We now know how to analyze pure compounds, but what if we have a mixture? Spectrophometry becomes quite complex when dealing with multiple species of compounds at once. In order to purify a compound we can separate if from a mixture based on its intrinsic chemical properties. Remember that fluorescein is negatively charged at a pH above pKa of the carboxyl group. We can take advantage of this fact and use its attraction to positive charges to separate it from other molecules. In ion-exchange chromatography, we will use a stationary phase with a positive charge, allowing negatively charged molecules to bind and positively charged species to flow through. We can then disrupt this interaction and retrieve our now-purified molecule, and use spectrophotometric analysis of our purified fractions to determine how well we were able to separate our molecules.
We are all well aware of the composition of the world -atoms form molecules, compound become more complex, and the organization of these atoms into materials with unique structures is what brings about life. As scientists though, we must study these substances , which presents a challenge. How do we study something so incredibly small? One of the simplest methods is spectrophotometry. Different molecules will interact with light in different ways. By studying this, we can quantitatively say both how much light a compound absorbs as well as what kind of light. Certain functional groups tend to absorb light at certain wavelengths, giving "peaks" to the spectrum of light absorption. This lab demonstrates basic principles of absorbance, measured using spectrophotometers.
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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.