Introduction to new chromatography technique - UPLC
Snehal V.Chopade, Dr.V.R.Patil - Principle
TVES College of pharmacy,
Faizpur accredited by North Maharashtra University,
UPLC can be regarded as new invention for liquid chromatography. UPLC refers to Ultra Performance Liquid Chromatography. UPLC brings dramatic improvements in sensitivity, resolution and speed of analysis can be calculated. It has instrumentation that operates at high pressure than that used in HPLC & in this system uses fine particles(less than 2.5µm) & mobile phases at high linear velocities decreases the length of column, reduces solvent consumtion & saves time.
This review introduces the theory of UPLC, and it can summarizes some of the most recent work in the field.
Reference Id: PHARMATUTOR-ART-1206
Chromatography is a non-destructive procedure for resolving a multi-component mixture of traces, minor or constituents in to individual fractions.It is a method of separating a mixture of components in to individual components through a porous medium under the influence of solvent 
For many years, researchers have looked at “fast LC” as a way to speed up analyses.[4,5] The need for speed , the availability of affordable and easy to use mass spectrometers. Smaller columns and faster flow rates (amongst other parameters) have been used. Elevated 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, limitations are soon reached and compromises must be made, sacrificing resolution.
High performance liquid chromatography (HPLC) is approved technique as it has been used in laboratories worldwide over the past 30-plus years. HPLC technology simply doesn’t have the capability to take full advantages of sub-2µm particles..
UPLC can be regarded as new invention for liquid chromatography. UPLC refers to Ultra Performance Liquid Chromatography. UPLC brings dramatic improvements in sensitivity, resolution and speed of analysis can be calculated. It has instrumentation that operates at high pressure than that used in HPLC & in this system uses fine particles(less than 2.5µm) & mobile phases at high linear velocities decreases the length of column, reduces solvent consumption & saves time. This review introduces the theory of UPLC, and it can summarizes some of the most recent work in the field.
According to the van Deemter equation, as the particle size decreases to less than2.5 µm, there is a significant gain in efficiency, while the efficiency does not diminish at increased flow rates or linear velocities.
Therefore by using smaller particles, speed and peak capacity (number of peaks resolved per unit time in gradient separations) can be extended to new limits, termed Ultra PerformanceLiquid Chromatography, or UPLC .
The technology takes full advantage of chromatographic principles to run separations
Using columns packed with smaller particles(less than2.5µm) and/or higher flow rates for increased speed, this gives superior resolution and sensitivity [7,8].Now a days in industrial area UPLC refers for some of the most recent work field.
The UPLC is based on the principal of use of stationary phase consisting of particles less than 2.5 μm (while HPLC columns are typically filled with particles of 3 to 5 μm). The underlying principles of this evolution are governed by the Van Deemter equation, which is an empirical formula that describes the relationship between linear velocity (flow rate) and plate height (HETP or column efficiency) [8,11].
A, B and C are constants
v is the linear velocity, the carrier gas flow rate.
*The A term is independent of velocity and represents "eddy" mixing. It is smallest when the packed column particles are small and uniform.
* The B term represents axial diffusion or the natural diffusion tendency of molecules.This effect is diminished at high flow rates and so this term is divided by v.
* The C term is due to kinetic resistance to equilibrium in the separation process.The kinetic resistance is the time lag involved in moving from the gas phase to the packing stationary phase and back again. The greater the flow of gas, the more a molecule on the packing tends to lag behind molecules in the mobile phase. Thus term is proportional to v.
Therefore it is possible to increase throughput, and thus the speed of analysis without affecting the chromatographic performance. The advent of UPLC has demanded the development of a new instrumental system for liquid chromatography, which can take advantage of the separation performance (by reducing dead volumes) and consistent with the pressures (about 8000 to 15,000 PSI, compared with 2500 to 5000 PSI in HPLC). Efficiency is proportional to column length and inversely proportional to the particle size.
As shown in Figure 1, smaller particles provide increased efficiency as well as the ability to work at increased linear velocity without a loss of efficiency, providing both resolution and speed. Efficiency is the primary separation parameter behind UPLC since it relies on the same selectivity and retentivity as HPLC. In the fundamental
resolution (Rs) equation :
Resolution is proportional to the square root of N. But since N is inversely proportional
to particle size (dp):
as the particle size is lowered by a factor of three, from, for example, 5 µm (HPLC-scale)
to 1.7 µm (UPLC-scale), N is increased by three and resolution by the square root of three or 1.7N is also inversely proportional to the square of the peak width
eg: (Fig 1)UPLC stability indicating assay.
1. Column: 2.1 x 30 mm 1.7μm ACQUITY BEH C18
2. Temp: 30 °C. A 45 s, 5–85%B linear gradient,
3. flow rate: 0.8 mL/min was used.
