9: Dilutions and Spectrophotometry
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- Understand the concept and purpose of serial dilution.
- Learn how to perform a 2-fold serial dilution accurately.
- Recognize the role of spectrophotometry in measuring solution concentrations.
- Identify the function of a blank in spectrophotometric analysis.
- Predict how dilution affects absorbance readings in a spectrophotometer.
Before coming to the lab, read the assigned chapter on dilutions and spectrophotometry. Pay close attention to:
- The mathematical principles behind serial dilution.
- How dilution factors are calculated.
- The operation and principles of spectrophotometry.
- The relationship between absorbance and concentration (Beer’s Law).
- Why is serial dilution an important technique in laboratory science?
- If you begin with a 0.5M solution and perform a 1:2 dilution three times, what will be the final concentration?
- How does a spectrophotometer measure concentration, and why do we use a blank solution?
- Predict how the absorbance values will change as the concentration decreases in your dilution series.
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Chapter Nine
Dilutions and Spectrophotometry
Now, we'll focus on two key techniques: serial dilution and spectrophotometry. These methods are critical for the precision in the lab! By carefully diluting solutions and using spectrophotometric analysis, scientists can precisely determine concentrations. This information is vital for various applications, from pharmaceutical development to environmental monitoring. As we explore these methods further, we uncover their importance in scientific research and experimental analysis.
Well, let's start with dilution. The purpose of dilution is pretty straightforward: it's all about systematically reducing a solution's concentration to just the right level. This is essential because many reagents can be too high in concentrations, which can lead to inaccuracies or adverse effects if used directly. Imagine trying to measure out medication in a hospital without diluting it properly – it could lead to serious overdoses and harm to patients. That's why dilution is our trusty ally in ensuring accuracy and safety in various applications, from pharmaceutical development to environmental monitoring. Now, let's talk about serial dilution. This technique involves stepwise dilution to create a series of decreasing concentrations, each one reducing the concentration of the solution even further. When we visualize serial dilution, we see this beautiful gradient forming – kind of like watching colors fade away gradually, just like in Figure 9.1. This process isn't just for show, though; it's how we create solutions with pinpoint precision for all sorts of experiments and analyses in the lab.
Let's break down the steps of serial dilution using a practical example:
- Begin with a known volume of a concentrated dye solution, such as 1 mL, in a labeled test tube.
- Add 9 mL of solvent (typically water) to the original solution and thoroughly mix. This results in a 1:10 dilution, meaning the concentration is one-tenth of the original.
- Take 1 mL from the 1:10 dilution and add it to another 9 mL of solvent. Now you have a 1:100 dilution.
- Repeat this process, diluting the solution by a factor of 10 each time.
- After multiple dilutions, you'll have a series of test tubes with decreasing concentrations, forming a concentration gradient from the original to highly diluted solutions.
This stepwise dilution process allows scientists to create solutions with precise concentrations suitable for various experiments, ensuring accurate and reliable results in analytical chemistry.
One way we use serial dilution is to compare an unknown solution to a concentration gradient. For example, let's consider a solution of red dye. By observing where it falls within the color gradient, we can estimate its concentration. This method isn't always precise, though, so we often need more accurate analytical techniques. Say you're experimenting to determine the concentration of a sugar solution. You prepare a series of sugar solutions with known concentrations using serial dilution. Then, you measure the absorbance of each solution using a spectrophotometer. By plotting the absorbance against concentration, you create a standard curve. When you measure the absorbance of an unknown sugar solution and compare it to the standard curve, you can accurately determine its concentration. Now, let's put this into context with a real-lab scenario. Imagine a researcher needs to quantify the concentration of an enzyme or molecule in an assay. By using serial dilutions, they can create a range of concentrations. Then they can compare their unknown enzyme concentration to this gradient. This approach is both cost-effective and efficient, minimizing waste while ensuring precise measurements.
Spectrophotometry is a powerful analytical technique used to quantify substances in solutions. Spectrophotometers operate on the principles of transmission and absorbance. They measure the amount of light that passes through a sample and the extent to which it absorbs light, respectively. Absorbance values, typically represented as peaks at specific wavelengths, directly correlate with the concentration of the substance being analyzed. Now, let's break down how spectrophotometry works. Spectrophotometers emit light of a specific wavelength through a sample solution. A photodetector then detects the amount of light absorbed by the solution. The instrument measures the absorbance of the solution, which is the logarithm of the ratio of incident light intensity to transmitted light intensity. In essence, spectrophotometry is at the core of quantitative analysis, allowing scientists to elucidate the presence and concentration of dissolved chemicals in solutions.
