Need help with Milestone 2 pre-lab questions 1-7
5 AAS Analysis of Mercury in Fish Page | 1 3 AAS Analysis of Total Mercury Content in Fish Flesh Quantitation by Atomic Absorption Spectroscopy (AAS) Experiment Overview In this 7-session experiment you will utilize atomic absorption spectroscopy (AAS) to quantify the total mercury bioaccumulated in a fish flesh sample. Significant components of this experiment, as expected for most any quantitative experiment, are method validation and sample preparation. While guidance for the method validation is presented below, it is your responsibility to consult published literature to produce the sample preparation process and plan from the following three publications: ‘A Comparison of Techniques for Preparing Fish Fillet for ICP- AES Multielemental Analysis and the Microwave Digestion of Whole Fish’ ‘Optimization of a Microwave-Pseudo-Digestion Procedure by Experimental Designs for the Determination of Trace Elements in Seafood Products by Atomic Absorption Spectrometry’ ‘Field Sampling, Preconcentration and Determination of Mercury Species in River Waters’. which are all posted on the D2L site. It is intended that the first two references be consulted for guidance in developing a sample digestion protocol (or analyte leaching protocol – i.e. pseudo-digestion). The third reference should be consulted specifically to aid you in constructing a pre-concentration protocol. As the extracted analyte will be at a concentration well below the methods lower limit of detection (LOD), a pre- concentration process will need to be employed. This third reference should help direct your protocol development for mercury complexation (sodium diethyldithiocarbamate (SDDC) is the complexing agent used in this experiment), C18 solid phase extraction (SPE) column conditioning, concentration/enrichment of complexed mercury species with C18 SPE columns, and subsequent elution of complexed species from SPE columns. In other words, these references should guide you in your protocol to step through the sample preparation “flow diagram” illustrated in Figure 3.2 You will not analyze the total mercury via the techniques discussed in these references, you will use these references to guide your sample preparation, and then perform the quantitative analysis via AAS. This experiment requires significant sample preparation due to the facts that i) samples can only be introduced to the AAS as aqueous, fully-dissolved solutions (which is quite different state than the provided ‘native sample’) Figure 3.1: Model for biomagnification of mercury in the food chain. Image courtesy of Wikipedia: https://en.wikipedia.org/wiki/Mercury_in_fish#/me dia/File:MercuryFoodChain.svg AAS Analysis of Mercury in Fish Page | 2 and ii) the total mercury concentration in the digested fish solutions will be below the method/instrument detection limit. As the concentration of mercury in the digested fish solutions will be lower than the detection limit for AAS, the mercury species must be pre-concentrated to enable quantitative analysis; procedures for such are discussed in the second reference listed above. Figure 3.2 Flow diagram for the sample preparation necessary for this experiment For your initial calculations and method development plans, you may presume the concentration limit of detection for mercuric aqueous solutions with AAS to be on the order of 5 ppm (by mass). You should also expect to find on the order of 1000 ng of mercury per gram of dried fish in the samples. This experiment is divided into three discrete milestones as detailed below. Suggested Timeline Session Action Before Lab Session 1 complete Milestone 1 pre-laboratory assignment Lab Session 1 complete experimental for Milestone 1 Before Lab Session 2 complete Milestone 2 pre-laboratory assignment Lab Session 2-4 perform experimental for Milestone 2 Before Lab Session 5 complete Milestone 3 pre-laboratory assignment Lab Session 5-7 perform experimental for Milestone 3 AAS Analysis of Mercury in Fish Page | 3 Milestone 1: Instrument and Sample Quantity Validation Milestone 1 Assessment Milestone 1 Pre-laboratory assignment : 39 points Milestone 1 Experimental and Technical Report : 45 points Milestone 1 Overview Proper and thorough experimental design requires recognition that any time a new analytical technique or instrument is enlisted, such as flame AAS, some reference standard should initially be analyzed before attempting analysis of a real and unknown sample. This initial analysis enables one to: 1) become familiar with new instruments and software 2) develop protocols and determine optimal instrument and software settings 3) estimate detection limits, sensitivity, and resolution for the technique 4) establish rules-of-thumb for volumes or masses of sample required for analysis 5) measure a known system to verify proper functionality As this atomic absorption (AA) spectrometer and corresponding software are likely new to you, for this first milestone you will prepare a series of aqueous mercury standards (as detailed in the experimental section) and analyze them via AAS in order to accomplish the above goals. Both standard and “blank” solutions, you will determine estimates for the lower concentration limit of detection, the resolution, and the sensitivity for the method – and also confirm that a linear response (or determine the model for the response) to concentration (i.e. Beer’s Law) is attained that can be used for interpolation. One drawback for employing AAS as a quantitative chemical analysis technique is that the method typically requires a relatively large volume of sample solution (on the order of 10 mL), and thus a relatively large quantity of analyte, for analysis. This can be problematic since the analytical chemist is often tasked with quantifying trace quantities of analyte; quantities of analyte which can be unwittingly diluted to concentrations below the instrument’s detection limit. Thus two more key outcomes for this first milestone are development of a procedure for analysis via AAS that utilizes minimal sample volume and determination of what this minimum volume is. Developing such protocol is critical for your subsequent analysis of the fish sample in that it will dictate the amount of fish tissue and extent of pre-concentration required for analysis. Milestone 1 Pre-lab Assignment Recall that it is mandatory that your pre-lab assignment for the specific milestone be approved by your TA before you begin performing any experimental work. 1) The prep-room provides you a 10% HNO3 (by volume) calibrated standard solution with a Hg2+ concentration of 1000.0 ppm (by mass). Construct a detailed plan for how you will produce the series of four 50.0 mL mercuric standards as specified in the experimental section for this milestone. Note: you have access to 50.0 and 100.0 mL volumetric flasks, 0.5, 1.0, 2.0, 5.0, 10.0 mL volumetric transfer pipettes, and concentrated HNO3. Recognize, it is not essential that your standard be exactly equal to, for example, 1.0 ppm; it must only be near the desired value but, more importantly, a value that you know exactly. For ppm calculations for this question you can assume the density of solutions to be exactly 1.0 g/mL. Do not neglect the use of HNO3 in your standards preparation – the matrix must be AAS Analysis of Mercury in Fish Page | 4 the same for all of your solutions or your calibration curve would be meaningless due to matrix effects. For this experiment you will have access to both 20 – 100 uL and 100 – 1000 uL micropipettes in addition to the volumetric transfer pipettes. Note: if you use the micropipettes, you should verify their accuracy and precision using the density of water to verify that they are acceptable for the level of accuracy you desire. 2) Assume you have determined the lower concentration limit of detection (LOD) for mercury via your AAS protocol to be 5.0 ppm; i.e. the method cannot statistically distinguish solutions with mercury concentrations of 5.0 ppm or lower from that of a “blank” solution containing 0 ppm of mercury. In your protocol development you also determine that the minimum volume of sample solution that you can analyze via AAS is 20.0 mL. Assuming the dried fish sample contains 1000 ng of mercury per gram of dried tissue, what is the minimum mass of dried fish tissue that you would have to employ in order to prepare a solution with a mercury concentration above the LOD? For this question you can assume the density of all solutions to be 1.0 g/mL and all ppm concentrations are by mass – thus 1 ug / mL = 1 ppm. 3) While in the previous question you assumed a 5.0 ppm LOD, in this question you will utilize sample data provided to calculate the sensitivity, resolution, and LOD for the method; providing an opportunity to mirror the experimental and analysis process that you will employ to complete the first milestone. For this question you will need to download and analyze two .csv data files posted on the D2L site titled “Hg_AAS_Standards.csv” and “Hg_AAS_Blanks.csv”. The “Hg_AAS_Standards.csv” file contains two columns of data. The first column is the concentration (in parts-per-million by mass) of prepared mercury standards. The second column is the measured absorbance from the AA spectrophotometer for each standard. Construct a calibration curve for this data and find the best-fit linear model for the response. The “Hg_AAS_Standards.csv” file contains a single column of data; specifically 25 absorbance measurements, obtained from the AA spectrophotometer, for a mercury-free “blank” solution. You should use this set of data to determine the sample-to-sample measurement variation which is necessary for calculating the lower concentration LOD and resolution. Utilize these two sets of data to calculate and report i) the method sensitivity – i.e. the slope of the calibration curve (include units!), ii) the lower concentration limit of detection (LOD) for this method – see your quantitative chemical analysis textbook for assistance, and iii) the resolution of the method. It is typically important to know the minimum difference in sample concentration that can be resolved with a certain method – i.e. the resolution of the method. For calculating the resolution, utilize: Resolution ~ (sample standard deviation of signal from “blank”) / (method sensitivity or slope ) (Eq. 1) 4) Based on goals of this first milestone (consider the Overview, Pre-laboratory assignment, and Technical Report sections), construct a detailed list specifying the data that you must acquire in order to complete this first milestone – determine the sensitivity, resolution, and LOD. Make sure to use this list in the laboratory