Measurement: Straight Lines/ Standard Curves

Transcription

1 Measurement: Straight Lines/ Standard Curves URL: Learning Outcomes After completing this module you should be able to: Describe the linear relationships between two parameters Construct and use a standard curve for determining the concentration of a unknown solution using spectroscopy Use Beer s Law and the gradient of a standard curve to determine the concentration of an unknown solution using spectroscopy Why do we use line graphs in biology? Line graphs are one of the most common ways that trends or relationships are shown between one or more variables in experiments involving numbers. They allow us to immediately determine the effect of changing one of these numerical variables on another numerical variable you can measure. Though this sounds complicated don t panic, you have come across this before. For example, on the The Biggest Loser TV show, it would be interesting to show the rate of weight loss of contestants. This could be easily displayed by plotting the weight loss of the contestants at the various weigh ins. As all the data collected is numerical (i.e., time, since starting the show and weight) a line graph could be used to show the differences in the rate of weight loss between contestants. As the show s producers decide when the weigh ins are going to happen, time is known as the independent variable. That is, the variable that the experimenter controls. The weight of the contestant is dependent on what day is chosen so is known as the dependent variable. Convention says that we plot the independent variable (in this case, time) on the bottom or x- axis and the person s weight loss on the y - axis (dependent variable). Ok now, we have all been on diets. I personally would want my graph of weight loss to be as steep as possible and never flattening out until my goal weight, but of course this does not happen. Line graphs can therefore show when a person is losing weight quickly (fast rate) and when they are plateauing (zero rate). Line graphs are used because it is very easy to interpret rates of change between variables. 1

2 As you are now aware, line graphs are not always straight lines. If you do collect data points that allow you to draw a straight line, you can say there is a correlation between the two variables or a linear relationship. In science, there are lots of linear relationships, for example the length of the femur (a leg bone) and the height of humans, the excretion of Vitamin B 2 in urine vs dose etc. In biology, often the first time you deal with linear graphs is using spectroscopy where at low concentrations of chemicals there is a linear relationship between the absorbance of light through a solution and its concentration. Straight Lines-you have met them before! A linear relationships means that as one thing increases or decreases, the other variable increases or decreases by the same proportion. If you are paid an hourly rate in your job you are acutely aware of this relationship because: If you work: twice as many hours in a week, your pay is twice as much. only 50 % of the hours, you get 50% of your usual pay. no hours, you don't get paid. The relationship between the hours worked (independent variable) and your pay (dependent variable) is linear and if graphed would give you a straight line. The steeper the graph, the higher your hourly rate. Have a look at the graph to the right: In all cases, the person s pay is directly proportional to the work hours double the hours, double the pay. But some people are accumulating money a lot faster than other people. That is reflected in the steeper slope/gradient of some lines (like Bill Gates). 2

3 In fact, you can look at a graph such as the one below and work out how much each person makes per hour. (In other words, the change in money per time or rate of pay). From the graphs above can you work out the hourly rate of a Pizza guy and a PC tech: Pizza Guy: $ 20 per hour PC tech: $ 45 per hour You should have recognized that the "change in money per time" as the same as the change in y value divided by the change in x, which is the classic formula for slope or gradient of a line. If you can engrave this on your brain, you'll have a head start interpreting lots of graphs whether they are straight lines or not. The slope at any part of a graph tells you how fast the process is happening right then. This graph represents the sales of pizza slices over a period of 4 hours. At what point in time is pizza income increasing at the fastest rate? Click the buttons to see how slope and rate are related: 3

4 A straight line has only one slope or gradient A straight-line graph is a special type of curve. It has the same slope everywhere so when determining the slope it does not matter where in the line you calculate it. For example, to find out how much a Lab Tech makes per hour, there are several ways to do it. You could measure how much they made in any single hour such as they made $60 in two hours therefore $30 dollars an hour. If you want to get fancy, you could pick a different hour, but it seems more work for the same answer. For example, between hours 2 and 3, the Lab Tech earned $90 - $60 = $30 extra dollars: Remember when people want to find a slope, they often draw a figure that looks like that indicated in the graph below. This is will result in the same value for the rate of pay. Hint: As long as the line goes through (0,0), it s much easier to find the slope by just finding the value of y at a particular x value and dividing y by x. Remember what you are determining is the slope or gradient of the line. What is the pay rate for the forest conservation worker? How about the veterinarian? (All wage data taken from Monster.com) 4

5 You may remember from high school that the equation of a straight line is: The slope (known as m ) is that rate or gradient we were talking about. The intercept (b) is just the value for y where the line crosses the y axis. Linear relationships and Spectros A common linear relationship used in biology is correlation of the amount of light that some chemical solutions absorb with their concentration. You see examples of the linear relationship of light absorbance and concentration all the time. If a parent makes up a red cordial drink at a low concentration, children are very quick to complain. That s because children immediately use their eyes as a spectrophotometer and realize that the amount of light absorbed by the drink is different to what they were expecting. In fact, if the parents have halved the cordial concentration, then the absorbance of the solution would be half, with the drink looking less dark. A spectrophotometer (affectionately known as a spectro ) does this same job as the children s eyes but puts a number on the absorbance (ahh, a numerical variable we can measure!!). The higher the absorbance value, the less light that can get through the solution. 5