Consider an experiment where you're analyzing the concentration of a protein solution. You prepare a series of protein solutions with known concentrations and measure their absorbance at a specific wavelength using a spectrophotometer. The higher the protein concentration, the more light is absorbed, resulting in a higher absorbance value. By plotting absorbance against concentration for the standard solutions, you create a standard curve. Then, when you measure the absorbance of an unknown protein solution, you can accurately determine its concentration using the standard curve.
Next, we have to discuss the role of the blank solution. This blank, typically made of the solvent without any analyte, helps us account for any interference from the solvent or other components. By measuring the absorbance of the blank, we ensure accurate measurements of the target substance without any unwanted influences.
Now, how do you interpret unknown concentrations? To determine the concentration of an unknown solution, we use serial dilution to create a standard curve. This involves preparing a series of solutions with known concentrations and measuring their absorbance. By establishing a linear relationship between concentration and absorbance, we create a reference standard curve. Then, we compare the absorbance of the unknown solution to this curve, allowing us to precisely determine its concentration.
In summary, serial dilution and spectrophotometry are fundamental tools in biotechnology, enabling precise quantification of substances in solutions. Whether in pharmaceutical assays or environmental monitoring, these techniques empower scientists to uncover concentration mysteries with unparalleled accuracy.
LAB ACTIVITY - PART 1 - Serial Dilution
Materials
- One Cuvette holder (Grainger, 12C181)
- Six Uvettes (Fisher Scientific, E4099100008)
- 500mL of 0.5M of Copper (II) Sulfate (Flinn Scientific, C0108)
- One p1000 Micropipette.
- One box of p1000 Micropipette tips
Methods for 2-Fold Serial Dilution:
Today we will perform serial dilutions using copper (II) sulfate solution. This process is vital for various applications in scientific research, including accurately measuring the concentrations of substances in solution.
- Take six cuvettes and label them from 1 through 6.
- In cuvette 1, we'll add 2 milliliters (mL) of the 0.5M copper (II) sulfate solution.
- For cuvettes 2 through 6, we'll add 1 mL of water to each.
- Now, we'll start diluting our solution:
- Take 1 mL of the solution from cuvette 1 and transfer it to cuvette 2. Repeat this transfer from cuvette 2 to 3, 3 to 4, and 4 to 5.
- We won't transfer anything to cuvette 6. This cuvette serves as our blank.
- Check if each cuvette has a final volume of 1 mL to maintain consistency.
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LAB ACTIVITY - PART 2 - Spectrophotometry
Materials
- One Spectrophotometry
- Samples in Uvettes
- One Cuvette holder (Grainger, 12C181)
- One p1000 Micropipette.
- One box of p1000 Micropipette tips
Spectrophotometry:
Now, let's move on to the next step of our experiment, where we'll utilize spectrophotometry. This tool allows us to measure the amount of light absorbed by our samples, giving us valuable insights into their properties.
- Set the spectrophotometer to read at a wavelength of 662 nanometers (nm). This specific wavelength is the optimal absorbance for measuring Copper (II) Sulfate.
- Examine the spectrophotometer and identify where the light source.
- Check each cuvette to ensure that the clear side faces the light source.
- Start by placing cuvette 6, our blank, into the cuvette reader.
- Blank the spectrophotometer using cuvette 6 as our reference point.
- Next, measure the absorbance of cuvettes 1 through 5, one by one.
- As you measure each cuvette, carefully record the absorbance values.
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LAB ACTIVITY - PART 3 - Standard Curve
Standard Curve:
Now that we've completed the serial dilution and obtained absorbance values using spectrophotometry, it's time to take our analysis to create a standard curve. This curve will be generated using Excel spreadsheets.
- Input your data, including the concentration values and their corresponding absorbance values, into a Google Sheet. It's crucial to include the label above these values with "Concentration (M)" and "Absorption (662nm)" to avoid graphing complications.
- Highlight the entire dataset, including both values and labels.
- Next, navigate to the "Insert" dropdown menu and select "Chart." This will bring up both the chart and the chart editor options.
- In the chart editor, under "Setup", ensure the chart type is set to “scatter chart”.
- Moving to the "Customize" section, locate the "Series" option. Here, we'll select "Trendline" to add a trendline to our graph.
- Scroll down to the "Label" section and check the box to show the R2 value. This value indicates how well our data fits the trendline, serving as a measure of the reliability of our results.
