Material characterization techniques HPLC

Imagine a race where the runners are molecules from a mysterious mixture. HPLC sets the stage for this separation marathon. The sample, dissolved in a solvent, acts as the starting line. A pressurized stream of solvent pushes the molecules through a packed column, like an obstacle course. Each molecule interacts uniquely with the obstacles and the solvent, causing them to separate along the way. Finally, a detector acts as the finish line, identifying and measuring each molecule as it emerges. This powerful technique allows scientists to unmask unknown competitors (compounds), determine the number of each type of runner (quantify components), and even check if anyone cheated (assess purity).

1. MATERIAL CHARACTERIZATION TECHNIQUES High Performance Liquid Chromatography Presenters Amina Khan – 2020-CH-07 Muhammad Hamza – 2020-CH-11 Salman Mohsin – 2020-CH-09

2. CONTENTS What is MCT ? 1 What are materials What is characterization Classification of techniques 2 3 4 5 6 Chromatography What is Chromatography What is HPLC Working Principle & Construction Working Principle Components of HPLC Classification Types of HPLC Comparison of Different Types Applications Applications of HPLC In Various Fields Results Interpretation Form in which result is obtained The interpretation of the results

3. Materials Derived from term MATTER Matter is anything that has mass and occupy some space Materials are the building blocks of our world. Everything around us, from the clothes we wear to the buildings we live in, is made from materials. In simple words, materials is the MATTER IN USE .

4. Natural Materials Occurring in nature without human intervention (e.g., wood, stone, metals like gold or copper) Classification On the basis of Nature Synthetic Materials Man-made materials created through chemical processes (e.g., plastics, nylon, advanced ceramics) 1 2

5. Classification On the basis of Engineering

6. Key Points Properties: Each material has unique properties that determine its suitability for different applications. These properties can be: Physical: Density, strength, hardness, conductivity (heat and electricity), melting point, etc. Chemical: How a material reacts with other substances Mechanical: How a material responds to forces (deformation, fracture, etc.)

7. Key Points Selection: Choosing the right material for a specific job is crucial. Engineers and designers consider factors like: Required properties for the application (e.g., strength for a bridge, insulation for a building) Cost of the material Availability of the material Environmental impact of the material (sustainability)

8. Characterization Material characterization is the process of determining the physical, chemical, and structural properties of a material. It involves a variety of techniques to analyse the material's composition, microstructure, and performance.

9. Importance Material characterization is essential for several reasons: Material selection: Helps engineers choose the right material for a specific application based on its properties. Material development: Enables scientists to develop new materials with improved properties. Quality control: Ensures that materials meet the required specifications. Troubleshooting: Helps identify the cause of material failures. Understanding material behavior: Provides insights into how materials will perform under different conditions.

10. Classification On the basis of Information TO be Obtained Chemical Composition X-Ray Fluorescence (XRF): Identifies and quantifies elements present in a material. Energy-Dispersive X-ray Spectroscopy (EDS): Analyzes the elemental composition of a specific region within a sample (often used in conjunction with Scanning Electron Microscopy - SEM). Mass Spectrometry (MS): Determines the mass of molecules in a sample, aiding in chemical identification . Microstructure: Scanning Electron Microscopy (SEM): Creates high-resolution images of a material's surface, revealing its morphology and topography. Transmission Electron Microscopy (TEM): Provides even higher resolution images, allowing visualization of a material's internal structure at the atomic level. X-Ray Diffraction (XRD): Analyzes the crystal structure of a material by identifying the arrangement of atoms and molecules. P hysical and Mechanical Properties: Mechanical Testing: Measures a material's strength, stiffness, hardness, and other mechanical properties through techniques like tensile testing, compression testing, and hardness testing. Thermal Analysis: Evaluates a material's response to changes in temperature, including techniques like Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA). Surface and Interfacial Properties: Atomic Force Microscopy (AFM): Creates high-resolution images of a surface, revealing its topography and measuring forces at the atomic level. X-ray Photoelectron Spectroscopy (XPS): Analyzes the chemical composition and electronic state of elements at a material's surface.

11. HPLC High Performance Liquid Chromatography

12. Mobile Phase A solvent or solvent mixture that continuously flows through the system. Chromatography is a separation technique that separates components of a mixture based on their differential interaction with two phases Stationary Phase A solid material packed within a column. 1 2 Chromatography is a separation technique that separates components of a mixture based on their differential interaction with two phases: Mobile phase: A solvent or solvent mixture that continuously flows through the system. Stationary phase: A solid material packed within a column.

