Determination of Concentration

Using Spectrophotometry

Lab 8

Outline Purpose Qualitative Distinction Spectrophotometry Beer’s Law Calibration Curve Procedure Points of Interest Waste Important - printouts Next Lab Reminder

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.

A relationship can be observed between color intensity and the

concentration of a solution.

Qualitative Distinction

Chemical solutions owe their color to light-absorbing species in the solution, whether these are ions or complex molecules. For example: The blue color of today’s solution could

be due to Cu2+ (it is in fact food coloring, not Cu2+).

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 blue because the solution absorbs orange wavelengths and what’s left over appears blue.

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.

The spectrophotometer calculates the percentage of light transmitted.

It then uses an algorithm (formula) to convert percent transmittance to absorbance.

Spectrophotometry

Light Source

Wavelength Selector Sampl

eDetector

I0 I

b

Incident Light

Transmitted Light

b = 22.45 mm or 2.245 cm

Spectrophotometry

Transmittance: %T = x 100%

Absorbance: Abs = log

I

I

100

%T

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 absorb!

Calibration Curve

Plotting Abs vs. [colored solution] gives:

Abs vs. [colored solution]

y = mx + b

0

0.1

0.2

0.3

0.4

0.5

0 0.1 0.2 0.3 0.4

[colored solution], M

Ab

s

This is called a “calibration curve.”For y = m x + b

Abs = m [colored solution] + b

Abs = ε b c

After the calibration curve is established with your standard solutions, the equation is used to calculate the concentration of the unknown solution, given the absorbance of the unknown solution.

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. Using the calibration curve equation, the

concentration of the unknown solution can be calculated, given the %T or absorbance of the unknown solution:

Unk Abs = m [unk cs] + b, therefore, [unk cs] =

Unk Abs b

m

Points of Interest

MicroLAB™ colorimeter operation Your instructor will run through the

basics. Cuvet handling

Wipe down the cuvet with a KimWipe before insertion into the interface.

Safety Concerns – Food Dye

Avoid contact with skin and eyes. Do not inhale vapor or spray. Do not ingest.

Waste

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

Important:

Before you leave the lab today, print off the following:

Colorimeter spreadsheet Unknown Abs vs. [ ] graph Spectrum Profile #1

Lab 9 Reminder Lab 9 next week.