تحليل HPLC & Polarimetry PDF

Okay, here is the conversion of the supplied material into a structured markdown format. # مصور الطالب # تحليل ألي **DR.ANISA ELHAMİLİ** Separation Techniques (HPLC) Optical activity (polarimetry) **LAB5&6** النسخة الجديدة 12/8/2024 Pharmaceutical Instrumental Analysis Lab 5 Separation Techniqu...

Okay, here is the conversion of the supplied material into a structured markdown format. # مصور الطالب # تحليل ألي **DR.ANISA ELHAMİLİ** Separation Techniques (HPLC) Optical activity (polarimetry) **LAB5&6** النسخة الجديدة 12/8/2024 Pharmaceutical Instrumental Analysis Lab 5 Separation Technique (HPLC) Dr. Anisa Elhamili Department of Medicinal & Pharmaceutical Chemistry ## Separation Techniques **Chromatography** Its Greek roots chroma (color) and graphein (to write), it was first developed by Russian botanist, he produced a colorful separation of plant pigments through a column of calcium carbonate. It has since developed into as Lab tool for the separation of mixtures of substances into their components and identification of compounds. ## Separation Techniques All forms of chromatography work on the same principle. They all have a **stationary phase** (a solid, or a liquid supported on a solid) and a **mobile phase** (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates. ## High Performance Liquid Chromatography (HPLC) HPLC is a physical separation technique conducted in liquid phase. A sample is separated into its components by distributing between the st. p and m. p (a flowing liquid pumped at high pressure). The flowing liquid can be an organic solvent and the stationary phase can be porous silica particles packed in a column. **The essential components of a complete HPLC system are:** Solvent delivery system (pump), fixed volume injector loop or autosampler, packed column, solvent reservoirs, detector, data system and recorder. The image is a diagram of an HPLC setup including pump, injector, pressure gauge detector etc.. ## High Performance Liquid Chromatography (HPLC) The column is the heart of HPLC system, the HPLC columns are particle packed columns, the key of column selection is knowing the chemistry of the sample. The solvent delivery system or as it is commonly called the pump to deliver the m.p. Samples are usually introduced by syringe injection or by the use of an autosampler. ## High Performance Liquid Chromatography (HPLC) The important aspects in sample introduction are reproducible and precise injections. This is especially important with quantitative analysis where the reproducibility of the peak response is dependant on the precision of the sample introduction. HPLC detectors include for instance UV-Visible, fluorescence, electrochemical, mass spectrometry. Chromatographic efficiency expressed as the number of theoretical plates (N). $N = 16 \frac{(tR)^2}{Wb}$ where: * N = number of theoretical plates, * tR = retention time, * w = peak width at base. Resolution calculated using the equation: $Rs = \frac{t2-t1}{\frac{Wb2-Wb1}{2}}$ Asymmetry factor (AF) = A/B at 10% of peak height (A and B are the two half widths at each side of the peak centre). Image description: The image is a chromatography plot showing variables tR Area, W1/2 Wb, h1/2 and h Image description: The image is a graph showing the variables AU and Minutes. And the formula is $ N = 16 (\frac{tR}{Wb})^2 = 16 (\frac{1.67}{0.15})^2 = 1983$ Image description: The image is a graph showing the variables AU, and minutes. It also has the following formulas in it: $R_s = \frac{tR2-tR1}{(W_{b2}+W_{b1})/2}=1.8$ Atr= 0.27 min, Wb1 = 0.14 min , Wb2 = 0.16 min Image description: The image displays several graphs, labeled with Rs values of 0.6, 0.8, 1.0, 1.25, 1.5 and 2.0, showing separation of peaks. Image description: The image has a graph showing the retention factor using the formula bellow Retention factor: $k = \frac{t_R-t_M}{t_M}$ and $k = (1.67 - 0.34)/0.34$ and $k = 3.9$ ## High Performance Liquid Chromatography (HPLC) **Types of Chromatography** **Normal phase chromatography:** characterized by the use of stationary phase with polar functional groups and a non-polar mobile phase. **Reversed phase liquid chromatography (RPLC):** characterized by the use of non-polar stationary phase and polar mobile phase. Retention in RPLC is occur by hydrophobic interaction of the solute with the st. p. Almost all organic compounds have hydrophobic regions in their structure and are capable of interacting with the st. phase and this explain the wide range application. ## High Performance Liquid Chromatography (HPLC) A wide variety of RP-HPLC columns are available, most columns are silica based which offers good mechanical stability. A typical st. p is formed by chemically bonding a long-chain hydrocarbon group to porous silica. Typical ligands are n-octadecyl (C18), n-octayl (C8), n-butyl (C4). Separation in RPLC is affected by stationary phase type, column length, type and % of organic solvent in the m. p and mobile phase pH. Flow rate and temperature could also affect separation in RPLC. **Types of elution modes in HPLC** **Isocratic elution:** in which a single solvent is used (one solvent or a mixture in one reservoir) as a mobile phase. **Gradient elution:** in which two or more solvents are used separately as a mobile phase pumped into the LC system using and the ratio of solvents is varied in programmed way. **Advantages and disadvantage:** effective and less expensive separtion technique, however, the use of gradient elution takes longer run time for column equilibrium and cannot be used with some LC detectors e.g. refractive detector and baseline problems are also common. Q1: The obtained retention times for pyridine and t-butylbenzene peaks by HPLC were 1.3 min and 3.5 min respectively and the peak widths were 0.4 min and 0.6 min respectively, calculate the following.. * Resolution between the two peaks. * Retention factor for pyridine (tm 0.45) * Effeciency of butylbenzene Lab 6 Polarimetry ## Optical activity (Polarimetry) When plane-polarized light passes through a medium it is retarded to an extent which is indicated by the refractive index of the medium. When the medium is optically inactive both circularly polarized components are retarded to some extent and the beam emerge from the medium polarized in the same plane as the incident beam. If the medium is optically active the components are retarded to different extent because the refractive indices of the medium for left circularly polarized light (nī) and right circularly polarized light (np) differ. ## Optical activity The rotation of plane polarized light is inclined at an angle a degree to the plane of polarization of the incident beam, given by: $\alpha = \frac{1800}{\lambda} \Delta_n $ l: is the light path, 1: wavelength, a: optical rotation of the medium is positive when the plane of polarization is rotated clockwise relative to that of the incident beam when viewed looking towards the light source (dextrorotation) and negative when rotation is anticlockwise (levorotation). * Measurement of optical activity are frequently made with sodium D light and usefully at 20°C. * Temperature should be controlled for precise work. * Visual Polarimeter * Spectropolarimeter * Optical rotation: can be used to study the impurity of optically active materials. The image shows a Visual Polarimeter.