6 Key Knowledge: A spectrophotometer is a device that measures how much light a solution absorbs, which often has a linear relationship to the concentration of some chemicals in the solution. For a standard curve (i.e., a line graph that can used to find out the concentration of an unknown solution), the experimenter decides on the concentrations to be measured (independent variable) and the output from the spectrophotometer is the value for the dependent variable (called Optical Density or OD). Thus you end up with a graph like this: Yeah, a linear relationship!! If at higher concentrations the curve does flatten off, don t panic as this is often is due to the limitations of the instrumentation used at high absorbance values. You should not use data in this region of the graph for calculations. So there you go, if your parents are trying to rip you off when making up your favorite drink, you now have the science to do an experiment and come back with some objective data to prove your case! In other words, the OD increases by 0.2 units for every additional 1 mm of cordial. What is the slope of the graph above? Before you can even start to answer this, you need to think about units. So, y is measured in OD units, and x is concentration, which ranges from 0 to 5 mm. A useful unit for x would be "mm". Thus the units of the slope are "OD / mm" Don t stress if you are lost at this point below is the nuts and bolts of using standard curves which does not involve much maths. You don t have to understand all the maths behind standard curves to be able to use them!! As biologists, we must be able to be able to construct or set up experiments to generate data for a standard curve and use them appropriately. 6

7 Standard curve: Choosing a wavelength of light Before we use the spectro there is one other thing we need to know: the wavelength of light the compound (in this case the cordial) absorbs. In most experiments, this is known and is provided in the methods. The wavelength can be different for different chemicals. Do you remember the spectrum of light?? Water doesn't absorb any light, so all wavelengths (colors) get through, and mixed together, they look like white (= colorless) light. Our cordial solutions look red because the solution absorbs more of the other colors of light. The more concentrated the drink, the more non-red light that gets absorbed and correspondingly, what does get through looks redder. The online version of this module contains an interactive applet that allows you to practice with virtual spectrophotometer. To find this applet, go to: _TOC/page06.htm To test the concentration of our cordial, you would need to set your spectro to measure something other than a red wavelength. So in general, before you use a spectro, you have to figure out what wavelength works the best. If you remember from first year chemistry you could run an absorbance scan of the solution across all wavelengths to select the one that absorbs the best, or use a trial and error approach. Remember, unlike our cordial solution, some chemicals absorb UV light, so by the naked eye solutions look clear, however they can be measured by selecting a wavelength in the UV range. After you've figured out the best wavelength to use, you can proceed to the next step, actually gathering data to construct a standard curve. Standard curve making and using You need to produce a graph showing the linear relationship between the OD of the solutions (e.g. cordial) at various concentrations. That means you will get a line graph similar to that shown below (it may flatten out at higher ODs). As the experimenter, you decide what concentrations will be tested and the spectro will measure the ODs of those samples. You should also measure the OD of the unknown (in the case the diluted cordial drink made up by the parents) at the same time. Then plot the OD values for the concentrations you have chosen on a graph (do not use the data on your unknown sample at this point). You need to draw a line of best fit through the data points. 7

8 Now, when I say "draw a line of best fit", please DON'T think this means simply connecting the dots as if you were doing a dot-to-dot puzzle. Instead, you want a single straight line that goes approximately though the center of your group of dots (see graph above), so some dots are above and some are below the line, but all are as close as possible. (In statistical jargon, this is known as "doing a linear regression ) So back to constructing a standard curve for cordial. As we don t know the concentration of the cordial in molarity, and the manufacturers say you should be using a 20% (v/v) solution of the concentrate, the units of the x axis in this case is in %. You would take a sample of cordial, dilute it to 10% (v/v), read the absorbance using the spectro, and mark your data point on the graph. Then you need to do the same with a 20% (v/v) sample, a 30% (v/v) sample, and a 40% (v/v) sample. Your graph should look something like that shown below. So, for every 20% (v/v) change in concentration, OD increases by about 1.0 unit. The slope of the line is 1.0/20, or 0.05, which is also the value of "e", the extinction coefficient (more on that later). So if the kids want to accuse their parents of diluting the cordial too far, they can take their drinks, measure and OD and use the standard curve to determine the concentration of the kid s drinks. 8

