CHEM 1001 Lab 4: Spectrophotometry I - Protocol PDF

CHEM 1001 Lab 4: Spectrophotometry I: Intro, Wavescans & Calibration Curves A spectrophotometer is a piece of equipment used to measure the amount of light that a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a dete...

CHEM 1001 Lab 4: Spectrophotometry I: Intro, Wavescans & Calibration Curves A spectrophotometer is a piece of equipment used to measure the amount of light that a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. The beam of light consists of a stream of photons. When a photon encounters an analyte molecule (the analyte is the molecule being studied) there is a chance the analyte will absorb the photon. This absorption reduces the number of photons in the beam of light, thereby reducing the intensity of the light beam. First, the intensity (number of photons per second) of light (I0) passing through a blank is measured. The blank is a solution that is identical to the sample solution except that the blank does not contain the solute that absorbs light (i.e. the blank contains everything but what you are trying to measure). The blank is a type of control and is used to set a baseline for your sample readings. Second, the intensity of light (I) passing through the sample solution is measured. In practice, instruments measure the power rather than the intensity of the light. The power is the energy per second, which is the product of the intensity (photons per second) and the energy per photon. Third, the experimental data is used to calculate one of two quantities: the transmittance (T) or the absorbance (A). The transmittance scale is 0 – 100% whereas the absorbance scale is 0 – 2 absorbance units and is inversely proportional to the transmittance scale (i.e. 100% (T) = 0 (A)). The transmittance is simply the fraction of light in the original beam that passes through the sample and reaches the detector. The remainder of the light, 1 - T, is the fraction of the light absorbed by the sample. (Do not confuse the transmittance with the temperature, which often is given the symbol T.) In most applications, one wishes to relate the amount of light absorbed to the concentration of the absorbing molecule and therefore the absorbance rather than the transmittance is most useful for this purpose. If no light is absorbed, the absorbance is zero (100% transmittance). Each absorbance unit corresponds with an order of magnitude in the fraction of light transmitted. For A = 1, 10% of the light is transmitted and 90% is absorbed by the sample. For A = 2, 1% of the light is transmitted and 99% is absorbed. For A = 3, 0.1% of the light is transmitted and 99.9% is absorbed. Beer’s Law Beer’s Law (also referred to as the Beer-Lambert law) states that the concentration of a solution is directly proportional to the amount of light that it absorbs. * Note: Beer’s Law is not obeyed at very high or very low concentrations (non-linear relationship). Beer’s Law is written as: A=εlc where: A = Absorbance (no units) ε = Molar absorptivity (L g-1cm-1) l = Path length (cm) c = concentration of compound in solution (g L-1) 1  CHEM 1001 Lab 4: Spectrophotometry I: Intro, Wavescans & Calibration Curves Wavescans (otherwise known as: Absorption Curves) If light of different wavelengths is passed through a coloured solution, some wavelengths will be absorbed more than others. The amount of light absorbed can be plotted and this is called an ‘absorption’ curve. From this curve, we can calculate the wavelength that shows the maximum absorbance for the solution. Calibration Curves (otherwise known as: Standard Curves) Plotting the absorbance readings and concentrations of various coloured solutions yields a calibration curve (also known as a standard curve). This curve can then be used to determine the concentrations of unknown samples. The figure shown below is a plot of concentration vs. absorbance for calibration standards of increasing concentration. Notice, as the standard concentration increases (darker colour) the absorbance readings increase. Most coloured solutions obey Beer’s Law which means that the concentration of a coloured solution is directly proportional to the amount of light that it absorbs. In today’s lab you will be reading the absorbances of various red dye solutions. Once collected, these absorbances will be plotted against concentrations to produce a calibration curve as shown in the figure below. Most coloured solutions obey Beer’s Law which means that the concentration of a coloured solution is directly proportional to the amount of light that it absorbs. 2  CHEM 1001 Lab 4: Spectrophotometry I: Intro, Wavescans & Calibration Curves MATERIALS:  10% w/v stock solution of Red Dye #40  Distilled water  Vortex mixer  Spectrophotometer  Pipettes (2x 5 mL Mohr or Serological)  13x100mm test tubes PROTOCOL: Part I: Standard Preparation (per bench pair) 1. Calculate the volume of 10% (w/v) stock dye solution (C1) required to produce the concentrations (C2) outlined in the table below.  