Lab 3 and 4-AAp PDF

Lab 4: Calculations, dilutions and solution preparation Dr. Asmaa Abu Obaid Dr. Asmaa Abu Obaid 2 Dilution Factor It is the ratio of the aliquot volume to the final volume 1:5 dilution factor means? 1 part of solute + 4 parts of the solvent, so the final volume is...

Lab 4: Calculations, dilutions and solution preparation Dr. Asmaa Abu Obaid Dr. Asmaa Abu Obaid 2 Dilution Factor It is the ratio of the aliquot volume to the final volume 1:5 dilution factor means? 1 part of solute + 4 parts of the solvent, so the final volume is 5 Note that some solutions have in total slightly less volume than their components. As such, one has to start with the solute volume and add solvent to bring the mixture to a final indicated volume Dr. Asmaa Abu Obaid 3  Serial Dilution It is the stepwise dilution of a substance in solution the dilution factor at each step is constant The purpose of a serial dilution is to estimate the concentration of a sample, or to obtain the desired concentration of a reagent, chemical or compound Dr. Asmaa Abu Obaid 4 Preparation of buffer solutions A buffer is a solution that can resist pH change upon the addition of an acidic or basic components so the pH does not change significantly Buffers are used to maintain solutions at constant pH values PH stands for Hydrogen potentials. It refers to the concentration of the hydrogen ions in a solution. Why is it physiologically important? The internal pH of most living cells is close to 7 pH homeostasis should be maintained constant because all chemical reactions of the cell are very sensitive to changes in the concentrations of hydrogen ions (H+) and hydroxide ions (OH-) are very sensitive to change in pH Dr. Asmaa Abu Obaid 5 In pure water, the concentrations of these ions are equal. In a neutral solution like pure water, the hydrogen ion concentration is 10-7 molar, so the pH of a neutral solution is pH 7-- the midpoint of the pH scale. In a neutral solution, the H+ and OH- concentrations are equal. An acid is any substance that increases the H+ concentration of a solution. A base is any substance that reduces the H+ concentration of a solution. The pH scale is used to express the concentration of hydrogen ions in a solution. pH is defined as the negative logarithm of the hydrogen ion concentration. Dr. Asmaa Abu Obaid 6  Henderson–Hasselbalch equation This equation defines the relationship between pH and the ratio of acid and conjugate base concentrations it can be used to calculate the pH of a solution if the molar ratio of buffer ions ([A-]/ [HA]) and the pKa of HA are known the molar ratio of HA to A- that is necessary to prepare a buffer solution at a specific pH can be calculated if the  If base is added to the buffer solution it would be pKa is known. neutralized by reaction with HA  If acid (H+) were added to the solution, it would Dr. Asmaa Abu Obaid 7 be neutralized by A- in solution  The most effective buffering systems contains equal concentrations of the acid and the conjugate base when [A-] is equal to [HA] pH equals pKa Therefore, the pKa of a weak acid- base system represents the center of the buffering region Effective range for a buffer = pKa ± 1  The main buffer system in human blood is the CO2/bicarbonate system Dr. Asmaa Abu Obaid 8  Buffer systems: Blood buffer system CO2/HCO3- Phosphate buffer Plasma proteins Hemoglobin Dr. Asmaa Abu Obaid 9  pKa of this buffer system is 6.1 Respiratory Regulation of Blood pH: The respiratory system can reduce blood pH by removing CO2 from the blood. Dr. Asmaa Abu Obaid 10 Step 1: Sodium ions are reabsorbed from the filtrate in exchange for H+ by an antiport mechanism in the apical membranes of cells lining the renal tubule. Step 2: The cells produce bicarbonate ions that can be shunted to peritubular capillaries. Step 3: When CO2 is available, the reaction is driven to the formation of carbonic acid, which dissociates to form a bicarbonate ion and a hydrogen ion. Conservation of Bicarbonate in the Kidney. Tubular Step 4: The bicarbonate ion passes into the cells are not permeable to