Ultra Performance Liquid Chromatography (UPLC) - A Review

    

    

    Figure 1:  van Demeter plot, illustrating the evolution of particle sizes over the
last three decades.
    
    

Researcher scholars are study many decades at “fast LC” to speed up analyses. In drug discovery the “need for speed” has been came from by the selecting so many of samples in different laboratories, and the availability of sophisticated instrument like UPLC with detector as mass spectrometers. The unique features of column that is smaller columns and faster flow rates (amongst other parameters) have been used. During analysis higher temperature, having the dual advantages of lowering viscosity, and increasing mass transfer by increasing the diffusivity of the analytes, has also been investigated. However, using conventional particle sizes and pressures, limit-actions are soon reached and compromises must be made, sacrificing resolution for time.

It found that the analysis by UPLC method and the classic separation method is of HPLC (High Performance Liquid Chromatography). There are many advantages in HPLC like robustness, ease of use, good selectivity and adjustable sensitivity. But its main limitation is the lack of efficiency compared to gas chromatography or the capillary electrophoresis due to low diffusion coefficients in liquid phase, involving slow diffusion of analytes in the stationary phase. In UPLC main advantage is better efficiency with speedy analysis and this achieved by only smaller particle size. The Van Deemter equation shows that efficiency increases with the use of smaller size particles but this leads to a rapid increase in back pressure, while most of the HPLC system can operate only up to 400 bars. That is why short columns filled with particles of about 2μm are used with these systems, to accelerate the analysis without loss of efficiency, while maintaining an acceptable loss of load to improve the efficiency of HPLC separations, the following can be done [10-15].

• Work at higher temperatures- allows high flow rates by reducing the viscosity of mobile phase which significantly reduces back pressure.

• Use of monolithic columns- contains polymerize porous support structure that provides lower flow resistances than conventional particle-packed columns.

By above two parameter UPLC analysis improves in three areas.

1. Produced Chromatogram with resolved peak.

2. Fast analysis

3. Sensitive analysis

It uses fine particles and saves time and reduces solvent consumption. The new method make a very big difference by retains the same analytical separation method as HPLC while other things which drastically changes are speedy analysis, sensitivity and high resolution. Today’s pharmaceutical industries are focusing for new path to reduce cost and less time for development of drugs and in mean time the quality of their products are not suffer analytical laboratories maintain the whole things. Speed allows a greater number of analyses to be performed in a shorter amount of time thereby increasing sample throughput and lab productivity. These are the benefits of faster analysis and hence the ultra performance liquid chromatography. A typical assay was transferred and optimized for UPLC system to achieve both higher sample analysis throughput and better assay sensitivity. Analysis of operation cost and sample throughput found, UPLC cost advantageous over HPLC [16-20].

Chemistry of Small Particles whatever as shown in Figure 1, smaller particles create not only increased efficiency, parallel also the ability to work at higher linear velocity without a loss of efficiency, providing both resolution and speed. Efficiency is the fundamental separation parameter behind UPLC since it relies on the same selectivity and retentivity as HPLC. In the fundamental Resolution (Rs) equation:

Rs = √N/4 (α-1/ α) (k/k+1)

Resolution is proportional to the square root of N. But since N is inversely proportional to particle size (dp):

N α 1/dp

As the particle size is lowered by a factor of three, from, for example, 5 m (HPLC scale) to 1.7 m (UPLC-scale), N is increased by three and resolution by the square root of three or 1.7 N is also inversely proportional to the square of the peak width [20-24].

N α 1/ w²

This illustrates that the narrower the peaks are, the easier they are to separate from each other. Also, peak height is inversely proportional to the peak width:

H α 1/w

So as the particle size decreases to increase N and subsequently Rs, an increase in sensitivity is obtained, since narrower peaks are taller peaks. Narrower peaks also mean more peak capacity per unit time in gradient separations, desirable for many applications, e.g., peptide maps. Still another equation comes into play when migrating toward smaller particles:

F opt α 1/dp

This relationship also is revealed from the Van Deemter plot. As particle size decreases,

The optimum flow ( Fopt). To reach maximum N increases. But since back pressure is proportional to flow rate, smaller particle sizes require much higher operating pressures, and a system properly designed to capitalize on the efficiency gains . A system that can both reliably deliver the requisite pressures and that can maintain the separation efficiency of the small particles with tightly managed volumes. Higher resolution and efficiency can be leveraged even further, however when analysis speed is the primary objective. Efficiency is proportional to column length and inversely proportional to the particle size [17-20].

