7. Backg_Lab Assign_ Instrumentation (1)

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Instrumentation Background Throughout the quarter (and in other Biology lab courses, if you take them), you will use a variety of different instruments to measure different things. This week will be an introduction to several commonly used laboratory instruments and protocols. Before you begin, you should read through the supplemental Units and Measurements section near the back of this manual, in order to have an idea of what you are measuring. Getting to Know the Lab Any time you enter a laboratory space, there are often chemicals, tools, organisms, equipment, etc. that may be hazardous to you and those around you if used incorrectly. So, it is imperative to familiarize yourself with lab you will be working in. Be intentional about noticing the safety equipment first. Where is the eye wash station? Where is the fire blanket? Where are the MSDS sheets? And so on… For the first part of today’s lab you will take some time to learn about the space and equipment you will be working with. Complete Section 1 of your Lab Assignment before moving on to the experimental procedure below. Concentrations in Solutions In this lab, you will be creating solutions and performing dilutions. A key part of this is calculating the amount of substance you have in a given volume. This is known as Molarity. Molarity (shown as M) is a measurement of the concentration of a substance and is defined as the moles of a substance in 1 single Liter of solution. In order to better understand Molarity, let's define a mole. One mole (shown as mol) of a substance represents a very specific amount of that substance, specifically 6.02x10 23 . This is a large number, so rather than scientists always having to spell out this large number, it is represented as 1 mole. So, 1 mole = 6.02x10 23 particles of a substance much in the same way we use the phrase 1 century to represent 100 years and 1 dozen represents 12 of something. Now that you know a mole = 6.02x10 23 particles of a substance, you can see that Molarity is how many times the particles of a substance equals 6.02x10 23 in exactly 1 Liter of solution. This is a 1

uniform and standardized way for scientists to observe or calculate the measurement of a substance. In this lab, you will need a few equations to help you calculate the concentrations of the solutions you will create: M = Molarity mol = mole L = Liter mL = milliliter g =gram Molarity (M or mol/L) = Number of moles of solute (mol) Volume of solvent (L) Number of moles (mol) = Mass (g)__ Molar mass (g/mol) **The Below Equation is Specific to Percentages : 2 grams sodium chloride = 0.02 g/mL 100 mL water (This Equation is Specific to Percentages) The units here are g/mL. These units do not technically cancel out but since 1 mL of pure water has a mass of 1 g, we could say that: 0.02 g/mL = 0.02 g/g = 2% sodium chloride solution Procedure Making a Solution First, you will need to use an electronic balance to weigh out 5.8 grams of sodium chloride (which is simply table salt). Before you can do this, you will need something to put the salt on. Putting this and other chemicals directly on the metal of the balance can damage the metal over time and can result in contamination of what you are weighing out with stuff that was weighted before. What you need is a thin, lightweight weighing boat. We keep a stock of these pre-made but if we run out, it’s pretty easy to make 2

your own: tear out a ~6 inch strip of aluminum foil from a roll. Fold it once to form an approximate square. This will be your weigh boat for the salt. Now, you want 5.8 g of salt, but you will not just be putting salt on the balance; you will be putting your weigh boat on the balance too and you don’t want the mass of the foil to count as salt. To prevent this from happening and to get an accurate measurement of salt, put your weigh boat on the electronic balance and then press the button marked “tare”. This tells the machine to set the current mass to zero, essentially ignoring the mass of the weight boat. Now, you are ready to measure out the salt. Five grams is a pretty small amount, so directly pouring salt onto the weigh boat will likely result in far too much salt. Use a metal scoopula (yes, that is a technical term—it’s the tiny metal spatula thing) to transfer small amounts of the salt from your salt container to the weigh boat. If you put on too much, simply transfer a small amount back to the salt container. Importantly, you might see the mass fluctuate slightly, especially in the hundredths digit. This is normal for a balance like this and you should not fret over getting 5.80 grams: 5.82 or 5.78 g is accurate enough. Get your TA to verify that you have accurately measured 5.8 g of salt and have them initial in the appropriate area on the assignment page before you proceed to the next step! Measure 100 mL of water (tap water is fine for this, but for other, more sensitive applications in the laboratory, we will use distilled or deionized water). Be sure that you use the appropriate vessel for this (see the supplemental Units and Measurements section near the back of this manual)! Pour this water into a beaker. Then, add the 5.8 g of salt that you measured out in the previous section. Stir this solution using a stirring rod. The Spectrophotometer Spectrophotometers are instruments that measure how much light is absorbed by a sample. See the Units and Measurements section (near the back of this manual) for a more thorough explanation of how these instruments work. After reading this section, you should now know that you need a blank for the spectrophotometer. Since the solutions we will be working with will be water-based, your blank should be pure water. Get a small beaker (250 mL or so) and fill it with de-ionized water (you will use this water again in a few minutes, which is why you’re getting more than you need now). Pipette 5 mL of water into a glass tube that you have been supplied. Label this with a marker and lab tape. Do not put this label near the bottom of the tube—put it as high up as possible. One of the more common uses for a spectrophotometer is to calculate the concentration of a substance in a sample of unknown concentration. In this section, you will be making dilutions of green 3