- Switch the option under "Label" to "Use Equation." This will display the equation of the trendline, which follows the form y = mx + b. This equation will help us determine the concentration of unknown samples.
- See the graph:
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LAB ACTIVITY - PART 3 - Determining Concentration
Determining the Unknown Concentration:
Now that we've established our chart with the equation and R2 value, let's dive into interpreting our data and determining unknown concentrations.
- First, let's observe the R2 value. This value indicates how well our data fits the trendline. The closer it is to 1, the more reliable our results are. Typically, we trust the graph if the R2 value is above 0.95. If it falls below this threshold, it’s recommended to consider redoing the serial dilution and make changes to your pipetting method.
- Next, let’s apply the Equation. Let's say our example equation is y = 0.3x + 0.1, where y represents absorbance and x represents concentration. Suppose we measured an unknown Copper (II) sulfate solution via spectrophotometry and obtained an absorbance value of 0.5. Since absorbance is graphed on the Y-axis and concentration is graphed on the X-axis, we'll plug in 0.5 for Y in our equation: 0.5 = 0.3x + 0.1.
- Solving for x, we find that x = 1.67 M. This is our unknown concentration. We can apply this same equation to determine the concentrations of other unknown samples, providing valuable insights into their composition.
LAB ACTIVITY - PART 4 - Dilution Maths
Alright, let's dive into dilution math and understand how to calculate the volumes of stock solution and water needed to achieve the desired concentration. Imagine you have purchased a therapeutic solution with a stock concentration of 100 grams per milliliter (g/mL). However, through previous data analysis, you've determined that the most effective therapeutic concentration is 1 g/mL. Now, the question is: How can you create more solutions at this optimal concentration when you only have the stock solution at 100 g/mL available?
First, let's discuss the formula we'll be using: C1V1=C2V2, where C1 and C2 are the initial and final concentrations, respectively, and V1 and V2 are the initial and final volumes, respectively. Now, let's say we purchased 10 mL of a therapeutic stock solution with a concentration of 100g/mL, and we want to dilute it to a final concentration of 1g/mL. Our initial volume V1 is 10mL, and our final volume V2 is what we're trying to find. So, let's plug the numbers into our formula: C1V1=C2V2
(100g/mL)×(10mL)=(1g/mL)×V2
Now, we can solve for V2, which should be 1000mL total volume. So, we need to add approximately 990mL mL of water to dilute our 10 mL of stock solution to achieve a final volume of 1000mL and the final concentration of 1g/mL.
Now, let's move on to Example 2: Let’s say we have a therapeutic stock solution with a concentration of 100g/mL, but we only need a volume of 200mL with a concentration of 1g/mL. We'll use the same formula, C1V1=C2V2, but we need to find V1 this time. V1 is the volume of the stock solution we'll use. So, let's plug the numbers into our formula: C1V1=C2V2
(100g/mL)×V1 =(1g/mL)×200mL
Now, we can solve for V1, which should be 2mL total volume. So, we need to use 2 mL of the stock solution and add 198 mL of water to achieve a final volume of 200 mL with a concentration of 1g/mL.
It's important to understand why we might choose bulk dilution or aliquots. In bulk dilution, like in Example 1, we dilute the entire stock solution to save time and convenience. However, in aliquot dilution, like in Example 2, we dilute smaller portions to avoid denaturation. This is because as some molecules become more diluted, they can become less stable, so diluting smaller portions helps maintain stability and long-term storage of our chemical stocks and reagents.
Go ahead and practice answering the problem sets below with an activity:
Materials
- Obtain three 50-mL beakers and fill it with 5mL of 0.5M CuIISO4
- Obtain one 50-mL beaker and fill it with 50mL of 0.5M CuIISO4
- Obtain one larger beaker and fill it with 500mL of water.
Practice Problem #1: How much water must you add to the stock to obtain a new final concentration?
STOCK
- You are given three beakers containing 5mL of 0.5M CuIISO4.
Final product
You want to create three different concentrations from these stocks:
- 50mM CuIISO4
- 100mM CuIISO4
- 250mM CuIISO4
Practice Problem #2: How much stock and water must you mix to obtain a new final concentration?
STOCK
- You are given one beaker containing 50mL of 0.5M CuIISO4.
Final product
You want to create three different concentrations with a total volume of 50mL from the stock solution:
- 50mL of 50mM CuIISO4
- 50mL of 100mM CuIISO4
- 50mL of 250mM CuIISO4
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LAB ACTIVITY - PART 5 - Application of Serial Dilution
Today we're going to perform serial dilutions using copper (II) sulfate solution. We'll start with a 2-fold dilution in six cuvettes, followed by a linear dilution in ten cuvettes.