N α L/dp

Therefore, 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 smaller particles and shortening the column by one third (again due to the smaller particles), the separation is completed in 1/9 the time while maintaining resolution. So if speed, throughput, or sample capacity is a concern, theory can be further leveraged to get much higher throughput. But the design and development of sub-2μm particles is a significant challenge, and researchers have been active in this area for some time, trying to capitalize on their advantages. Although high efficiency nonporous 1.5-μm particles are commercially available, they suffer from poor loading capacity and retention due to low surface area. Silica-based particles have good mechanical strength but can suffer from a number of disadvantages, which include a limited pH range and tailing of basic analytes. Polymeric columns can overcome pH limitations, but they have their own issues including low efficiencies, limited loading capacities, and poor mechanical strength. In 2000, Waters introduced XTerra^®, a first generation hybrid chemistry that took advantage of the best of both the silica and polymeric column worlds. XTerra^®, columns are mechanically strong, with high efficiency, and operate over an extended pH range. They are produced using a classical sol-gel synthesis that incorporates carbon in the form of methyl groups [21- 22].

Instrumentation

A. Sample Injection

B. UPLC Columns

C. Detectors

A. 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 pulsefree 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 sensitivity. There are also direct injection approaches for biological samples.

B. UPLC Columns

Resolution is increased in a 1.7μm 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:

(i) ACQUITY UPLCTM BEH C8 (straight chain alkyl columns),

(ii) ACQUITY UPLCTM BEH C18 (straight chain alkyl columns),

(iii) ACQUITY UPLC BEH Shield RP18 (embedded polar group column) and

(iv) ACQUITY UPLC BEH Phenyl (phenyl group tethered to the silyl functionality with a C6 alkyl),

ACQUITY UPLC BEH Phenyl (phenyl group tethered to the silyl functionality with a C6 alkyl).

Each column chemistry provides a different combination of hydrophobicity, silanol activity, hydrolytic stability and chemical interaction with analytes. ACQUITY UPLC BEH C18 and C8 columns are considered the universal columns of choice for most UPLC separations by providing the widest pH range. They incorporate tri functional 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. ACQUITYUPLC BEH Shield RP18 columns are designed to provide selectivity that complements the ACQUITYUPLC BEH C18 and C8 phases. ACQUITY UPLC BEH Phenyl columns utilize a tri functional C6 alkyl tether between the phenyl ring and the silyl functionality. This ligand, combined with the same proprietary end capping processes as the ACQUITY UPLC BEH C18 and C8 columns, provides long column lifetimes and excellent peak shape. This unique combination of ligand and end capping on the 1.7μm BEH particle creates a new dimension in selectivity allowing a quick match to the existing HPLC column. An internal dimension (ID) of 2.1 mm column is used. For maximum resolution, choose a 100 mm length and for faster analysis, and higher sample throughput, choose 50 mm column. Half-height peak widths of less than one second are obtained with 1.7μm particles, which gives significant challenges for the detector. In order to integrate an analyte peak accurately and reproducibly, the detector sampling rate must be high enough to capture enough data points across the peak. 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 promote increased source ionization efficiencies. The ACQUITY UPLC System consists of a binary solvent manager, sample manager including the column heater, detector, and optional sample organizer.

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. The sample manager also incorporates several technology advancements. Using pressure assisted sample introduction, low dispersion is maintained through the injection process, and a series of pressures 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.

C. Detectors

The detectors are use in UPLC analysis is UV/Visible detector. Detection of analytes is conventionally based on absorbance that is concentration sensitivity detectors. In UPLC the flow cell volume would have to be reduced to maintain concentration and signal. Based on 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 fiber. 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 500mL. 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 [23-24] (Figure 2).

    

    

    Figure 2:  Detectors Flow Cell.