UV/Vis Spectroscopy Lab PDF

Here is the transcription of the document in markdown format: ## 8/7/2024 # قصور نضر # تحليل ألي Every hardworking person has a share....! ### د.أنيسة ## LAB 3&4 Scan QT code OR Telegram ### مع تمنياتنا لجميع الطلية بالنجاح والتوفيق ## Pharmaceutical Instrumental Analysis ### Lab 3 ### Effect of...

Here is the transcription of the document in markdown format: ## 8/7/2024 # قصور نضر # تحليل ألي Every hardworking person has a share....! ### د.أنيسة ## LAB 3&4 Scan QT code OR Telegram ### مع تمنياتنا لجميع الطلية بالنجاح والتوفيق ## Pharmaceutical Instrumental Analysis ### Lab 3 ### Effect of solvent on UV absorbance Dr. Anisa Elhamili Department of Medicinal & Pharmaceutical Chemistry ## Instrumental Analysis ### Introduction Instrumental analysis is a field of analytical chemistry that investigates samples using instruments. - Analytical Methods can be classified into: - **Qualitative** instrumental analysis is that measured property indicates presence of analyte in matrix. - **Quantitative** instrumental analysis is that magnitude of measured property is proportional to concentration of analyte in matrix. --- ## Instrumental Analysis ➤Analytical methods are generally classified into instrumental and non-instrumental category. ➤ In instrumental analysis, measurement of physical property is made to determine the contents or composition of a substance. | Physical properties measured | Instrumental method Adopted | | ------------- |:-------------:| | 1. Electrical Potential | Potentiometry | | 2. Electrical conductance | Conductometry | | 3. Electrical current | Polarography, Amperometry | | 4 Absorption of radiation | Uv, visible, IR, Atomic absorption Spect. | | 5. Emission of radiation | Emission spectroscopy, Flame photometry, | | 6. Mass to charge ratio | Mass spectrometry | | 7. Thermal properties | Thermogravimetric analysis (TGA), Differential scanning calorimetry (DSC) | --- ## Instrumental Analysis Calibration methods Basis of quantitative analysis is magnitude of measured property in proportional to concentration of analyte. The image is a graph with axes labeled "Instrument Response (Signal)" and "Dynamic Range". A curve starts at the origin and gradually increases, then levels off in the dynamic range. DL, LOQ and LOL are marked on the curve. (larger slope of calibration curve, more sensitive measurement). Calibration expression is Absorbance = slope [Analyte (ppm)] + intercept Instrumental analysis are unique in their function. They do not give direct quantitative data but supplies information which can be converted into a suitable form which correlates with the structure or the content. Thus it acts as a communication device. It consists of the following steps: 1. Generation of signal. 2. Transformation of signal into measurable form or unit. 3. Amplification of the transduced signal. 4. Read out system --- ## Spectroscopic Methods Spectroscopic methods of analysis are based on the interaction of *LIGHT* with matter. (LIGHT is an Electromagnetic (EM) radiation). The image is a diagram illustrating the electromagnetic spectrum, showing different types of radiation, including the following: |HIGH ENERGY |LOW ENERGY | | ----------- | ----------- | | yrays | microwaves | | x rays | radiowave | | uv vis | | | Ir | | Wavelength (nm) values are provided on the x-axis, ranging from $10^{-3}$ to $10^{11}$. ## Ultraviolet–Visible (UV/Vis) Spectroscopy ### Principle It refers to absorption spectroscopy in the ultraviolet-visible region, wavelength range (UV 200-400nm, Vis 400-700nm). When radiation of certain wavelength is passed through a sample solution, there will be excitation of electrons and transitions to a high energy level (the sample absorbs energy from the radiating field). Different molecules absorb light at different wavelengths. The intensity of the absorption varies as a function of wavelength and this variation is the absorption