SCI110 LAB 5:

Using a Spectrophotometer to Generate a Standard Curveand Solve a Practical Problem

About this laboratory

In this lab,you will usegraduated pipettes to prepare dilutions of potassium permanganate. You will also use a spectrophotometer to build a standard curve. The latter will allow you to estimate the concentration of “mystery” solution samples.

During lab you will be evaluated on the application of skills acquired in the use of:

  • Graduated pipets to measure variable volumes
  • Organizing and generating data
  • Making appropriate lab notebook entries
  • Proper use of a spectrophotometer

After lab, you will write a laboratory report following the Laboratory Report Guidelinesthat assesses your ability to:

  • Interpret data
  • Use a standard curve to interpolate and extrapolate data.
  • Use statistics to evaluate the reliability of the data
  • Write scientifically following the conventions within the scientific community for communicating a scientific study in writing.

The Laboratory Report Scoring Rubric will be used to evaluate the ability to write scientifically. Follow the scoring rubric to ensure you have included all the required components of a laboratory report. As you are writing, evaluate the quality of what you are writing for both content and the use of college-level writing to express your ideas.

Pre Lab Activities

  1. Review the SOP on the use of graduated pipets.
  2. Review the SOP on the use of micropipettes.
  3. Review the SOP on the use of spectrophotometers.
  4. Review information on how to make a dilution.
  5. Review the PowerPoint: Excel- Preparing a Standard Curve.

Introduction

Generating a standard curve gives chemists, biologists and environmental scientists, for example, the ability to interpolate and extrapolate data to determine the concentration of an unknown solution.

The spectrophotometer is the instrument used to generate the data and designed to measure the amount of light absorbed by a solution. Using the spectrophotometer we can quantitatively measure absorbance and this information can be used to determine the concentration of the solution.

Potassium permanganate (KMnO4) is an inorganic chemical compound with interesting oxidizing properties. As a result, it is used to treat water, as a skin disinfectant or even as a treatment for some fungal infections. Aqueous solutions have an intensely pink or purple color; even diluted solutions will stain permanently oxidizable substances such as skin, paper, and clothing.

Laboratory Procedures

The lab student / technician will:

  • Prepare a dilution using a graduated pipette
  • Use a spectrophotometer to generate data
  • Calculate the concentration of a dilution
  • Use Excel to create a standard curve for each dilution set from spectrophotometer data

A. Making Dilutions

Materials:

  • 20ml of 2.00x10-3 M KMnO4standard solution
  • Distilled H2O
  • Two Mohr (graduated) pipettes and a pipette bulb[MA1]
  • 5 test tubes (20ml volume)

Procedure:

1.Properly label 5 test tubes as A, B, C, D and E.[MA2]

  1. Obtain two clean Mohr or serologicall pipettes and bulbs/pumps. One will be used for pipetting the standard solution and the other for pipetting water.
  2. Obtain the 2.00x10-3 M KMnO4standard solution.
  3. Following the SOP for Graduated Pipettes, pipet 1ml of the 2.00x10-3 M KMnO4standard solution in the A test tube, 2ml in the B test tube, 3ml in the C test tube, 4ml in the D test tube and 5ml in the E test tube.
  4. Using a clean Mohr pipette, complete the dilutions by adding 4 ml of water in the A test tube, 3ml of water in the B test tube and continue doing the appropriate dilutions.
  5. Carefully mix each dilution using a vortex mixer.

B. Using a Spectrophotometer to Generate a Standard Curve

Materials needed to generate a standard curve:

  • Spectrophotometer
  • Dilution series
  • Cuvettes
  • Computer with Excel

Procedure:

1.Follow the pre-operation SOP for spectrophotometers. Turn on the spectrophotometer; the lamp inside it will need to warm up.

2.Set the mode to Absorbance and the wavelength to 520nm.

AFTER the spectrophotometer has warmed up:

For the dilution set perform the following spectrophotometer analysis. Be sure you have an organized way to record data in the notebook. Follow all SOP’s for the proper use of the pipettes and spectrophotometer.

  1. Pipet 1-2[MA3]ml of distilled water into a clean cuvette, place it into the cell compartment of the spectrophotometer with the arrow facing forward.
  2. Close the lid and zero/blank the instrument (this subtracts any absorbance from the water allowing only absorbance of the standard at 520nm).
  3. Pipet 1-2ml of the first A dilution into a clean cuvette and replace the water with the sample in the cell compartment. Close the lid and record the Absorbance reading from the display.
  4. Repeat Step 3 for each dilution and record the data in your lab notebook making sure each sample is correctly identified.
  5. Calculate the concentration for each dilution (A – E) made.
  6. Using an Excel spreadsheet, enter the data into the spreadsheet and graph the data using an x-y scatter plot adding a trendline and R2. Make sure to label each graph and both axes.

C. Solving a Practical Problem

A pet shop owner has received two costly cichlid fish from Lake Malawi. Unfortunately, they are sick with the “white spot” or “Ich” disease. Thisprotozoan infection can be treated by immersing the animals for a few hours in a potassium permanganate solution.

If the solution is not concentrated enough, the parasite will survive and kill the fish. Lake Malawi cichlids, however, can die if immersed in a too concentrated solution of potassium permanganate. The safe range for treatment is 1.00-6.00 x10-6 M.

After mixing the purple crystals in the treatment tank, the shop-owner is having doubts about his calculations. He brings you a sample of tank water to evaluate by spectrophotometry, wondering if he should proceed with the treatment. At 520nm, your absorbance reading is 0.15. Explain how you are using your standard curve, how reliable it is to take a decision – and whether or not the fish should be put in the treatment tank.

Revised 04/2017

[MA1]Can they use either Mohr OR serological pipette? A pump is a better option than the bulb. Most of the students can’t get the accuracy needed with the bulb.

[MA2]Can we have them label them as A-E? The students get really confused as to why they can’t just use the 1/5 in the tables and on the graph.

[MA3]Depending on the cuvettes that are used this can be too much. Mike can you fill a couple cuvettes to see what an appropriate amount would be? I’m thinking I’ve used 1 mL.