able 1: Comparison between UPLC and HPLC

Characteristics

HPLC

UPLC Less than 2m

Particle size Maximum backpressure Analytical column

3 to 5m

35-40 MPa

103.5 MPa

Alltima C18

Acquity UPLC BEH C18

Column dimensions 150 X 3.2 mm Column temperature Injection volume

150 X 2.1 mm

30 °C 5μL(Std.In100% MeOH)

65 °C 2μL(Std.In100% MeOH)

Advantages: • Decreases run time and increases sensitivity • Provides the selectivity, sensitivity, and dynamic range of LC analysis • Maintaining resolution performance. • Expands scope of Multiresidue Methods • UPLC’s fast resolving power quickly quantifies related and unrelated compounds • Faster analysis through the use of a novel separation material of very fine particle size

25]. So far performance similar or even higher has been demonstrated by using stationary phases of size around 2 μm without the adverse effects of high pressure. In addition.• Operation cost is reduced • Less solvent consumption • Reduces process cycle times. failed batches.23] • Delivers real-time analysis in step with manufacturing processes • Assures end-product quality. speed & sensitivity for liquid chromatography therefore due to UPLC new chemistry & instrumentation technology can provide more information per unit of work. so that more product can be produced with existing resources • Increases sample throughput and enables manufacturers to produce more material that consistently meet or exceeds the product specifications.UPLC has main advantage over others is reduction of analysis time which also helps to reduce solvent consumption. References: . UPLC gives increased resolution. sensitivity and resolution compared with conventional HPLC. or the need to re-work material [22. potentially eliminating variability. including final release testing Disadvantages: Due to increased pressure requires more maintenance and reduces the life of the columns of this type. the phases of less than 2 μm are generally nonregenerable and thus have limited use [24.A negative aspect of UPLC could be the higher backpressure than in conventional HPLC.Butit seems that UPLC can offer significant improvements in speed. This backpressure can be reduced by increasing the column temperature.

High performance liquid chromatography (HPLC) is approved technique as it has been used in laboratories worldwide over the past 30-plus years. Elevated temperature. UPLC brings dramatic improvements in sensitivity. has also been investigated .UPLC Introduction to new chromatography technique . and increasing mass transfer by increasing the diffusivity of the analytes. Reference Id: PHARMATUTOR-ART-1206 Introduction: Chromatography is a non-destructive procedure for resolving a multicomponent mixture of traces. resolution and speed of analysis can be calculated. having the dual advantages of lowering viscosity.V. sacrificing resolution. the availability of affordable and easy to use mass spectrometers. and it can summarizes some of the most recent work in the field. reduces solvent consumtion & saves time. Smaller columns and faster flow rates (amongst other parameters) have been used. UPLC refers to Ultra Performance Liquid Chromatography.5] The need for speed .Principle TVES College of pharmacy. HPLC technology simply doesn’t have the capability to take full advantages of sub- .R. Faizpur accredited by North Maharashtra University.It is a method of separating a mixture of components in to individual components through a porous medium under the influence of solvent [3] For many years.Patil .[4. minor or constituents in to individual fractions. Jalgaon Abstract: UPLC can be regarded as new invention for liquid chromatography.5µm) & mobile phases at high linear velocities decreases the length of column. This review introduces the theory of UPLC.You are hereIntroduction to new chromatography technique .Chopade. limitations are soon reached and compromises must be made.UPLC About Authors: Snehal V. researchers have looked at “fast LC” as a way to speed up analyses. Dr. using conventional particle sizes and pressures. It has instrumentation that operates at high pressure than that used in HPLC & in this system uses fine particles(less than 2. [6] However.