9 Using a standard curve minimum maths method So if the kids want to accuse their parents of diluting the cordial too far, they can take their drinks, measure and OD and use the standard curve to determine the concentration of the kids' drinks. For example, if the OD of the drink were 0.8, you would move along the y axis to an OD of 0.8 and then draw a line across to the standard curve, then a perpendicular line and read the value of the x axis. You will get the answer approximately 15% (v/v). So if you have a spectro in your garage and do the above experiment you now can make a very solid case that your parents are over diluting your drink. Beer's Law: explains the relationship between cordial concentration and light Beer s Law uses a maths approach to explain the experimentally obtained standard curve. Luckily, the relationship is linear, and all we need to figure out is the slope from the standard curve. So let's write the equation of a line, keeping in mind that... Instead of x, we say C (for concentration). Instead of y, we say OD (for optical density, which is the technical way of saying how much light the cordial absorbed ). Instead of m, we say e (which stands for extinction coefficient, i.e., the amount of light that is extinguished by each extra bit of cordial). We know that if there is no cordial in the solution then there is no absorbance so the intercept (b) equals zero Just to make it a little trickier, the amount of light absorbed also depends is the distance the light has to pass through the cordial solution. So if I had a glass of 10 cm diameter, then the absorbance would be twice that of a glass with 5 cm diameter (if I was looking through the side of the glass). Another linear relationship! 9

10 So, to be really correct, we have to write the equation like this: where "l" stands for the length of the path that light follows. Luckily, those people who designed the spectros were pretty smart: they keep the distance that light has to travel constant at 1 cm, which means in practice we can just use the easier equation, OD = ec (that is why the cuvettes have a 1 cm width!) The equation above is known as Beer's Law. Yes, there was a person named Beer (Herr Professor Beer, actually). In most cases we don t know the rate at which light gets absorbed for compounds in solution at different concentrations ( e value or extinction co-efficient ). We usually experimentally determine the e value via a standard curve. This is where the graph bit comes into play!! The slope/gradient or rate of a standard curve is the e value. Are you lost? Let s recap!! The e value in the equation above is commonly known as the extinction coefficient that we can use to determine the concentration of a chemical in solution., The value of e is the slope/gradient/rate of a line graph of an OD vs concentration graph i.e. a standard curve. This is why we make standard curves. The only way to find the correct rate/gradient/slope is by measuring it. Oh, now I know why we do so many standard curves in biochemistry!! So now we have a second way to determine the cordial concentration using the standard curve. The plug and chug method. It is as easy as determining the slope of the line from the standard curve and using it in the formula: OD=ec So using this method the kid s cordial had an OD of 0.8. The slope of the line is.05 so 0.8 =.05 c so c=0.8/.05 = 16% (v/v) rather than the 20% (v/v) recommended by the manufacturer. The almost the identical result as when we read it off the graph. 10

11 It is exactly the same in the lab-try it yourself John and Suzy want to measure the concentrations of a protein (Bovine Serum Albumin, BSA) in solution. After finding the proper wavelength and the ODs of 1 mg/ml, 2 mg/ml, 4 mg/ml, and 8 mg/ml BSA solutions were obtained. They then measured the ODs of two unknown BSA solutions. What were the concentrations of the protein solutions? Table1: Data collected for ODs of solutions at various BSA concentrations BSA concentration OD (280 nm) (mg/ml) Unknown Unknown First, what is e? Remember that e is the rate at which stuff in the water absorbs light. Slope = rate Find the slope of the graph. How much does the absorbance change as you go from 0 to 8 mg/ml protein? Since Beer's Law says the relationship is DIRECTLY PROPORTIONAL, then the absorbance will change 1/10 th as much from 0 to 1mg/mL as from 0 to 10 mg/ml. Answer: e = 0.3 OD /mg/ml. So far, we know that e = 0.3 OD/mg/mL. Fitting that into our equation, we get OD = 0.3 c 11

12 Now that you have e, what are the concentrations of the three protein samples (their ODs were 0.7 and1.5) you can write an equation for concentration based on absorbance. To get the equation for concentration, do this: OD = e c c = OD / e c = OD / 0.3 Use Google as a calculator! Answer: 0.7/0.3 = 2.3 mg/ml, 1.5/0.3=5 mg/ml, Summary You have learnt how to construct and use a standard curve to determine the concentration of an unknown solution. When you use a spectrophotometer, you need to know the wavelength of light your chemical of interest absorbs at. You need to determine the e value for your compound. This process of calibration is also called creating a standard curve. That is standard as in something you can measure against, and curve as in a function drawn on a graph. Once you have the standard curve, you can use it in one of two ways: Simply read the concentration from the curve itself, by measuring the OD of the unknown and using this value to find the x value. Use Beer s Law (OD = ecl) once you have determined the slope of your line on a standard curve Learning Outcomes After having completed this module you should now be able to: Describe the linear relationships between two parameters Construct and use a standard curve for determining the concentration of a unknown solution using spectroscopy Use Beer s Law and the gradient of a standard curve to determine the concentration of an unknown solution using spectroscopy. 12