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food dye and then measuring the absorbance of these solutions using a spectrophotometer. There will be a container of “stock” food dye. This has been prepared by your TA beforehand and should be on one of the setup benches. In your calculations, treat this starting solution as “100%” food dye. Pipette 10 mL of this 100% food dye into a glass tube that you have been supplied. Label this as you did above. Now, to make dilutions. You will make 50%, 25%, 12.5% and 6.25% solutions of dye. Fill in the table associated with question 14) in the Lab Assignment section to plan out how you will make these dilutions. Use the technique of serial dilutions! Once you have filled in the table, create the solutions by combining the appropriate amounts of dye solutions and water. Most dilutions you will start with a stock solution (very high concentration of a specific substance) and add it into a solvent to reduce the total amount of substance in the new solution (lower concentration). However, a serial dilution is a series of repeated dilutions where you are diluting by the same constant for every dilution. For a serial dilution, you will not dilute using the original stock solution, but ecause this is a series of dilutions, you will always pull from a previous dilution. Take a look at the diagram below for a visual example: You can make these dilutions directly in the glass tubes that you already have. Mix them well using your pipette as you prepare them and be sure to label them as described previously, so you do not mix up your samples. 4

Finally, you will to get a sample of dye of an unknown concentration. This has been prepared by your TA beforehand and should be on one of the setup benches. Pipette 5 mL of this into a glass tube. At this point, you should have 7 total tubes: water, 100%, 50%, 25%, 12.5%, 6.25% and unknown. You can probably predict what its concentration might be like by simply comparing how dark of green it is to your other tubes. However, this is not very precise and if this were a different compound (like DNA or protein, for example) you would not be able to see it by eye. Measure the absorbance aka optical density of your solutions using a spectrophotometer. Your TA should demonstrate to you how to use this instrument appropriately. Importantly, you will blank/zero the instrument first using your tube of water. After that, the order in which you read your samples will not matter. 5

Lab Assignment: Instrumentation Due at the end of lab 40 points total (Number in parenthesis is point value) Student Name: ________________________________________________________________________ As you go through the assignment instructions on the previous pages, fill out the tables and answer the questions below. Tear out the following pages of questions, staple them together and then turn them in to your TA before you leave. Section 1: Safety/Get to Know the Lab Locate the numbered items around the lab. There, you will find information on the use/importance of each item. This information will be vital for your continuance in the lab and locating safety items and specific equipment before beginning an experiment is a good lab practice. Use the information to fill in the blanks. 01 ) Locate #7. What is the name of this piece of safety equipment? ___________________________. 02 ) Locate #5. This is the _____________ container. What is the ONLY kind of item that should be disposed of here? _________________________________________________________________________________. 03 ) Locate #3. This is where the __________________________ kit is located in the lab. 04 ) Locate #1. What is the ONLY item that should be disposed of here? ________________________. 05 ) Locate #4. What can be disposed of in this waste can? __________________________________________________________________________________. 06 ) Locate #2. What is this instrument called? What is this instrument used for? __________________________________________________________________________________. 07 ) Locate #6. What is this instrument called? __________________. The green pipette is used specifically for ____________ mL of liquid and the blue pipette is for _______________mL of liquid. 08 ) Locate #8. What does MSDS stand for? What is the purpose of having these in a lab? __________________________________________________________________________________. 09 ) Locate #9. Which two pieces of safety equipment are located here? When would these be necessary to use? ______________________________________________________________________________. 10 ) Locate #10. When should these be used? _____________________________________________ 11 ) Check your answers from Section 1 with your TA before moving on: ___________ TA Initial (6). You MUST have your TA’s initial to receive ANY credit for this section!! 6