and it can summarizes some of the most recent work in the field. B and C are constants v is the linear velocity. this gives superior resolution and sensitivity [7. * The B term represents axial diffusion or the natural diffusion tendency of molecules. This review introduces the theory of UPLC.5 μm (while HPLC columns are typically filled with particles of 3 to 5 μm).This effect is diminished at high flow rates and so this term is divided by v. which is an empirical formula that describes the relationship between linear velocity (flow rate) and plate height (HETP or column efficiency) [8. H=A+B/v+Cv Where. or UPLC [8].8] . reduces solvent consumption & saves time. Principle: The UPLC is based on the principal of use of stationary phase consisting of particles less than 2. UPLC refers to Ultra Performance Liquid Chromatography.[7]. Therefore by using smaller particles. *The A term is independent of velocity and represents "eddy" mixing. UPLC brings dramatic improvements in sensitivity.Now a days in industrial area UPLC refers for some of the most recent work field.5 µm.2µm particles. A. resolution and speed of analysis can be calculated. as the particle size decreases to less than2. there is a significant gain in efficiency. It has instrumentation that operates at high pressure than that used in HPLC & in this system uses fine particles(less than 2. The underlying principles of this evolution are governed by the Van Deemter equation. speed and peak capacity (number of peaks resolved per unit time in gradient separations) can be extended to new limits.5µm) & mobile phases at high linear velocities decreases the length of column. while the efficiency does not diminish at increased flow rates or linear velocities[8].11]. the carrier gas flow rate. termed Ultra PerformanceLiquid Chromatography. 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. It is smallest when the packed column particles are small and uniform. UPLC can be regarded as new invention for liquid chromatography. According to the van Deemter equation.

the more a molecule on the packing tends to lag behind molecules in the mobile phase.7 µm (UPLC-scale).The kinetic resistance is the time lag involved in moving from the gas phase to the packing stationary phase and back again. compared with 2500 to 5000 PSI in HPLC). 5 µm (HPLC-scale) to 1. The advent of UPLC has demanded the development of a new instrumental system for liquid chromatography.000 PSI. providing both resolution and speed. and thus the speed of analysis without affecting the chromatographic performance. But since N is inversely proportional to particle size (dp): as the particle size is lowered by a factor of three. The greater the flow of gas. for example. smaller particles provide increased efficiency as well as the ability to work at increased linear velocity without a loss of efficiency.* The C term is due to kinetic resistance to equilibrium in the separation process. . Efficiency is the primary separation parameter behind UPLC since it relies on the same selectivity and retentivity as HPLC.7N is also inversely proportional to the square of the peak width eg: (Fig 1)UPLC stability indicating assay. N is increased by three and resolution by the square root of three or 1.[18] As shown in Figure 1. from. In the fundamental resolution (Rs) equation [8] : Resolution is proportional to the square root of N. Efficiency is proportional to column length and inversely proportional to the particle size. Thus term is proportional to v. which can take advantage of the separation performance (by reducing dead volumes) and consistent with the pressures (about 8000 to 15. Therefore it is possible to increase throughput.

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). peak height is inversely proportional to the peak width: Column: Temp: flow rate: Mobile phase: 2. an increase in sensitivity is obtained.14. As earlier said efficiency is proportional to column length and inversely proportional to the particle size [8]: Therefore. desirable for many applications. the column can be shortened by the same factor as the particle size without loss of resolution. B was acetonitrile.7μm ACQUITY BEH C18 30 °C. 4-amino-6chloro-1. 5–85%B linear gradient. So as the particle size decreases to increase N and subsequently Rs.Also.4. UV detection at 273 nm and 40 an increase in sensitivity is obtained. A was 10 mm ammonium formate.6-triaminopyrimidine. triamterine.1 x 30 mm 1. The application of UPLC resulted in the detection of additional drug metabolites. pts/s 5. pH 4. superior separation and improved spectral quality [13. the separation is completed in 1/9 the time while maintaining resolution. 3. hydrochlorthiazide. .UPLC conditions: 1. Peaks in order: 5-nitroso-2. 5 µL injection.1 mg/mL each.8].0. A 45 s.8 mL/min was used. Narrower peaks also mean more peak capacity per unit time in gradient separations. 0. and methylbenzenesulfanamide. since narrower peaks are taller peaks. 2. peptide maping.3-benzenesulfanamide. 4. 0. eg.

