pKa of Fluorescein
Lab 2: pKa of Fluorescein
Previously, we showed how different compounds absorb light. The chemical structure of a molecule determines exactly how much light it absorbs, as well as which wavelengths are absorbed. It stands to reason then, that by removing an atom from a molecule, we can change the way it absorbs light. In this experiment, we will relate these two concepts by measuring the absorbance of a molecule under acidic and basic conditions. The changing pH will allow us to find how strongly a specific hydrogen is attached to our molecule, and we will observe how the changing chemical structure affects the observed absorbance. Afterwards, using mathematical analysis, we can experimentally determine the pKa, or affinity of our hydrogen to our parent molecule.
Learning Objectives
- To understand how changes in structure can impart changes on the properties of a molecule, and
- To use various data fitting models to experimentally determine the
pKa of fluorescein
Materials
Handout 2.1: Discussion of Simple Linear Regression (PDF)
Handout 2.2: Using the LINEST Function in Spreadsheets (PDF)
- Spectrophotometer with UV lamp turned on at the beginning of lab
- Micropipettors
- pH 4 Buffer
- pH 10 Buffer
- Parafilm®
- Solutions of fluorescein SDS
Objective
To learn how to determine experimentally the pKa of fluorescein using linear, nonlinear, and 2nd derivative fitting of spectrophotometric data.
Protocol
Important: Before coming to lab, you will need to read the handout "pH,
Handout 2.3: pH,
Part I. Preparing the buffers. In medium test tubes, prepare your buffers by combining the pH 4 and pH 10 buffers as indicated in table 2.1. When you have added the solution components to a tube, tightly cover the tube with a square of Parafilm®, invert to thoroughly mix, and then label the tubes 1, 2, 3, etc. Measure and record (in your lab notebook) the pH values of the resulting buffer solutions according to the instructions provided by the TA. You can use Table 2.1 as a model. For anticipated acidic solutions, standardize your pH meter from 4 - 7; for anticipated basic solutions, standardize your pH meter from 7 - 10. Try to keep the electrode centered in the test tubes when you measure the pH of each solution.
Figure 2.1 Chemical structure of fluorescein
In a basic solution, which proton will be lost first? How do you know?
Part II. Spectrophotometric determination of the
Part III. Analyzing the data In Excel, plot as functions of pH both your
Table 2.1 Making buffers with pH values from 4 - 10
Solution | |
|
|
Total volume( |
Approx. pH | Actual pH |
1 | 200 | 0 | 1800 | 2000 | 4 | |
2 | 190 | 10 | 1800 | 2000 | 4.3 | |
3 | 180 |
20 | 1800 | 2000 | 4.6 | |
4 | 170 | 30 | 1800 | 2000 | 4.9 | |
5 | 160 | 40 | 1800 | 2000 | 5.2 | |
6 | 150 | 50 | 1800 | 2000 | 5.5 | |
7 | 140 | 60 | 1800 | 2000 | 5.8 | |
8 | 130 | 70 | 1800 | 2000 | 6.1 | |
9 | 120 | 80 | 1800 | 2000 | 6.4 | |
10 | 110 | 90 | 1800 | 2000 | 6.7 | |
11 | 100 | 100 | 1800 | 2000 | 7 | |
12 | 90 | 110 | 1800 | 2000 | 7.3 | |
13 | 80 | 120 | 1800 | 2000 | 7.6 | |
14 | 70 | 130 | 1800 | 2000 | 7.9 | |
15 | 60 | 140 | 1800 | 2000 | 8.2 | |
16 | 50 | 150 | 1800 | 2000 | 8.5 | |
17 | 40 | 160 | 1800 | 2000 | 8.8 | |
18 | 30 | 170 | 1800 | 2000 | 9.1 | |
19 | 20 | 180 | 1800 | 2000 | 9.4 | |
20 | 10 | 190 | 1800 | 2000 | 9.7 | |
21 | 0 | 200 | 1800 | 2000 | 10 |
Some caveats: When you fit these data, let the
Image 2.1 Fluorescein under UV light
Why does fluorescein "glow" under ultraviolet light? How is this different from the color we see when we look at fluorescein under visible light?