Institute of Chemical Sciences Ultra Performance Liquid Chromatography (UPLC) Presented by Mazhar Abbas Roll No. MPH-12-20 Semester 1st Institute of Chemical Sciences Bahauddin Zakariya University, Multan, Pakistan.

Types of Liquid Chromatography (TLC) Paper Chrom. Gravity Chrom. Tsvett, 1903 Flash Chrom. 1978 HPLC 1952 UPLC 2004

About UPLC By 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 We see that H decreases drastically as dp becomes smaller, and there is an added benefit because the right side of the curve becomes more flat, thus enabling higher speeds without loss of column efficiency. Van Deemter plot illustrating the evolution of particle sizes

I:It is an overlay of both conventional (3 µm) HPLCand 1 I:It is an overlay of both conventional (3 µm) HPLCand 1.7 µm 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.

Principle of UPLC Reducing the particle diameter of a packing material, column length. The back pressure requred 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: Resulation (Rs) is directly propotional to the square root of N. But N is inversely propotional to the particle size (dp) As the particle size is lowered by a factor of three, from, for example, 5 micro m (HPLC-scale) to 1.7 micro 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 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 sepration

Method optimization on UPLC Applying the scaling factor to the mobile phase. 2. The mobile phase flow rate was increased until limited by column back-pressure. 3. Reducing the total run time by increasing organic solvent content was more economical.

Comparison between HPLC and UPLC HPLC Assay UPLC Assay Column C18, 50 x 4.6 mm, 4 µm particles BEH C18, 50 x 2.1 mm, 1.7 µm particles Flow rate 3.0 ml / min 0.6 ml / min Injection volume 20 µl 3 µl partial loop fill OR 5 µl full loop fill. Total run time 10min 1.5min Total solvent consumption (including 0.5 min of delay time in between injections) Acetinitril:10.5ml Water: 21.0 ml Acetinitril:0.53ml Water: 0.66 ml Plate count 2000 7500 USP resolution 3.2 3.4 Lower limit of quantitation (LOQ) ~ 0.2 µg/ml ~ 0.054 µg/ml

Ethylene Bridged Hybrid [BEH] The 1.7 µm Ethylene Bridged Hybrid [BEH] particle is one of the key enablers behind UPLC® technology. It is available in three different pore sizes [130Å, 200Å and 300Å]

Temperature Control in UPLC Temperature control is needed to get Reproducibility Solubility Stability

Temperature control system 1. Oven 2. Heater Block 3. Water bath Temperature control system

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

Schematic diagram of instrumentation of UPLC

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 pump b. Quaternary gradient pump

Gradient vs. Isocratic Conditions • 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. Purity of the solvent b. Detector compatibility c. Chemical inertness d. Low viscosity 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: 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: 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

Types of columns in UPLC IV. The columns Types of columns in UPLC 1. 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 µm)

a. Porous, polymeric beds: Column packing a. Porous, polymeric beds: based on styrene-divinyl benzene 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 µm) with narrow size range. Particles of diameter > 20 µm are usually dry packed, while particles of diameter < 20 µm 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. Analytes change the refractive index of the light in a proportional amount to the concentration. Heat can change the RI of the mobile phase so thermo control important RI is ideal for analyzing complex sugars and carbohydrates which have no chromophores, fluorescence or electrochemical activities

Refractive Index detector

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 (BEH C18 columns) can be used at broad range of pH ranging from 2 to 12.

10. Applications 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

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

12. Conclusion Now a day’s 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

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