Laboratory 4 Determination of Phosphate Using Two Different Techniques, Optical Spectroscopy and Nuclear Magnetic Resonance Nuclear Magnetic Resonance: Introduction: Protons and Neutrons, like electrons, also have the quantum property of spin. Depending on the total number of protons and neutrons in the nucleus, the nucleus may have unpaired spins. In this case, when the atom with a nuclear spin is placed in a magnetic field, the spin may either align with the magnetic field or opposed to the magnetic field. The spin, which is opposed to the magnetic field, is at higher energy than the spin which is aligned with the magnetic field. The separation between the two states is proportional to the strength of the field. Magnetic field B The greater the magnetic field, the greater the separation between the two states. The separation may be expressed as the frequency of radiation with that energy, ν. If the strength of the magnetic field is B, the equation which relates them is: ν = γB where γ is the gyromagnetic ratio of the particular nucleus. Different nuclei will have a different resonant frequency in the same magnetic field. Nuclei H 2 H 31 P 23 Na 14 N 13 C 19 F 1 (MHz/T) 42.58 6.54 17.25 11.27 3.08 10.71 40.08 27 In NMR spectroscopy, the sample is placed in a strong magnetic field. This separates the nuclear spin states (aligned with the field or opposed to the field). Radiating the sample with of the correct frequency will cause nuclei in the lower energy state (aligned with the field) to move into the excited state (opposed to the field). The sample will then relax over time to the lower energy state. NMR spectroscopy is such a powerful tool for structural information because not only do the different nuclei have different resonance frequencies, but also nuclei in different chemical environments have measurably different frequencies. We will study the phosphorous nucleus. In all the samples in this experiment phosphorus appears as either HPO4-2 or H2PO4-. These two ions rapidly convert from one form to the other during the time frame of the experiment. This means that only one peak is observed in the final spectrum. The area of the peak is proportional to the total phosphate concentration. The Bruker NMR and software: The NMR consists of a large magnet which holds the sample and the probe. The probe is a radio frequency (RF) detector. There is also a computer to control the console, and an electronic console, which controls the temperature of the instrument, the RF pulses, the shim magnet system, and detects the RF signal. The instrument is a Fourier Transform NMR, which means that the RF frequencies are collected all at once. The original data from the instrument comes in the form of a Free Induction Decay (FID). An example of an FID is shown in figure 1. 28 Amplitude of Signal Free Induction Decay Time Intensity The signal naturally decays with time, the oscillations correspond to a resonant frequency. The data is transformed from the frequency domain by a Fourier Transform. After this process a more traditional spectrum is obtained. Frequency The integrated peak area is proportional to the number of nuclei, which absorb at that frequency. 29 For the NMR spectroscopy section, we will work as one big group. The instructors have made a set of NMR standards with the following concentration of phosphate: 50 ppm, 100 ppm, 150 ppm, 200 ppm, and 250 ppm. The standards have been placed into NMR tubes and labeled. The instructor will demonstrate filling the tubes. You will place 0.5 mL of your sample in the tube and then add 0.1 mL of D2O to the same tube. The tubes are fragile and expensive. Be careful. 2. Collect the NMR data for your unknown. The software is designed to be read and implemented from left to right. a. Open a data file from the folder labelled CHM 152. Then click the start button. In this menu create new data set. You will need to advance the exp # by one. Then click OK. b. Now open the Acquire menu. Go to sample, pull down to turn on airlift to remove the sample currently in the NMR. Carefully remove the sample from the NMR and replace it with your sample. The instructor will demonstrate. 30 c. Lower the sample into the NMR wait for it to click. Then click Lock, the lock finds the signal of solvent to calibrate the RF frequency. d. Next click Tune. This sets the RF frequency for the sample. e. Then turn on the spin. f. Next click on Shim. This makes fine adjustments to the magnetic field. g. Next Click prosol, then gain then go. Now wait for the spectrum to be collected. It may look like this: 31 This is a Free Induction Decay. When that is done, click Process. This converts the FID to a conventional spectrum. There should be one phosphate peak. To integrate the peak first click on find peaks automatically, then integrate automatically: Open the integration table. It may look like this: 32 The peak has a chemical shift close to 0.0, The value needed is the Integration [ABS]. Record this number to share with your classmates. Plot the standard curve and determine the amount of phosphate in the unknowns. Questions: 1. Our NMR machine is designed to have the 1H signal resonate at 400MHz. What is the size of the magnetic field in Telsa? 33 2. What is the resonant frequency of the 31P atom in this magnetic field? What region of the electromagnetic spectrum is it? Part 2: UV/VIS method to measure phosphate in beverages: (Do this while waiting to take your NMR spectrum) We will use in this experiment the unit ppm or part per million. One ppm = 1 part per million, or 1 milligram solute/kg solvent. In water this is 1 mg/liter. The method Phosphate reacts with ammonium molybdate, (NH4)2MoO4 to form molybophosphoric acid, 12MoO3 • H3PO4 . This is reduced by stannous chloride to form molybdenum blue, which consists of mainly MoO2, and is a highly colored compound which absorbs at 650 nm. Procedure: 1. From the stock solution of 20 ppm phosphate solution make five standard solutions of 0.5 ppm, 1 ppm, 1.5 ppm, 2 ppm and 2.5 ppm. The total volume of each solution should be 25 mL. Determine the volume of the 20 ppm solution needed to make each diluted solution. Volume of 20 ppm solution added mL 0 Concentration of the standard ppm 0 0.5 1.0 1.5 2.0 2.5 2. Prepare the Spec 20 to read the samples at 650 nm. 3. Color develop the standards and the samples. To each of the 25 mL standard samples add 1.00 mL of the ammonium molydate solution. Add 2 drops of the stannous chloride solution. And mix. Do the same for your unknown samples. 