13. Historic Timeline First Ever Presented 1903 The official date of birth of chromatography is the 21 March 1903 in Warsaw when Mikhail Semenovitch TSWETT has presented at the Congress of the Polish Natural Sciences Society a communication entitled : « A new class of adsorption phenomena and their applications in biochemical analysis » about the separation and purification of vegetal pigments (a mixture of chlorophylls and xantophylls ) on a chalk column 1938 Elution Theory REICHSTEIN proposes a theory for the elution and separation of solutes on a column 1967 HPLC beginning of HPLC after the works of HUBER and HUZSMAN , this technique was first named « High Speed Liquid Chromatography » then « High Pressure Liquid Chromatography » and finally « High Performance Liquid Chromatography » 1969 Progress after the 5 th International ymposium International « Advances in Chromatography » the development of HPLC was very fast

14. Classification On the basis of Separation Technique Adsorption Chromatography Stationary phase is polar. Mobile phase is nonpolar. Separation based on the differing adsorption strengths of components onto the polar stationary phase. Partition Chromatography Stationary phase is nonpolar. Mobile phase can be polar or nonpolar. Separation based on the partitioning of components between the polar mobile phase and the nonpolar stationary phase. Ion- Exchnage Chromatography Stationary phase contains charged groups. Separation based on the ionic interaction between charged components in the sample and the oppositely charged groups on the stationary phase. Size-Exclusive or Gel Permeation Chromatography Stationary phase is porous with pores of different sizes. Separation based on the size and shape of the sample components. Larger components are excluded from the smaller pores and elute first, while smaller components can enter the pores and elute later.

15. High Performance Liquid Chromatography It is also referred to as high pressure liquid chromatography ,is a technique in analytical chemistry used to separate, Identify, and quantify specific components in mixtures. The mixtures can originate from food, chemicals , pharmaceuticals, biological, environmental and agriculture etc. which have been dissolved into liquid solutions. High performance means high resolution: ability to distinguish between two components

16. 01 - BRANDING 02 - WEBSITE 03 - SOCIAL MEDIA High pressure liquid chromatography(HPLC): • Type of liquid chromatography • Conducted in a column • Characterized by the use of high pressure & small particle size to push a mobile phase solution through a column of stationary phase allowing separation of complex mixtures with high resolution.

17. Basic principle Based on distribution of solute between a liquid mobile phase and a stationary phase. • The small diameter particles are used as stationary phase support.

18. 1 2 3 4 5 6 Components of HPLC: Solvent Reservoir Pumps Sample Injection System Columns Detectors Computer Waste collector

19. 1 2 3 4 5 6 HPLC PUMP • To produce an appropriate pressure to push solvent & sample into the column. • Ideal pump Deliver high pressure ( upto 50MPa) Deliver pulse free flow Constant volume delivery Deliver high volumes (flow rates) of solvent (to 10 mL/min) Solvent replacement is easy

20. 1 2 3 4 5 6 Types of HPLC Pump Constant Pressure • A steady pump pressure • (usually about 1000–2000 psi) is needed to ensure reproducibility & accuracy. Constant displacement Pump Reciprocating pump: constant flow rate through the column Slight cyclical variation in pressure→pulse dampeners.

21. 1 2 3 4 5 6 Analyte Columns in HPLC: • Considered the “heart of the chromatograph” the column’s stationary phase separates the sample components of interest using various physical and chemical parameters. • The small particles inside the column are what cause the high back pressure at normal flow rates. • The pump must push hard to move the mobile phase through the column and this resistance causes a high pressure within the chromatograph. • Standard Column : 3-25cm long, ID(4.6 mm) ;optimum flow volume = 1 ml/min. • Narrow Bore : 3 mm(ID); flow volume 0.6 ml/min • Microbore /Open tubular : 25-50cm long, 1 mm(ID); 50 microliter/min.

22. 1 2 3 4 5 6 HPLC columns Within the Column is where separation occurs. Key Point –Proper choice of column is critical for success in HPLC Materials of construction for the tubing • Stainless steel (the most popular; gives high pressure capabilities) • Glass (mostly for biomolecules) • PEEK polymer (biocompatible and chemically inert to most solvents Packing material: The packing material is prepared from SILICA particle, ALUMINAparticle and ion exchange RESIN. Porous plug of stainless steel or Teflon are used in the end of the columns to retain the packing material. According to the mode of HPLC , they are available in different size , diameters, pore size or they can have special materials attached ( such as antigen or antibody ) for immuno affinity chromatography.

