Ultra Performance Liquid Chromatography

Introduction to chromatographyChromatography is regarded as an analytical technique employed for the purification and separation of organic and inorganic substances.

Mikhail Tswett (1906), in his experiment, a leaf extract sample in petroleum ether was allowed to pass through a column of CaCO3, pure ether was continued to flow through the column as a result of which various chlorophyll pigments, etc., were separated into a series of differently coloured and easily distinguished zones.

Classification in chromatography Chromatography is divided into two classes: 1. Gas chromatography 2. Solution chromatography Chromatography can also be divided into the following general areas: 1. Adsorption chromatography 2. Partition chromatography 3. Exclusion chromatography 4. Ion exchange chromatography

About UPLCBy using smaller particles, speed and peak capacity can be extended to new limits. It also provides good resolution.

According to Van Deemter, decrease in particle size less than 2.5 mm, a significant gain in efficiency is achieved.

Van Deemter plot illustrating the evolution of particle sizes

I:It is an overlay of both conventional (3 m) HPLCand 1.7 m UPLC for a five component sample mixture. II:It is an expansion of the first 0.6 minutes of the overlay to show the increased speed of UPLC, while resolution is still maintained.

Principle1. Reducing the particle diameter of a packing material, column length. 2. The back pressure required for the use of the small particle column. 3. Monolithic columns provide lower flow resistance than conventional columns. 4. Columns can be operated at high flow rates.

Theory of separations using small particles The fundamental resolution equation

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

HPLC Assay C18, 50 x 4.6 mm, Column

UPLC Assay BEH C18, 50 x 2.1 mm,

4 m particles1.7 m particles Flow rate Needle wash 3.0 ml / min Methanol 0.6 ml / min Methanol 3 l partial loop fill Injection volume 20 l OR 5 l full loop fill. Total run time Total solvent consumption (including 0.5 min of delay time in between injections) Plate count USP resolution Lower limit of quantitation (LOQ) 10min Acetinitril:10.5ml Water: 21.0 ml 2000 3.2 ~ 0.2 g/ml 1.5min Acetinitril:0.53ml Water: 0.66 ml 7500 3.4 ~ 0.054 g/ml

Delay volume

~ 720 l

~ 110 l

Separation Modes of UPLC1. Reversed-phase chromatography 2. Normal-phase and adsorption chromatography

3. Ion exchange chromatography4. Size exclusion chromatography

Temperature Control in UPLCTemperature control is needed to get Reproducibility

SolubilityStability

Temperature control system1. Oven 2. Heater Block

3. Water bath

Temperature control system

InstrumentationI. Pump II. Solvent system III. Sample injector IV. Column V. Detector

Schematic diagram of instrumentation of UPLC

UPLC (Ultra Performance Liquid Chromatography)

I. Pumps The most important advantages of pumps are: higher resolution, faster analyses, and increased sample load capacity. Pump Module types Isocratic pump - delivers constant mobile phase composition; solvent must be pre-mixed Gradient pump - delivers variable mobile phase composition;

a. Binary gradient pumpb. Quaternary gradient pump

Gradient vs. Isocratic Conditions Isocratic

Best for simple separations Often used in quality control applications that support and are in close proximity to a manufacturing process. Gradient Best for the analysis of complex samples Often used in method development for unknown mixtures. Linear gradients are most popular

II. Solvent system A. Mobile phases several common properties:

a. Purityb. Detector compatibility c. Chemical inertness B. Mobile phase reservoir The most common type of solvent reservoir is a glass bottle. Most of the manufacturers supply these bottles with special caps, Teflon tubing and filters to connect to the pump inlet and to the purge gas (helium) used to remove dissolved air.

III. Sample Injection Manual Injector:

1. User manually loads sample into the injector using a syringe. 2. And then turns the handle to inject sample into the flowing mobile phase which transports the sample into the beginning (head) of the column, which is at high pressure.

Auto sampler:1. User loads vials filled with sample solution into the auto sampler tray (100 samples) 2. And the auto sampler automatically a. Measures the appropriate sample volume, b. Injects the sample, c. Then flushes the injector to be ready for the next sample, etc., until all sample vials are processed for unattended automatic operation

IV. The columns Types of columns in UPLC1. Analytical column [internal diameter (i.d.) 1.0 - 4.6-mm; lengths 15 250 mm] 2. Preparative column (i.d. > 4.6 mm; lengths 50 250 mm) 3. Capillary column (i.d. 0.1 - 1.0 mm; various lengths) 4. Nano column (i.d. < 0.1 mm, or sometimes stated as < 100 m)

Column packing a. Porous, polymeric beds: based on styrene-divinylbenzene co-polymer.

b. Porous layer beds: silica or modified silica on an

spherical inert core (e.g. glass beds). c. Totally porous silica particles: (diameter < 10 m) with narr size range.

Particles of diameter > 20 m are usually dry packed, while particles of diameter < 20 m are slurry packed, in which particles are suspended in a suitable solvent and the slurry so obtained is driven into column under pressure.

V. Detector 1. Spectroscopic Detection 2. Refractive Index Detection 3. Fluorescence Detection

1. Ultraviolet (UV) Absorption An ultraviolet light beam is directed through a flow cell and a sensor measures the light passing through the cell. If a compound elutes from the column that absorbs this light energy, it will change the amount of light energy falling on the sensor. The resulting change in this electrical signal is amplified and directed to a recorder or data system.

2. Refractive Index (RI) Detection The ability of a compound or solvent to deflect light provides a way to detect it.

3. Fluorescence Detection Compared to UV-Vis detectors fluorescence detectors offer a higher sensitivity and selectivity that allows quantifying and identifying compounds and impurities in complex matrices at extremely low concentration levels (trace level analysis).

VI. Data system

Since the detector signal is electronic, using modern data collection techniques can aid the signal analysis. In addition, some systems can store data in a retrievable form for highly sophisticated computer analysis at a later time.

9. Advantages of UPLC High resolution

High speed It is up to 9 times faster, has up to twice the resolution and three times the sensitivity than that of HPLC. Quick and ease of instrument handling High sensitivity.Even neon particles can be separated easily. UPLC (BH3 columns) can be used at broad range of pH ranging from 2 to 12.

10. ApplicationsA. One of the most common compromises in HPLC is sacrificing resolution for speed. The UPLC increase the resolution in shorter run times and it generates more information faster without sacrificing resolution. These newer technique fulfils the market needs. B. Together with an UPLC System merged with a mass spectrometer, it enables scientists to attain more information about the identity of pharmaceutical, biological, industrial and environmental compounds than conventional UV/Vis-based detector systems

C. UPLC overcomes such problems, as illustrated in Figure 6, which shows a separation of eight diuretics in less than 1.6 minutes. The same separation on a 2.1 by 100mm 5m C18 HPLC column yields almost identical resolution, but takes ten minutes. For some analyses, however, speed is of a secondary importance, and peak capacity and resolution are the priority.

11. Other applicationsFrom injection of a poly-drug reference standard and whole blood extract, separation and identification of amphetamine, methamphetamine, ephedrine, pseudoephedrine, phentermine and ketamine in less than 3 min using the UPLC method. Determination of Coumarone in UPLC-Electro sprayTandem Mass Spectrometry. UPLC used in bioanalysis, metabolite identification and method development.

12. ConclusionNow a days various pharmaceutical companies are going to use HPLC and UPLC as separation techniques to increase the marketing needs. The new technology in chemistry and instrumentation provides more information per unit of work as UPLC begins to fulfill the promise of increased speed, resolution and sensitivity predicted for liquid chromatography