4. After 5 minutes measure the absorbance of the standards and the samples. Make a Beer’s Law plot of the standard concentration versus absorbance and 34 use this to determine the concentration of the unknown samples. If the concentration of your unknown sample is greater than 2.5 ppm you will need to dilute it until it is less than 2.5 ppm. If you do not yet known the concentration (but your absorbance reading exceeds the absorbance of the 2.5 ppm sample) make a 1 to 10 dilution and a 1 to 100 dilution and then repeat the experiment for your known from part 3. Determine the concentration of the unknowns. Remember to include any dilutions. Questions Discuss the differences and similarities of the 2 techniques. Safety and Disposal: Place the waste in the container provided. 35 Measurement of Physical Properties In any measurement it is important to know the precision of the measurement and also its accuracy. All physical measurements should be made as precisely and accurately as possible. Maximizing the precision of a measurement is accomplished by using the most precise equipment available, and using it properly. If possible it is also wise to compare your result with either a known or theoretical value. Significant Figures When calculating a result from more than one measurement it important to retain the uncertainty information from all the measurements. There is an entire field of mathematics devoted to this topic. In this course we use the relatively simple method of significant figures. A summary of the rules with examples: Addition and subtraction: line up the numbers to be added or subtracted; the answer is truncated to the decimal place of the least precise number. Ex. 12.1 + 2.345 = 14.4 15.678 - 2.2 = 13.5 (notice I rounded up) Multiplication and Division: Significant Figures in the answer are equal to the number of significant figures in the least precise number. 15.6 x 2.1 = 31 16.789 ⎟ 25.67432 = 0.65392 25.1 x 3.00 = 75.0 Note zeroes before another number as in 0.65392 do not count. In the middle and the end they count. Laboratory Notebooks In this course you will be required to keep a laboratory notebook. A good laboratory notebook is an accurate record of everything, which occurred in the lab. In patent disputes a good lab book versus an inaccurate lab book can mean millions of dollars. In this course it may mean hundreds of points. Before the lab you will be required to prepare a lab report outline to be completed during the lab session. Each lab report will contain: 7 Title and Purpose 1. Procedure and Observations 2. Data and Calculations 3. Results and Conclusions 4. Answers to questions in manual An example of a lab report is shown below: 1. Title: Density of liquid and a solid. Purpose to measure the density of a liquid and an unknown solid. 2. Procedure: Observations Part 1 Liquid Unknown # 5 smells like gasoline Weigh an empty 10.0 mL volumetric flask Fill with unknown liquid. Weigh filled volumetric flask Mass of empty flask = 12.032 grams Mass of full flask = 18.685 grams Part 2 Solid Part 2 Unknown #12 Shiny orange color Fill a graduated cylinder with about 25 mL of water Measure precisely volume of water. Volume of water = 24.83 mL Volume of water + metal = 28.53 mL Mass of Dry metal = 46.409 grams weigh dry solid sample Data and Calculations: Part 1 Liquid: Density = Mass / volume Mass of liquid = mass of liquid + flask - mass of flask = 18.685 grams - 12.032 grams = 6.652 grams. Density = 6.652 grams / 10.000 mL = 0.6652 g/mL Part 2 solid volume of solid = volume of solid + water - volume water = 28.53 mL - 24.83 mL = 3.70 mL density = 46.409 g/3.70 mL = 12.5 g/mL Results and Conclusions: The density of the liquid was determined to be 0.6652 g/mL by comparison with the density table in the CRC it appears the sample could be hexane, which has a density of 0.660 g/mL The density of solid was 12.5 grams / mL. The solid looked like copper, but the density of copper from the CRC is: 8.94 g/mL, which is significantly less than my unknown sample. Therefore although the sample looks like copper it must be something else. 8 In this example, the data is recorded in the section with the observations, and the procedure is recorded in one column and the observations are recorded in an adjoining column. This allows you to record your observations with the correct section of the procedure. In some experiments, the type and volume of data is better recorded in a table. In this case it should follow the procedure section. You should still leave room in the procedure section for observations. One of the objectives of this course is for students to learn how to determine what data they need to collect, and how to organize it. For some experiments explicit instructions for organizing the data and calculations will be given, but for other experiments you will need to determine this for yourself before class. In the case of repetitive calculations tables are necessary. A spreadsheet such as Excel can be used, and instructions are included for the Reaction Rate experiment. All your calculations must follow the rules for significant figures and every value must have a correct unit. A spreadsheet or calculator will not determine the correct number of significant figures; it is up to you. When determining the results and conclusions, there are some things to keep in mind. The results should relate back to the purpose. Address directly if the purpose was fulfilled. If the result is a number clearly restate what it is and the unit for the number. If possible compare your result with a literature value. If you received no result or an unexpected result, give some scientific explanation of this. Human error is not a good explanation, because the experiment or section, which was in error, should be repeated. Thoroughness is important but it is not necessary to write everything you know about density or volume etc. To be ready to use all the lab time efficiently, before lab class you should have completed the purpose, procedure and arranged the data table or written down what you need to measure. Lab Instructors may have additional report requirements. 9
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