spectrum. Groups in a molecule which absorb light are known as chromophores. The amount of light absorbed is proportional to concentration (Beer's Law). --- ## Instrument UV-Vis spectrophotometer consist of: (1) Radiation Sources: Tungsten Lamp (Visible), Deuterium lamp (UV) (2) Sample Containers: Cuvettes (3) Wavelength selector: Monochromator (4) Radiation detector (5) Signal processor and readout The image shows a diagram of a spectrophotometer, including components such as a lamp, lens, monochromator, sample cuvette, detector, amplifier, and readout. For good analysis the sample must have the following properties: 1. Stability in solution. 2. Adherence to Beer's law. 3. Large molar absorptivities. 4. Sufficient separation of the desired analyte absorbance wavelength from interfering substances. If Not, the substance is usually converted into a new species suitable for quantitative spectrophotometry. SAMPLE + CHROMOGENIC REAGENT → UV-VIS ABSORBING PRODUCT --- ## Terms describing UV absorption 1. Chromophores: functional group of a molecule responsible for the absorption of light. 2. Auxochromes: functional group that doesnot itself absorb in the UV region, but when attached to chromophore alters both the wavelength and intensity of an absorption. 3. Bathochromic shift : shift to longer wavelength or lower frequency also called red shift. 4. Hysochromic shift : shift to shorter wavelength or high frequency also called blue shift. 5. Hyperchromism : increase in absorption intensity. 6. Hypochromism : decrease in absorption intensity. ## Experiment Measuring the absorption spectrum of acetone in water and in hexane Instrument - Spectrophotometer Glassware - Pipette - Volumetric flasks Chemicals - Distilled water - Hexane - Acetone --- ## Procedure 1- Prepare solution of acetone in water and acetone in hexane 0.5%v/v 2- Rinse one of the cuvettes with distilled water and fill it with water. Put the cuvette in the sample compartment (reference solution). Set the wavelength to 250nm, then set the absorbance to zero. 3- Rinse a second cuvette once with distilled water and once with acetone in water solution, then fill it with the solution. Pace the cell in the sample compartment, measure the absorbance at 250nm. 4- Repeat steps 2 and 3 for the two cuvettes at wavelength increaments of 5 up to 300nmand record the absorbance at each wavelength. 5- repeat step 2,3 and 4 for recording the absorbance of acetone in hexance (reference solution is hexane). 6- Put the resulted absorbance aganist wavelength and determine the maximum wavelength for both solutions. 7- Determine the shift happen in the UV spectrum. ## Explanation The image shows a diagram with axes labeled "Wavelength (nm)" and "Absorbance". Several labels are oriented around a cross in the center of the image: - Hypsochromic (Blue shifted) - Hyperchromic - Hypochromic -Bathochronse (Red shifted) --- ## Results |Wavelength nm | A (in water) | A (in hexane)| | ----------- | ----------- | ----------- | | 250| 0.48| 0.31| | 255| 0.62| 0.38| | 260| 0.75| 0.45| | 265| 0.89| 0.54| | 270| 0.8| 0.6| | 275| 0.64| 0.66| | 280| 0.51| 0.69| | 285| 0.41| 0.61| | 290| 0.32| 0.53| | 295| 0.25| 0.42| | 300| 0.19| 0.32| ## Lab **4** ## Atomic Spectroscopy --- ## Optical Atomic Spectroscopy 1- Atomic Absorption Spectroscopy (AAS) - Flame Atomic Absorption Spectroscopy (FAAS) - Electro thermal (Flame-less) Atomic Absorption Spectroscopy (EAAS) 2- Atomic Emission Spectroscopy (AES) 3- Atomic Fluorescence Spectroscopy (AFS) --- ## Atomic Absorption Spectroscopy The technique is like UV/Vis absorption spec. with some modifications 1. Special radiation source, called hallow cathode lamp, instead of broad wavelength source. 2. Analyte is not quantitated directly, but converted to gaseous atoms using hot flame, neubilizer is used to introduce sample into flame. 3. No speciation, all form of analytes is converted to gaseous atoms. 