Materials
- Microcentrifuge tubes
- One Cuvette holder (Grainger, 12C181)
- Six Uvettes (Fisher Scientific, E4099100008)
- 500mL of 0.5M of Copper (II) Sulfate (Flinn Scientific, C0108)
- One p1000 Micropipette.
- One box of p1000 Micropipette tips
Procedures:
- Get together with your group and decide on the diluted concentration of Copper (II) Sulfate. It should be between 100mM to 250mM. We'll need just 1mL of the solution.
- Write down the concentration you've chosen
- Dilute your solution and then measure its absorbance in the box below:
Chosen Concentration (M) | Expected Absorbance |
For the remainder steps, you will conduct a 2-fold serial dilution
- We'll begin with a 0.5M copper (II) sulfate solution and dilute it in deionized water. Each subsequent cuvette will be diluted two-fold from the previous one.
- Label seven (7) cuvettes from 1 through 7.
- Add 2mL of 0.5M copper (II) sulfate solution to cuvette 1.
- Add 1mL of water to cuvettes 2 through 7.
- Transfer 1mL of solution from cuvette 1 to cuvette 2, then from 2 to 3, 3 to 4, 4 to 5, and 5 to 6.
- Discard the 1mL from cuvette 6.
- Do not transfer any solution to cuvette 7, as it serves as a blank.
- After completing the dilution series, we'll analyze the resulting solutions using spectrophotometry, reading at 662nm, to generate graphs. Record your data using the table below:
Standard Curve for Cu(II)SO4
Concentration (M) | Absorbance (Choose measurements between 600nm - 700nm) |
0.500 | |
0.250 | |
0.125 | |
0.063 | |
0.031 | |
0.016 |
- We'll calculate the R-squared (R2) values to assess the linearity of our dilution series. Finally, we'll plug in the“Expected Absorbance” value into the y=mx+b equation from our graphs to determine the concentration by solving for x.
- We'll take the observed concentration value from the graph and compare that to your Chosen Concentration from the above table and solve to percent error:
- Percent Error = ((|Chosen Concentration - observed Concentration) / Chosen Concentration) x 100%
- Was your calculated concentration similar to your Chosen Concentration of Copper (II) Sulfate?
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LAB ACTIVITY - PART 6 - Skill Evaluation 2 (20pts)
The instructor will create a diluted UNKNOWN solution, ranging between 100mM to 250mM
For this skill evaluation, you will conduct a 2-fold serial dilution.
You will record two separate independent duplications for this run as only the best result will be input into your grade
- We'll begin with a 0.5M copper (II) sulfate solution and dilute it in deionized water. Each subsequent cuvette will be diluted two-fold from the previous one.
- After completing the dilution series, we'll analyze the resulting solutions using spectrophotometry, reading at 662nm, to generate graphs.
- We'll calculate the R-squared (R2) values to assess the linearity of our dilution series.
- Finally, we'll measure the absorbance of the diluted UNKNOWN solution from the instructor.
- We'll use the equation from our standard curve graphs to determine the concentration of the diluted UNKNOWN solution.
- We'll take the observed calculated concentration value and report that to the instructor
- The instructor will compare that to the expected value and solve to percent error:
- Percent Error = ((|Expected Value - Observed Value|) / Expected Value) x 100%
Grading rubric:
- The instructor will grade you based on your percent error. Only the lowest percent error from your two duplicate results will be part of your grade
- For example, if you obtained 8% error on one experiment and 5% error on another experiment, the instructor will only record the 5% error as part of your grade
- Grade calculation: if your percent error was 5%, the final grade will be calculated as: 20 - (5) = 15 points.
- A bonus of 2 points of extra credit will be given to any individual with a percent error under 1%.
- If both duplicates were under 1%, then you will be granted 4 additional points to your final grade.
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- Summarize the key steps performed in the lab for both serial dilution and spectrophotometry.
- Report the absorbance values obtained for each dilution.
- Discuss any trends observed in the relationship between concentration and absorbance.
- Based on your absorbance readings, create a standard curve by plotting concentration vs. absorbance.
- How well did your data align with Beer’s Law? Explain any deviations.
- If an unknown sample had an absorbance reading of 0.35 at 600 nm, use your standard curve to estimate its concentration.
- What challenges did you encounter when performing the dilutions? How did you overcome them?
- How does understanding dilution and spectrophotometry apply to real-world laboratory settings?
- What improvements could be made to increase the accuracy of your experiment