Determination of Concentration Using Spectrophotometry Lab 11

Outline Purpose Qualitative Distinction Spectrophotometry Beer’s Law Calibration Curve Procedure Points of Interest Waste Safety Concerns

Purpose This experiment demonstrates the linear relationship between the absorbance and the concentration of a colored solution. Beer’s Law will be used to determine the concentration of a sample for which the concentration is unknown.

Qualitative Distinction Chemical solutions owe their color to light-absorbing species in the solution, whether these are ions or complex molecules. For example: The green color of today’s solution could be due to Ni2+ (it is in fact food coloring, not Ni2+). The color of cranberry juice is due to anthocyanins.

Qualitative Distinction The color we see is the color of light transmitted, or “getting through” the solution. We see the color of light “left over” after some wavelengths have been absorbed. Today we will see green because the solution absorbs red wavelengths (650 nm) and what’s left over appears green.

Qualitative Distinction The intensity of the color is proportional to the concentration of the absorbing chemical species. We can do a qualitative distinction by eye, or We can do a quantitative measurement by spectrophotometry.

Spectrophotometry Spectrophotometers shine a light through the sample. Some detect light from only one wavelength and some detect light from all visible wavelengths. The MicroLAB™ spectrophotometer emits and detects light from sixteen different wavelengths. The light that is not absorbed by the sample, but transmitted instead, hits a light detector.

Spectrophotometry Transmitted Light Incident Light I0 I Light Source Wavelength Selector Sample Detector b b = 22.45 mm or 2.245 cm

Spectrophotometry The spectrophotometer calculates the percentage of light transmitted. It then uses an algorithm (formula) to convert percent transmittance to absorbance. Transmittance: %T = x 100% Absorbance: Abs = log

Standard Solutions Today you will make up several solutions of known concentration. There is a known relationship between the concentration of samples and the absorbance of samples at a given wavelength. This relationship is commonly referred to as Beer’s Law.

Beer’s Law Abs = ε b C where Abs = absorbance (no units) ε = molar absorptivity (M-1cm-1) b = path length (cm) C = concentration (M) These measurements all take place at the wavelength at which our absorbing species absorbs light. By plotting Beer’s Law on a graph, we can establish a calibration curve with our standard solutions.

Plotting Beer’s Law If we were to plot an extended range of Absorbance vs. [Colored Solution], M we would notice a linear response in a limited region of the plot only. (Why?) We pick this region for our calibration curve.

Calibration Curve Plotting Abs vs. [Colored Solution], M yields: This is called a “calibration curve.” For y = m x + b Abs = m [colored solution] + b Abs = ε b c Note: The “b” in y = mx + b refers to the y- intercept of the graph. The “b” in Beer’s Law refers to the path length light has to travel through your sample. DO NOT confuse them!

Calibration Curve After the calibration curve is plotted with your standard solutions, the calibration curve equation can be used to calculate the concentration of an unknown solution, IF: the unknown solution contains the same color-absorbing species as your known (“standard”) solutions the absorbance of the unknown solution is known or can be established the absorbance of the unknown solution falls within the absorbance range of your standard solutions (otherwise any concentration calculations are invalid)

Questions What should you do if your unknown solution has an absorbance value that falls above your calibration range? What should you do if your unknown solution has an absorbance value that falls below your calibration range?

Procedure To obtain the calibration curve: Prepare a series of colored solutions of known concentration (“standards”). The absorbance of each solution is measured. Absorbance versus concentration is plotted. To find the unknown concentration: Using the calibration curve equation and the absorbance of the unknown solution, the concentration of the unknown solution can be calculated: Unk Abs = m [Unk cs] + b, therefore, [Unk cs] =

Points of Interest MicroLAB™ colorimeter operation Cuvet handling Your instructor will run through the basics. Cuvet handling Wipe your cuvets down with a dry KimWipe before insertion into the interface. Blank Solution Selection See the next slide

Blank Solution A blank solution is used before the first sample is inserted into the spectrophotometer. The blank solution is most always the solvent of your solutions. The solvent may contain species that can absorb light at the same wavelength as the analytical wavelength for your analyte. The spectrophotometer is zeroed out with the blank solution (Abs = 0 or %Trans = 100%) The blank solution therefore corrects for the matrix effects of the solvent.

Safety Concerns Reagent: Health Considerations: Food coloring Avoid contact with skin and eyes. Do not inhale vapor or spray. Do not ingest.

Waste All waste solutions can be disposed down the sink, with plenty of water.

Next Week – Skill Evaluations Come prepared for the quiz. Study pages 289 – 310 carefully. Bring your lab manual and goggles. Submit your Lab 11 Report