4. AAS is used for metallic, not for non metals. 5. The precise wavelength associated with atomic spectral lines provide excellent identification of analytes. --- ## Atomic Absorption Spectroscopy ### Principle Based on the breakdown of a sample into atoms, followed by the measurement of the atom's absorption or emission of light. i. deals with absorbance, fluorescence or emission (luminescence) of atoms or elemental ions rather then molecules. atomization: process of converting sample to gaseous atoms or elementary ions. ii. Provides information on elemental composition of sample or compound. UV/Vis, IR gives molecular functional group information, but no elemental information. iii. Basic process the same as in UV/Vis, fluorescence etc. for molecules. ## Atomic Absorption Spectroscopy * Samples are solids, liquids and gases but usually not ATOMS! * Sample Introduction (Flame, Furnace, ICP) * Sources for Atomic Absorption / Fluorescence * Hollow Cathode Lamps * Sources for Atomic Emission (Flame, Plasma) * Wavelength Separators + Slits +Detectors --- ## Atomic Absorption Spectroscopy Sample Atomization, expose sample to flame or high-temperature. (Need to break sample into atoms to observe atomic spectra). Basic steps: - Nebulization – sample solution get into fine droplets by spraying via thin nozzle. - Desolvation - heat droplets to evaporate off solvent just leaving analyte or compounds. Volatilization – convert solid analyte particles into gas phase. - Dissociation – break-up molecules in gas phase into atoms. Ionization - cause the atoms to become charged. - Excitation – with light, heat, etc. for spectra measurement. The second image shows a diagram illustrating the process of atomic absorption, starting with a sample solution and progressing through nebulization, desolvation, volatilization, and resulting in free atoms and ions. --- ## Steps in experiment 1. The anayte is prepared in aqueous solution. 2. The solution is aspirated into flame using neubilizer. 3. The solvent evaporate, gaseous atoms formed. 4. The gaseous atoms absorb radiation from the hollow lamp. 5. The absorbance of the sample is determined. Because atomic spectra are lines, the absorbing and emitting wavelength are identical in contrast to molecular spectra. ## Atomic Absorption Spectroscopy (AAS) - commonly used for elemental analysis - expose sample to flame or high-temperature There is an image of a sample going into atomic cell and the detector measuring it. Light points to sample from the left. ## Flame AAS: - simple atomization of gas/solution/solid. - flame atomization best for reproducibility. - relatively insensitive - incomplete volatilization, short time in flame --- Flame Atomic absorption spectrometer, light from a source is directed through the sample to a detector Image shows the SpectrAA600 atomic absorption spectrometer. ## Sources for AAS 1- Hollow Cathode Lamps (HCL) - The source of light is a lamp whose cathode is composed of the element being measured. Each element requires a different lamp. - Multielement lamps are available. Image shows the Fisher Scientific atomic lamp. 2- Electrodeless Discharge Lamps (EDL). --- ## Experiment Measuring the concentration of heavy metal (Fe, Cu) in water sample ## Sample preparation 1ml of concentrated nitric acid ($HNO_3$) is added to each sample and the samples were filtered through a filter paper. 10 mL is taken for the following analysis. When not in use, the samples were kept refrigerated at +4 C°. ## Instrumentation Instrumental parameter used is burner 100 mm, lamp is HCL, optical mod is single beam, the technique used is flame is $C_2H_2 \Air$, the wave leng was adjusted at 248.3 and 324.7 for Fe, Cu, respectively Results The concentration of standard solutions (ppm) used for AAS measurements |Fe | Cu | |-----|-----| | 0 | 0 | | 0.5 | 0.25| | 1 | 0.5| | 2 | 1 | Image shows the calibration curve of Cu, described with the formula $y=10.62x$ and $R=0.998$.