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Section 2: Making a Solution 12 ) You will be measuring out 5.8 g of sodium chloride. How many milligrams of salt is this? _________________________________________________________________________________ (1). 14 ) Weighed out 5.8 g of sodium chloride: __________________ TA Initial (1) 13 ) Grams are a measurement of: (1) A. Volume B. Weight C. Mass D. Length 15 ) Although you combined the water and salt together in a beaker, what piece of labware did you use to accurately measure the amount of water? _________________________________________________________________________________ (1). 16 ) Milliliters (mL) are a measurement of: (1) A. Volume B. Weight C. Mass D. Length 17 ) You measured out 100 mL of water. How many Liters of water is this? _________________________________________________________________________________ (1). 18 ) You created a sodium chloride solution by combining 100 mL of water and 5.8 g of sodium chloride. Given that the molar mass of sodium chloride is 58 g/mol, what is the molarity of the sodium chloride solution that you have created? (3) 7

19 ) Let’s say you want to have a sodium chloride solution that is 0.1 M and you want 100 mL of it. You don’t actually have to perform this dilution, but how much of your stock sodium chloride solution (the one that you calculated the molarity of in question 7) would you combine with how much water to get this 100 mL of a 0.1 M sodium chloride solution? ____________________________________________________________ mL of stock NaCl solution (2). ____________________________________________________________ mL of water (2). 20 ) Here’s a hypothetical situation to give you a little more practice with calculating how to make solutions. The molar mass of sucrose (table sugar) is 342 g/mol. If you wanted to make 0.5 L of a 0.5 M sucrose solution, how many grams of sucrose would you dissolve in the 0.5 L of water? (2) 21 ) So, you have your 0.5 M sucrose solution. Now, you need 1 L of a 50 mM sucrose solution. How much of the 0.5 M stock solution would you combine with how much water? Hint: convert them both to the same units (either M or mM) first. ____________________________________________________ mL of 0.5 M stock sucrose solution (2). _______________________________________________________________________ mL of water (2). 22 ) So, now you have a 50 mM sucrose and you need 1 L of a 5 mM sucrose solution. How much of the 50 mM sucrose solution would you combine with how much water? ___________________________________________________ mL of 50 mM stock sucrose solution (2). _______________________________________________________________________ mL of water (2). 8

Section 3: The Spectrophotometer 23 ) Fill out the following table as you make your dilutions of the 100% dye solution. Remember that you are doing a serial dilution—review the Concentrations and Dilutions section at the back of this manual for an explanation and a diagram of what this should look like. (5) Tube 1 (Not diluted) Tube 2 Tube 3 Tube 4 Tube 5 Dye Solution 10 mL of 100% dye mL of 100% dye mL of 50% dye mL of 25% dye mL of 12.5% dye Water 0 mL mL mL mL mL Total volume 10 mL 10 mL 10 mL 10 mL 10 mL Final dye concentration 100% 50% 25% 12.5% 6.25% Optical density (O.D.) 24 ) Measure the optical density of the dye solution of unknown concentration (you can call this tube 6) _____________________________________________________. (1) 25 ) It is not likely that the dye solution of unknown concentration will have an optical density exactly the same as one of your four tubes of known concentration. However, it should have an optical density between two of the known concentrations. Based on its optical density and the optical densities of your tubes with known concentration, the green dye solution of unknown concentration is likely between: ____________________________________ % and ____________________________________ %. (4) 26 ) Clean up lab area _________________ (1) For credit, get your GA to initial above before you leave. You may only receive this point if you were present in lab at the time of the lab experiment. 9

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