You will be making the following concentrations (C2): 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, and 1% individually from the 10% (w/v) stock dye solution (C1).  The volume (V2) for each is 5mL.  Use C1V1=C2V2 to calculate the volume of stock (V1) required for each solution  Fill in the following chart to help set up all the dilutions. Calculations: Red Dye Concentration (C2) Volume of stock red dye (V1) Volume of distilled water (5 – V1) Total Volume (V2) (% (w/v)) (mL) (mL) (mL) 1.0 0.5 4.5 5 2.0 5 3.0 5 4.0 5 5.0 5 6.0 5 7.0 5 8.0 5 9.0 5 10 5 3  CHEM 1001 Lab 4: Spectrophotometry I: Intro, Wavescans & Calibration Curves 2. Label ten individual test tubes with one of the following concentrations: 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%  When labeling test tubes be sure to write close to the top of the tube, so it doesn’t interfere with the spectrophotometer’s beam of light. 3. Using a 5 mL Mohr or Serological pipette, add the appropriate amount of dH 2O (listed in the table above) to the corresponding test tubes.  Be sure to add the appropriate amount to the correct tube.  Add the dH2O to all test tubes prior to adding the 10% red dye stock solution. 4. Using a separate 5 mL Mohr or Serological pipette, add the appropriate amount of 10% (w/v) stock dye solution (listed in the table above) to the corresponding test tubes.  Be sure to add the appropriate amount to the correct tube. 5. Mix each solution by covering the end of the tube with your finger and vortex for approximately 5 seconds. 6. Label a 13x100 test tube with: BLANK. 7. Using the pipette that you used for the water above, add 5mL of distilled water to this BLANK test tube – this is your BLANK for zeroing the spectrophotometer. 4  CHEM 1001 Lab 4: Spectrophotometry I: Intro, Wavescans & Calibration Curves PART II: Wavescan  For your absorbance curve, you will be using your 5% red dye solution prepared in the previous section. 1. Each time you use a spectrophotometer, record the manufacturer, model and SLC equipment number Spectrophotometer Example Spectrophotometer Used Manufacturer: Agilent Model: Cary 60 SLC Equipment Number: SLC-1234 2. Prior to analysis by spectrophotometry, ensure that your solutions are thoroughly mixed (vortexed) and the outside of the test tube (or cuvette) Is wiped clean with a Kimwipe 3. Set the wavelength on the spectrophotometer to: 400nm. 4. Place the BLANK test tube into the spectrophotometer and close the lid. 5. Set the spectrophotometer’s absorbance to zero by pressing the button labelled: 0 ABS or 100%T. 6. Replace the BLANK test tube with your 5% red dye solution and close the lid. 7. Record the absorbance of the red dye solution at the current wavelength in the table below. 8. Set the wavelength to 410nm and repeat steps 4 – 7 and record the absorbance in the table. 9. Continue as above at 10nm intervals up to 650nm  Remember to blank the spectrophotometer each time you change the wavelength. Wavelength (nm) Absorbance Wavelength (nm) Absorbance 400 530 410 540 420 550 430 560 440 570 450 580 460 590 470 600 480 610 490 620 500 630 510 640 520 650 5  CHEM 1001 Lab 4: Spectrophotometry I: Intro, Wavescans & Calibration Curves PART III: Calibration Standard Curve 1. Adjust your spectrophotometer to the wavelength that showed the maximum absorbance for your solution from your Wavescan (Absorption Curve) (PART II). 2. Blank/zero your spectrophotometer Using your distilled water blank from Part I  You do not need to blank your spectrophotometer between samples because you are not changing the wavelength. 3. Read all ten (10) concentrations and record each absorbance value in the table below. Red Dye Concentration Absorbance (% (w/v)) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10 WASTE DISPOSAL Waste Type Disposal Location/Container Liquid Waste container in the fumehood Glass Test Tubes Dispose in the bucket labeled: ‘Broken glass Only’ 6  CHEM 1001 Lab 4: Spectrophotometry I: Intro, Wavescans & Calibration Curves Part IV: DATA ANALYSIS 1. Refer to the ‘Plotting with Excel’ and ‘Graphing Marking Scheme’ documents provided for instructions and expectations when using Excel to make graphs. 2. Using the data from Part II, produce an absorption curve for the 5% red dye solution by plotting Absorbance (y-axis) versus Wavelength (x-axis). 3. Using the data from Part III, produce a calibration curve for the red dye solutions by plotting Absorbance (y-axis) versus Concentration (x-axis). Lab #4 – Spectrophotometry I – Results Summary /12 marks  One Blackboard submission per group  Due date on Blackboard  Refer to the ‘Submitting Assignments Via Blackboard’ instructions Results Summary Requirements  Submit one PDF document containing two pages.  Page 1: o Wavescan: Plot of wavelength vs absorbance for your red dye solution (5 marks) o Summary table of your wavelength and absorbance readings (1 mark)  Page 2: o Calibration Curve: Plot of calibration concentration vs absorbance for your red dye solution (5 marks) o Summary table of your calibration concentration and absorbance readings (1 mark) 7