a second generation bridged ethane hybrid (BEH) technology was developed. high iffiest small particle has poor loading capacity and retention due to low surface area. sample manager(including the column heater).[20] In early 2004. In 2000. and researchers have been active in this area for some time to capitalize on t their advantages.[20. the first commercially available UPLC system that embodied these requirements was described for the separation of various pharmaceutical related small organic molecules. mixed under high pressure. Capitalizing on smaller particles: Only small particles are not responsible for fast resolution & sensitivity. detector.Chemistry of small particles: The design and developmentof sub-2 µm particles is a significant challenge. They are highly efficient & can be operate at wide range of pH.8] The ACQUITY UPLC System consists of a binary solvent manager.[8. The binary solvent manager uses two individual serial flow pumps to deliver a parallel binary gradient. pressure-tolerant particles High-pressure fluidic modules Minimized system volume Negligible carryover Reduced cycle times . in order to provide the kind of enhanced mechanical stability required for UPLC. some special instrumentation system should be design. easy-to-use software and specialized support services.[9.20] Instrumentation: The UPLC System has been holistically designed to match the performance needs of innovative column chemistries with robust hardware. [20].[10] These 1.hybride of silica & polymeric column was introduced which consist of classical sol-gel synthesis that incorporates carbon in the form of methyl groups. To maintain retention and capacity must use novel porous particles that can withstand high pressures.7 µm particles derive their enhanced mechanical stability by bridging the methyl groups in the silica matrix. proteins. and optional sample organizer. 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. these columns are mechanically strong. [20] But.14] A commercially available non-porous. it is called the ACQUITY UPLCTM System. and peptides. It consists of: · · · · · Small.

Uplc columns 3. 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. it increases unattended sample capacity by ten times. and monitored via a graphical system console interface. Detectors 5. Softwares 6. Connection insight service (if provided by mfg. . Sample injection 2. diagnosed. Accesories 7. providing instrument diagnostics. And when interfaced with the Sample Organizer. ACQUITY UPLC Systems are easily controlled.water provided it) 1.· · 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. both optical and mass. High-speed detectors. The console offers: · Quick and easy access to critical instrument parameters · Simple system start-up. contribute to increased sensitivity and help manage the heightened speed and resolution requirements of UPLC. Column maneger & heater or cooler 4. described the design of injection valves and separation reproducibility [15] and the use of a carbon dioxide enhanced slurry packing method onthe capillary scale for the separation of some benzodiazepines.Sample inject: Lee et al.company. 1.

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. 2. especially . Packing 1. which include a limited pH range and tailing of basic analytes. With 1.5 mm C18-modified particles for the analysis of proteins [16]. Conventional injection valves. Packed bed uniformity is also critical.a first generation hybrid chemistry that took advantage of the best of both the silica and Polymeric column worlds. while operating in both the gradient and isocratic separation modes is required [7]. XTerra columns are mechanically strong. To protect the column from extreme pressure fluctuations.5µm particles are commercially available. and researchers have been active in this area for some time. modified a commerciallyavailable HPLC system to operate at 17. Waters introduced XTerra®. a second generation bridged ethyl hybrid (BEH) technology was developed called ACQUITY BEH. In 2000. which in turn requires a high sample capacity. either automated or manual.7µm particles is approximately 15. There are also direct injection approaches for biological samples [18.herbicides. UPLC Columns: The design and development of sub-2 µm particles is a significant challenge. half-height peak In UPLC. and various pharmaceutical compounds [17]. trying to capitalize on their advantages 2–4.000 psi. nonporous 1. Although high efficiency. Polymeric columns can overcome pH limitations. 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. limited loading capacities. They are produced using a classical sol-gel synthesis that incorporates carbon in the form of methyl groups. and poor mechanical strength. Low volume injections with minimal carryover are also required to increase sensitivity [8]. with high efficiency and operate over an extended pH range.19]. are not designed and hardened to work at extreme pressure. But in order to provide the necessary mechanical stability for UPLC. sample introduction is critical. The calculated pressure drop at the optimum flow rate for maximum efficiency across a 15-cm long column packed with 1. but they have their own issues including low efficiencies.7µm particles.7µm particles derive their enhanced mechanical stability by bridging the methyl groups in the silica matrix.Jorgenson et al. these 1. A fast injection cycle time is needed to fully capitalise on the speed afforded by UPLC. Therefore.500 psi and used 22 cm longcapillaries packed with 1. Requirements include a smoother interior surface of the column hardware and re-designing the end frits to retain the small particles and resist clogging.7µm particles into reproducible and rugged columns was also a challenge that needed to be overcome. a pump capable of delivering solvent smoothly and reproducibly at these pressures and that can compensate for solvent compressibility.

● .if shorter columns are to maintain resolution while accomplishing the goal of faster separations. Fig. 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. ● LC-MS can be used to detect compounds from polyaromatic (non-polar) to peptide and proteins. 1: ACQUITY UPLC BEH Column chemistries  College Scholars hips GC-MS requires compounds to be volatile to be ionised – traditionally electron impact source is used.20].8.

GC-MS is still able to detect long chain aliphatic compounds (petroleum based anlaytes) and very low mass volatile material better than LC-MS .