bicarbonate; thus, peritubular capillaries and returns to the blood. bicarbonate is conserved rather than reabsorbed. The hydrogen ion is secreted into the filtrate, where it can become part of new water molecules and be reabsorbed as such, or removed in the urine. Dr. Asmaa Abu Obaid 11 Lab 3: Spectroscopy and λmax determination Dr. Asmaa Abu Obaid Dr. Asmaa Abu Obaid 12  In this experiment, you will learn how to prepare solutions using dilutions, and learn how to use a spectrophotometer Dr. Asmaa Abu Obaid 13  Spectroscopy: the study of how light interacts with matter Spectrophotometry works because light gets absorbed by matter Molecules absorb and emit light depending on the type of covalent bonds Why do we use the spectrophotometer? To quantify the amount of substance in a solution Monitor the change in concentration of a substance Examples: to determine the density of suspended cells (e.g. bacteria) or the concentration of a biological entity (e.g., proteins or DNA). The device is used in : physics, biology, biochemistry, material and chemical engineering, etc. Dr. Asmaa Abu Obaid 14 spectrophotometer spectrophotometer is an instrument that measures the amount of photons (the intensity of light) absorbed after it passes through sample solution. commonly used for the measurement of absorbance or transmittance of solutions at specific wavelengths (λ) that range between 200nm - 2500nm Dr. Asmaa Abu Obaid 15  Most of biological samples are detected in the range of UV- visible light UV-visible spectroscopy UV: 200-400nm Visible light: 400-800 nm IR: 800-15000 nm Dr. Asmaa Abu Obaid 16  Spectrophotometer principle a substance that transmits all visible wavelengths (i.e., absorbs nothing, it appears white. On the other hand, if a substance transmits none of visible wavelengths (i.e., absorbs light over all visible ranges), then it appears black Spectrophotometers use a prism or grating (monochromator) to make it possible for a particular beam of light to pass through a solution sample a certain range of wavelength is narrowed down (other wavelengths are filtered out) Dr. Asmaa Abu Obaid 17  spectrometer is a device that produces the desired range of wavelength of light photometer detects the amount of photons that is absorbed and then sends a signal to a digital display (measures the intensity of light) spectrometer photometer Dr. Asmaa Abu Obaid 18 Dr. Asmaa Abu Obaid 19 I=I0 * 10-ebc Dr. Asmaa Abu Obaid 20  The intensity of incident light on a sample is 0.5 w/m2 and the intensity of light entering the detector is 0.36 W/m2. a) calculate the transmittance. B) determine the absorbance. Dr. Asmaa Abu Obaid 21  A 0.25M solution in a test tube with a path length of 1 cm has the absorbance of 0.075 at 560 nm. A)what is the absorptivity of the solution? B) what will be the absorbance if the concentration of the solution is 0.65M? C) what is the concentration of the solution if the absorbance is 0.450? Dr. Asmaa Abu Obaid 22  A compound has a molar absorptivity of 40.9 cm -1 M-1 at 293. if a 1 cm cuvette is used, and the absorbance is found to be 0.111, what is the concentration of the sample? Dr. Asmaa Abu Obaid 23  Wavelength at maximum absorbance each solute has a range of absorbance values if the wavelength is varied, thus you should fix the wavelength before you measure Absorbance at different Concentrations at λmax solute gives the maximum absorbance Different solutes have different λmax values Dr. Asmaa Abu Obaid 24  prepare solutions using dilutions, and learn how to use a spectrophotometer Prepare a set of serial dilutions of the 2.0M CuSO4dilutions: 1000mM, 100mM,10mM, and 1mM. Measure the absorbance value for each. Perform absorbance scan for 10mM CuSO4 from 400 to 960 nm. Take reading every 20 nm. Record the absorbance of each wavelength. Do not forget to zero and reset (against water) - 100% T (TARE) after every wavelength change. After you have scanned the full range each 50nm (400-800nm) find the wavelength, λmax ,associated with the greatest absorbance. Find the concentration of the unknown CuSO4 solution Dr. Asmaa Abu Obaid 25