23. Types of columns in HPLC: • Guard Column • Fast Column • Preparative( i.d. > 4.6 mm; lengths 50 –250 mm) • Capillary( i.d. 0.1 -1.0 mm; various lengths) • Nano( i.d. < 0.1 mm, or sometimes stated as < 100 μ m) • Analytical[internal diameter ( i.d. ) 1.0 -4.6-mm; lengths 15 –250 mm]

24. Daniel Gallego “Lorem ipsum dolor sit amet, consectetur adipiscing elit. Etiam mattis, nunc vitae eleifend posuere, turpis mauris vestibulum purus, in pellentesque tellus elit vel nisl.” Lorna Alvarado Detectors in HPLC The detector can see (detect) the individual molecules that come out (elute) from the column. •A detector serves to measure the amount of thosemolecules so that the chemist can quantitatively analyze the sample components. •The detector provides an output to a recorder or computer that results in the liquid chromatogram(i.e., the graph of the detector response).

25. Common HPLC detectors: UV-VIS • DiodeArray •Multiple Wavelength •Variable Wavelength •Mass Spectrometers •Refractive Index •Fluorescence •Light Scattering •Electrochemical •Radioactivity •Conductivity

26. Daniel Gallego “Lorem ipsum dolor sit amet, consectetur adipiscing elit. Etiam mattis, nunc vitae eleifend posuere, turpis mauris vestibulum purus, in pellentesque tellus elit vel nisl.” Lorna Alvarado COMPUTERS: • Electronic signals generated by detectors are recorded inthe form of chromatograghic peak at varied function of time • Peak Area, height, retention time, base width of chromatograghic peak is measured to compute analyte concentration of each peak.

27. How can we analyze the sample? For example: 1.Carbohydrates 2. fructose 3. Glucose 4. Saccharose 5.Palatinose 6. Trehalulose 7. isomaltose

28. “Lorem ipsum dolor sit amet, consectetur adipiscing elit. Etiam mattis, nunc vitae eleifend posuere, turpis mauris vestibulum purus, in pellentesque tellus elit vel nisl.” Lorna Alvarado

29. 03 - SOCIAL MEDIA Working of HPLC: Equilibration stage: (to ready column for purification) Only buffer pass not sample, detector detect the buffer and straight line obtain called baseline. Hydrophilic curve Hydrophobic curve

30. 01 - BRANDING 02 - WEBSITE 03 - SOCIAL MEDIA Types of HPLC Normal phase HPLC Mobile phase is non polar Stationary phase is polar 2. Reverse phase HPLC: Mobile phase is polar Stationary phase is non polar 3. Size exclusion HPLC:

31. 4. Ion Exchange HPLC:

32. 01 - BRANDING 02 - WEBSITE 03 - SOCIAL MEDIA Factors which influence HPLC performance Internal diameter of column - the smaller in diameter, the higher in sensitivity Pump pressure - the higher in pressure, the higher in separation Sample size The polarity of sample, solvent. .Temperature - the higher in temperature, the higher in separation

33. Applications Of HPLC Pharmaceutical Analysis Biological Analysis Environmental Analysis Food And Beverages

34. Equipment Specification Operating Conditions Solvent A: This is a mixture of water and acetic acid in a 25:1 ratio by volume. Water is a polar solvent, while acetic acid is a weak acid with some polarity. Solvent B: Pure methanol, which is a more organic and less polar solvent compared to water and acetic acid. Binary Gradient: This indicates that the HPLC system uses two pumps to deliver a mixture of Solvent A and B. The proportion of each solvent changes gradually throughout the analysis (gradient elution). Flow Rate: 1.000 mL/min. This is the rate at which the mobile phase is pumped through the column. Pressure Limits: Maximum Pressure (P.Max): 400.0 kgf/cm² (kilograms-force per square centimeter). Minimum Pressure (P.Min): 0.0 kgf/cm². (Atmospheric Pressure) Column Oven: Temperature: 40 °C. Maintaining the column at this temperature can influence analyte retention times and separation efficiency. Detector: A UV/Vis detector model with designation A for identification within the system. Lamp: D2. This refers to the type of lamp used in the detector. Deuterium lamps emit light in the ultraviolet (UV) region, commonly used for detecting aromatic compounds that absorb UV light. HPLC-DAD: This refers to the analytical technique used. HPLC separates compounds based on their interaction with a stationary phase and a mobile phase. DAD allows detection of compounds at various wavelengths, providing a more comprehensive analysis. Flavonoid standards: The sample analysed likely contains known flavonoid compounds used for comparison with unknown samples. Gradient HPLC: This indicates that the composition of the mobile phase changed gradually during the analysis, potentially improving the separation of various flavonoids. C18 column: This refers to the type of stationary phase used in the HPLC system. C18 columns are commonly used for separating non-polar and moderately polar compounds, which many flavonoids are. Monitored at 280 nm: This specifies the wavelength of light used for detection. Many flavonoids absorb UV light around 280 nm, allowing for their identification.

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