4. Mobile phase: A was 10 mm ammonium formate, pH 4.0,
B was acetonitrile. UV detection at 273 nm and 40 pts/s
5. Peaks in order: 5-nitroso-2,4,6-triaminopyrimidine, 4-amino-6-chloro-1,3-benzenesulfanamide, hydrochlorthiazide, triamterine, and methylbenzenesulfanamide; 5 µL injection, 0.1 mg/mL each.
So as the particle size decreases to increase N and subsequently Rs, an increase in sensitivity is obtained.Also, peak height is inversely proportional to the peak width:
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. eg. peptide maping.
As earlier said efficiency is proportional to column length and inversely proportional to the particle size :
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. The application of UPLC resulted in the detection of additional drug metabolites, superior separation and improved spectral quality [13,14,8].
Chemistry of small particles:
The design and developmentof sub-2 µm particles is a significant challenge, and researchers have been active in this area for some time to capitalize on t their advantages.[9,14]
A commercially available non-porous, high iffiest small particle has poor loading capacity and retention due to low surface area. To maintain retention and capacity must use novel porous particles that can withstand high pressures. In 2000,hybride of silica & polymeric column was introduced which consist of classical sol-gel synthesis that incorporates carbon in the form of methyl groups, these columns are mechanically strong. They are highly efficient & can be operate at wide range of pH.
But, in order to provide the kind of enhanced mechanical stability required for UPLC, a second generation bridged ethane hybrid (BEH) technology was developed. These 1.7 µm particles derive their enhanced mechanical stability by bridging the methyl groups in the silica matrix..
Capitalizing on smaller particles:
Only small particles are not responsible for fast resolution & sensitivity, some special instrumentation system should be design. So some special kind of system capable of delivering the pressures required to realize the potential of UPLC have been reported in the literature and elsewhere.
In early 2004, the first commercially available UPLC system that embodied these requirements was described for the separation of various pharmaceutical related small organic molecules, proteins, and peptides; it is called the ACQUITY UPLCTM System.[20,8] 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. mixed under high pressure.[8,20]
The UPLC System has been holistically designed to match the performance needs of innovative column chemistries with robust hardware, easy-to-use software and specialized support services. It consists of:
· Small, pressure-tolerant particles
· High-pressure fluidic modules
· Minimized system volume
· Negligible carryover
· Reduced cycle times
· Last response detectors
· Integrated system software and diagnostics
The ACQUITY UPLC System’s high-pressure fluidics optimizes flow rates to make the most of small particle technology. The ACQUITY UPLC System’s sample-handling design is designed to ensure exceptionally low carryover and reduced cycle time. And when interfaced with the Sample Organizer, it increases unattended sample capacity by ten times. High-speed detectors, both optical and mass, contribute to increased sensitivity and help manage the heightened speed and resolution requirements of UPLC.
ACQUITY UPLC Systems are easily controlled, diagnosed, and monitored via a graphical system console interface. The console offers:
· Quick and easy access to critical instrument parameters
· Simple system start-up, elegant system status monitoring and predictive performance indicators to ensure maximum productivity
· Data management capabilities that are supported by both MassLynx™ and Empower™ software
The ACQUITY UPLC System is also supported by Intelligent Device Management technology with our Connections INSIGHT™ service, providing instrument diagnostics.
1. Sample injection
2. Uplc columns
3. Column maneger & heater or cooler
7. Connection insight service (if provided by mfg.company.water provided it)
Lee et al. described the design of injection valves and separation reproducibility  and the use of a carbon dioxide enhanced slurry packing method onthe capillary scale for the separation of some benzodiazepines, herbicides, and various pharmaceutical compounds .Jorgenson et al. modified a commerciallyavailable HPLC system to operate at 17,500 psi and used 22 cm longcapillaries packed with 1.5 mm C18-modified particles for the analysis of proteins .
The calculated pressure drop at the optimum flow rate for maximum efficiency across a 15-cm long column packed with 1.7µm particles is approximately 15,000 psi. Therefore, a pump capable of delivering solvent smoothly and reproducibly at these pressures and that can compensate for solvent compressibility, while operating in both the gradient and isocratic separation modes is required .
With 1.7µm particles, half-height peak 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 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 increase sensitivity . There are also direct injection approaches for biological samples [18,19].
2. UPLC Columns:
The design and development of sub-2 µm particles is a significant challenge, and researchers have been active in this area for some time, trying to capitalize on their advantages 2–4. Although high efficiency, nonporous 1.5µm 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. But in order to provide the necessary mechanical stability for UPLC, a second generation bridged ethyl hybrid (BEH) technology was developed called ACQUITY BEH, these 1.7µm particles derive their enhanced mechanical stability by bridging the methyl groups in the silica matrix. Packing 1.7µm particles into reproducible and rugged columns was also a challenge that needed to be overcome. Requirements include a smoother interior surface of the column hardware and re-designing the end frits 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 BEH columns also include eCord™ microchip technology that captures the manufacturing information for each column including the quality control tests and certificates of analysis [7,8,20].
Fig. 1: ACQUITY UPLC BEH Column chemistries
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