and 60 sec with a dual wash used to further decrease carry over. A variety of micro titer plate formats (deep well, mid height, or vials) can also be accommodated in a thermostatically controlled environment. Using the optional sample organizer, the sample manager can inject from up to 22 micro titer plates. The sample manager also controls the column heater. Column temperatures up to 65°C can be attained. To minimize sample dispersion, a “Pivot out “design allows the column outlet to be placed in closer proximity to the Source inlet of an MS detector. ¹⁴

UPLC COLUMNS:

The design and development of sub-2μm particles is a significant challenge, and researchers have been very active in this area to capitalize on their advantages ^15-16. Although high efficiency nonporous 1.5μm 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. Other alternative, polymeric columns can overcome pH limitations, but they have their own issues, including low efficiencies and limited capacities. In 2000, Waters introduced 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 produces using a classical sol-gel synthesis that incorporates carbon in the form of methyl groups. However, in order to provide the kind of enhance mechanical stability UPLC requires, a second generation hybrid technology¹⁷,was developed, called ACQUITY UPLC.ACQUITY 1.7μm particles bridge the methyl groups in the silica matrix as shown in fig1, which enhances their mechanical stability. Resolution is increased in a 1.7 μm particle packed column because efficiency is better. Separation of the components of a sample requires a bonded phase that provides both retention and selectivity. Four bonded phases are available for UPLC separations: 

ACQUITY UPLC BEH T M C18 and C8

(straight chain alkyl columns)

ACQUITY UPLC BEH Shield RP 18

(embedded polar group column)

ACQUITY UPLC BEH Phenyl

(phenyl group tethered to the silyl functionality with a C6 alkyl)¹⁸

ACQUITY UPLC BEH Amide columns

(trifunctionally bonded amide phase)

Each column chemistry provides a different combination and hydrophobicity, silanol activity, hydrolytic stability and chemical interaction with analytes.

ACQUITY UPLC BEH T M C18 and C18 Columns – These are considered as the universal columns of choice for most UPLC separations by providing the widest pH range. They incorporate trifunctional ligand bonding chemistries which produce superior low pH stability. This low pH stability is combined with the high pH stability of the 1.7μm BEH particle to deliver the widest usable pH operating range.

ACQUITY UPLC BEH Shield R18 Columns – These are designed to provide selectivities that complement theACQUITY UPLC BEH T M C18 and C8 Columns.

ACQUITY UPLC BEH Phenyl Columns – These utilize a trifunctional C6 alkyl ethyl between the phenyl ring and the silyl functionality.

ACQUITY UPLC BEH Amide columns-BEH particle technology, in combination with a trifunctionaly bonded amide phase, provides exceptional column life time, thus improving assay robustness. BEH Amide columns facilitate the use of a wide range of phase pH [2 –11] to facilitate the exceptional retention of polar analytes spanning a wide range in polarity, structural moiety and Pka.

DETECTORS:

For UPLC detection, the tunable UV/Visible detector is used which includes new electronics and firmware to support Ethernet communications at the high data rates. Conventional absorbance-based optical detectors are concentration sensitive detectors, and for UPLC use, the flow cell volume would have to be reduced in standard UV/Visible detectors to maintain concentration and signal. According to Beer’s Law, smaller volume conventional flow cells would also reduce the path length upon which the signal strength depends. A reduction in cross-section means the light path is reduced, and transmission drops with increasing noise. Therefore, if a conventional HPLC flow cell were used, UPLC sensitivity would be compromised. The ACQUITY Tunable UV/Visible detector cell consists of a light guided flow cell equivalent to an optical fibre. Light is efficiently transferred down the flow cell in an internal reflectance mode that still maintains a 10mm flow cell path length with a volume of only 500nL. Tubing and connections in the system are efficiently routed to maintain low dispersion and to take advantage of leak detectors that interact with the software to alert the user to potential problems ^19.

ADVANTAGES OF UPLC:^{20, 21}

1. Decreases run time and increases sensitivity.

2. Provides the selectivity, sensitivity, and dynamic range of LC analysis.

3. Maintaining resolution performance.

4. Expands scope of Multi residue Methods

5. UPLC’s fast resolving power quickly quantifies related and unrelated compounds.

6. Faster analysis through the use of a novel separation material of very fine particle size

7. Operation cost is reduced.

8. Less solvent consumption.

9. Reduces process cycle times, so that more product can be produced with existing resources.

10. Increases sample throughput and enables manufacturers to produce more material that consistently meet or exceeds the product specifications, potentially eliminating Variability, failed batches, or the need to re-work material.

11. Delivers real-time analysis in step with manufacturing processes. Assures end-product quality, including final release testing.

DISADVANTAGES:

1. Due to increased pressure requires more maintenance and reduces the life of the columns of this type. So far performance similar or even higher has been demonstrated by using stationary phases of size around 2μm without the adverse effects of high pressure.

2. In addition, the phases of less than 2μm are generally non-regenerable and thus have limited use.

APPLICATIONS OF UPLC: ²³

1. Analysis of natural products and traditional herbal medicine

2. Identification of metabolite.

3. Study of metabonomics/metabolomics.

4. Bioanalysis/bioequivalence studies

5. Dissolution testing.

6. Manufacturing/QA/QC

7. Impurity profiling

8. Computer library maintenance

9. Determination of amino acids composition of proteins, cell – culture media, food and nutritional analysis.

10. Purification and Analysis of oligonucleotides using C₁₈ columns

11. Determination of amino acids composition of proteins, cell – culture media, food and nutritional analysis.

12. UPLC – ELS detector has been specially designed for applications where analyte have poor or no UV and VISIBLE response, or don’t ionize by MS.

EXAMPLE: Antibiotic, Antivirals, Biomolecules, Natural products.

13. UPLC technique is widely used for peptide mapping.

14. This technique is used for purification and separation of polar analytes.

CONCLUSION:

UPLC is a proven technique that has been using 1.7 µm particles and properly holistically designed system can provide significantly more resolution while reducing run time and improved sensitivity for the analysis of many compounds. UPLC presents the possibility to extend and expand the utility of chromatography.

ACKNOWLEDGEMENTS:

We are thankful to the management of Anurag Pharmacy College, Ananthagiri, Kodad, Nalgonda Dist, Andhra Pradesh, India, for providing all facilities during this study and special thanks to Dr. M.Chinna Eswaraiah, principal of Anurag Pharmacy College for his valuable guidance and constant encouragement to complete this work.

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Received on 26.09.2012 Modified on 09.10.2012

Research J. Pharm. and Tech. 5(12): Dec. 2012; Page 1484-1489