INTRODUCTION TO HPLC

1. https://syukur16tom.wordpress.com/ Password: http://smtom.lecture.ub.ac.id/ Password: INTRODUCTION TOHPLC SEPARATION into components for IDENTIFICATION, QUANTIFICATION andPURIFICATION of mixtures with a high resolution

2. PRESENTATION OUTLINE 1. INTRODUCTION 2. TYPES OF HPLC 3. REVERSED-PHASE HPLC 4. ADVANTAGES OF HPLC 5. APPLICATION OF HPLC

3. 1. INTRODUCTION 1. Definition • High-performance liquid chromatography (HPLC) is a form of liquid chromatography (LC) used to separate compounds that are dissolved in solution. • HPLC is an analytical technique to SEPARATE, IDENTIFY, and QUANTIFYcomponents in a mixture. • It is the single biggest chromatography technique essential to most laboratories worldwide. • The different components in the mixture pass through the column at different rates due to their partition behavior between the mobile phase and the stationary phase.

4. 2. Discovery • M. Tswett, the Russian-Polish botanist, is generally recognized as the first person to establish the principles of chromatography. • Tswett described, in a paper presented in 1906, how he filled a glass tube with chalk powder (CaCO3) and, by allowing an ether solution of chlorophyll to flow through the chalk, the chlorophyll was separated into layers of different colors. • He called this technique “CHROMATOGRAPHY” (adsorption chromatography). • Chromatography, chroma (color) + graphein (to write) represents the premier analytical method of the 20th century for the advancement of a variety of the disciplines of science.

5. Invention of Chromatography by M. Tswett Ether Chromatography Colors Chlorophyll CaCO3 Chalk powder

6. HPLC and Pre-HPLC Techniques

7. 2. Work Principle • Chromatography can be often compared to the flow of a river. • A river consists of a stationary riverbed and water that continuously moves in one direction. • What happens if a leaf and a stone are thrown into the river? The relatively light leaf does not sink to the bottom, and is carried downstream by the current. On the other hand, the relatively heavy stone sinks to the bottom, and although it is gradually pulled downstream by the current, it moves much more slowly than the leaf. • If you stand watch at the mouth of the river, you will eventually be able to observe the arrival of the leaf and the stone. However, although the leaf will arrive in an extremely short time, the stone will take much longer to arrive.

8. This analogy represents the components of chromatography in the following way: • River: Separation field • Leaf and stone: Target components of sample • Standing watch at the river mouth: Detector

9. 2. TYPES OF HPLC The two most common variants of HPLC are normal-phase and reversed-phase HPLC 1. Normal-Phase HPLC • The column is filled with tiny silica particles (polar), and a non-polar solvent, for example, hexane. • A typical column has an internal diameter of 4.6 mm or smaller and a length of 150 to 250 mm. • Non-polar compounds (e.g. CO2, C6H6) in the mixture will pass more quickly through the column, as polar compounds will stick longer to the polar silica than non-polar compounds will.

10. 2. Reversed-Phase HPLC • The column size is the same. The column is filled with silica particles which are modified to make them non-polar. • This is done by attaching long hydrocarbon chains(8–18 C atoms) to its surface. • A polar solvent is used, for example, a mixture of water and an alcohol such as methanol and acetonitrile. • Polar compounds in the mixture will pass more quickly through the column because a strong attraction occurs between the polar solvent and the polar molecules in the mixture. • Non-polar molecules are slowed down on their way through the column.

11. 3. Solute, Stationary Phase and Mobile Phase Interaction • Differences in the interactions between the solutes and stationary and mobile phases enable separation. Solute Degree of adsorption, solubility, ionicity, etc. Stationary phase Mobile phase

12. 3. REVERSED-PHASE HPLC 1. Separation Mechanism • The stationary phase and mobile phase make contact via an interface. They do not intermingle, and are kept in a steady state of equilibrium. • In the river analogy, the riverbed corresponds to the stationary phase and the flowing water corresponds to the mobile phase. • Substance contains a component that is only weakly attracted by the stationary phase will be pulled along quickly by the flow of the mobile phase. • A component strongly attracted by the stationary phase will stick to the stationary phase and only move slowly.

13. 2. Mobile Phase/Stationary Phase • A site in which a moving phase (mobile phase) and a non-moving phase (stationary phase) make contact via an interface that is set up. • The affinity with the mobile phase and stationary phase varies with the solute. Separation occurs due to differences in the speed of motion.

14. 3. HPLC Components • HPLC instruments consist of (1) a reservoir of mobile phase, (2) a degassing unit, (3) a pump, (4) an injector, (5) a separation column, and (6) a detector (6). 6 1 5 2 4 3

15. Schematic representation of an HPLC unit. (1) Solvent reservoirs, (2) Solvent degasser, (3) Gradient valve, (4) Mixing vessel for delivery of the mobile phase, (5) High-pressure pump, (6) Switching valve in "inject position", (6') Switching valve in "load position", (7) Sample injection loop, (8) Pre-column (guard column), (9) Analytical column, (10) Detector (i.e. IR, UV), (11) Data acquisition, (12) Waste or fraction collector.

16. 4. Flow Channel Diagram of HPLC • The flow channel of HPLC starts from mobile phase (eluent) to detector and ends up at draining container.

17. 5. Reversed-Phase HPLC Columns • The heart of a HPLC system is the column containing particles that functions fro separation of co mponents. Reversed-Phase HPLC Columns

18. 2.1 mm ID Column, 0.35 mL/min, 50/50 MeOH/Water 50°C 45°C 40C 35°C 30°C 25°C 20°C 6. Column Oven • Column oven is used to reduce retention times which is enable to make higher flow rates.

19. 7. HPLC Detectors • HPLC detectors may consist of: UV-VIS, Diode Array, Refractive Index (RI), Fluorescence, Light Scattering, Conductivity, and Mass Spectrometer. • UV-VIS • Wavelength range 190-700 nm • D2 (deuterium) and W (tungsten) lamps • UV-VIS is most common HPLC detector for a variety of samples such as: • Proteins and peptides • Organic molecules • Pharmaceuticals • Monitor 2 wavelengths at one time

20. 7. Separation Process and Chromatogram • Components are separated and detected by a detector. Chromatogram Output concentration 20 Time

21. Chromatogram tR tR : Retention time Peak t0 Intensity of detector signal t0: Non-retention time h A: Peak area A h: Peak height Time

22. 4. ADVANTAGES OF HPLC • High separation capacity, enabling the batch analysis of multiple components. • Superior quantitative capability and reproducibility. • Moderate analytical conditions • Unlike GC, the sample does not need to be vaporized. • Generally high sensitivity. • Low sample consumption. • Easy preparative separation and purification of samples.

23. Bioscience proteins peptides nucleotides 5. HPLC APPLICATION Chemical polystyrenes dyes phthalates Pharmaceuticals tetracyclines corticosteroids antidepressants barbiturates Consumer Products lipids antioxidants sugars Environmental polyaromatic hydrocarbons Inorganic ions herbicides Clinical amino acids vitamins homocysteine

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25. Application • UV/Vis absorbance detectors can be used to detect any compound that absorbs at the wavelength being monitored • Common wavelengths: • 254 nm for unsaturated organic compounds • 260 nm for nucleic acids • 280 or 215 nm for proteins or peptides • Absorbance detectors can be used with gradient elution • wavelength being monitored is above the cutoff range of the solvents being used in the mobile phase • limits of detection for fixed and variable UV/Vis absorbance detectors are ~ 10-8 M • limits of detection for photodiode array detectors are ~ 10-7M