Ultra performance liquid chromatography

UPLC

1. Ultra Performance Liquid Chromatography Presented by Bindiya Patel

2. Agenda Definition Introduction Principal Advantages Disadvantages Instrumentation Application

3. Objective To focus on ultra performance liquid chromatography technique. To study its instrumentation. To know its various application .

4. What is chromatography ??? Chromatographic process can be defined as separation technique involving mass transfer between stationary and mobile phase. The separated species appeared as a colored bands on the column, which account for the name for the method, In Greek chromo meaning “color”, Graphic meaning “writing”

5. Introduction UPLC refers to Ultra Performance Liquid Chromatography. UPLC is one mode of chromatography, used in analytical techniques. It improves in three areas: chromatographic resolution, speed and sensitivity. UPLC is a rising chromatographic separation technique whose packing materials have smaller particle size lesser than 2.5μm. The technology takes full advantage of chromatographic principles to run separations using columns packed with smaller particles and higher flow rates.

6. Principle The principle of UPLC is based on Van Deemter equation which describes the relationship between flow rate and HETP or column efficiency H=A+B/v + Cv Where, A = Eddy diffusion B = Longitudinal diffusion C = Equilibrium mass transfer v = flow rate van Deemter equation, that describes the relationship between linear velocity (flow rate) and plate height (HETP or column efficiency)

7. Advantages… Decreases run time and increases sensitivity. Reducing analysis time so that more product can be produced with existing resources. Provides the selectivity, sensitivity, and dynamic range of LC analysis Maintains resolution performance. Fast resolving power quickly quantifies related and unrelated compounds. Operation cost is reduced. Less solvent consumption.

8. Disadvantages Due to increased pressure requires more maintenance and reduces the life of the columns of this type. In addition, the phases of less than 2 μm are generally non- regenerable and thus have limited use.

9. Comparison between HPLC and UPLC CHARACTERISTICS HPLC UPLC Particle size 3-5μm Less than 2μm Maximum back pressure 35-40 Mpa less 103.5 Mpa more Column Alltima C18 Acquity UPLC BEH C18 Column dimension 150 X 3.2 mm 150 X 2.1 mm Column temperature 30 °C 65 °C Volume injection 5μL (Std. In100% MeOH) 2μL (Std.In100% MeOH) Sample throughput less more Sample preparation simple tedious Column coagulation Does not takes place Takes place Analysis time more less Sensitivity less higher

10. UPLC Instrumentation

11. Instrumentation Pumping systems Achieving small particle, high peak capacity separations requires a greater pressure range. Both the gradient and isocratic separation modes are used. 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.

12. Cont… 2. 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 . Low volume injections with minimal carryover required to increase sensitivity.

13. UPLC columns 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 TM BEH C 18 & C 8 (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) ACQUITY UPLC BEH Amide columns (trifunctionally bonded amide phase).

14. Cont… ACQUITY UPLC TM BEH C 18 & C 8 . 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

15. Cont… Acquity UPLC BEH Shield RP 18 These are designed to provide selectivity's that complement the ACQUITY UPLC BEH T M C18 and C8 Columns. Acquity UPLC BEH phenyl columns These utilize a trifunctional C6 alkyl ethyl between the phenyl ring. Acquity UPLC BEH Amide columns BEH particle technology, in combination with a trifunctionally 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].

16. Acquity UPLC BEH Column chemistries

17. Detectors Detectors used are UV detectors Fluorescent detector Refractive index detector Light scattering detector Electrochemical detector Mass spectrometric detector

18. Phototube Consist of high sensitive cathode in a form of half cylinder in evacuated tube. Anode is also present along the axis of the tube. Inside layer is coated with light sensitive layer. When light is incident surface coating emits electron this is attracted and collected by anode. Current which is created between cathode and anode is regarded as measure of radiation falling on the detector.

19. Photomultiplier Ejected photoelectron strikes dynode, secondary e- released Voltage accelerates e- to next dynode and so Result is large charge packet hitting anode High Gain & detected

20. Fluorescence detector The light from an excitation source passes through a filter or monochromator, and strikes the sample. A proportion of the incident light is absorbed by the sample, and some of the molecules in the sample fluoresce. The fluorescent light is emitted in all directions. Some of this fluorescent light passes through a second filter or monochromator and reaches a detector, which is usually placed at 90° to the incident light beam to minimize the risk of transmitted or reflected incident light reaching the detector.

21. Evaporative light scattering detector

22.   This detector based on the deflection principle of refractometry, where the deflection of a light beam is changed when the composition in the sample flow-cell changes in relation to the reference side (as eluting sample moves through the system). As sample elutes through one side, the changing angle of refraction moves the beam. This results in a change in the photon current falling on the detector which unbalances it. The extent of unbalance (which can be related to the sample concentration) is recorded on a strip chart recorder. Refractive index detector

23. Common UPLC chromatographic conditions Columns: ACQUITY UPLC BEM C18, BEH Shield RP18, BEH C8 OR BEH Phenyl Column. Dimensions: 2.1 X 50mm 1.7 μ m. Mobile Phase A1: 20mM NH 4 COOH in H 2 O, pH 3.0. Mobile Phase A2: 20mM NH 4 HCOOH in H 2 O, pH 10.0. Mobile Phase B1: Acetonitrile . Mobile Phase B2: Methanol. Flow rate: 0.5ml/min. Injection Volume: 10.0 μ l. Week needle wash: 3% methanol. Strong needle wash: 90% acetonitrile. Temperature: 30°C. Detection: UV 254 nm. Sampling rate: 20pts/sec.

24. Application Analysis of natural products and traditional herbal medicine. Identification of metabolite. Study of metabonomics / metabolomics . Bio analysis/bioequivalence studies. Manufacturing/QA/QC Impurity profiling. Forced Degradation Studies. Dissolution Testing. Toxicity Studies.