If you were looking for the brotherhood of man...

No, not Brotherhood of Man, winners of Eurovision 1976 (you've got to admit that the tune is catchy), but the singular conncetion between all people, old or young, black or white, from the north or the south, you might look to oxidative metabolism as that connection. From one man to another and, as Sir Hans Kreb's said (who can argue with a knight?), from man to microbes, everyone does it. And does it more or less the same.

If it is so universal, what exactly is intermediary metabolism?

Have you eaten?

Why do I ask? You know the old adage

That's it, your diet is used to build the molecules that make your or oxidized for the extraction of energy to power what those molecules (and you) do.

But the beauty of intermediary metabolism is that energy is released not in one large bang (even Wikipedia references spontaneous human combustion as psuedoscience), but by a large number of small reactions, each involving managable amounts of energy. Gentle changes, no booms.

While intermediary metabolism is a large subject, today we will consider the citric acid cycle (the description of which led to the awarding of a Nobel Prize to Hans Krebs).

Alright, I know, an intense introduction, but let me briefly explain. At the top is pyruvate, a product of sugar decomposition (the pathway producing pyruvate, not pictured above, is glycolysis, the splitting of glucose), which combines (following decarboxylation) with an organic acid to form citric acid. And elucidation of that reaction was the key step in unifying the connection of carbohydrates to the many organic acids (citric, isocitric, α–ketogluteric, succinic, fumaric and oxaloacetic).

Other interesting things to note, three molecules of CO2 are released. Pyruvate is a three carbon molecule. Where do you make the carbon dioxide you exhale—in or around the citric acid cycle.

With oxidation to carbon dioxide, there must be matching reductions. Look for formations of NADH which feed the electron transport chain.

And don't worry, you can still make a number of products from the intermediates. Amino acids are the first that come to mind.

Our quest today is citric acid. Certainly it is a key metabolyte, but it is also very important commercial product. Citric acid is a key acidifier in a number of food products. The ingredients of a popular soda product (available in the cafeteria) are below.

Fizzy water, sugar and citric acid are the first three—wet, sweet and tart, the holy trinity of Sprite. Do note that near the end of the list is the salt of the conjugate base of citric acid, sodium citrate. Weak acid, congugate base ... everyone is thinking pH buffer, right?

If you were not allowed to have soda–pop as a child (no shame), maybe your treat beverage was Kool-Aid (made with less than one cup of sugar, of course). Guess what the first ingredient is in the unsweetened Kool-Aid packet (answer at kraftheinz.com).

Now, I've chosen to start with flavored beverages because more than half of the annual production of citric acid is used as acidifiers in beverage production. But it's not just drinks. You'll find a bit of citric acid in most prepared foods which have a touch of tart ... yes, you'll even find it in strawberry Pop-Tarts (but not brown sugar cinnamon).

So, what is citiric acid?

If you look left to right, the core of citric acid is 2–hydroxypropane (three carbons with an -OH on the central one). On each of the three carbons is one carboxylic acid (-COOH). So, citric acid is a six carbon molecule in which three of those carbons are carboxylic acids. You can see why it is a favorite molecule to use to add the tart, acidic flavor to beverages and foods.

The other thing that will become important later is that the citric acid cycle (above) is also referred to as the tricarboxylic acid cycle due to the first couple molecules which have three carboxylic acids. Some call it the CAC, some call it the TCA. They are both talking about the same thing. But that usually happens in biochemistry—the same thing gets a different name for each context in which it is discovered.

The most general approach is to precipitate calcium citrate, using calcium hydroxide to both titrate citric acid to citrate (as it is a strong base) and also precipitate citrate as calcium citrate (not very soluble in water). The major challenge with this approach is that Ca(OH)2 is not particularly soluble in water. To keep it smooth in lab, we'll first titrate citric acid and then precipitate with a calcium salt. The (unbalanced) reactions look like this.

$$C_6H_8O_7 (aq) + NaOH (aq) \rightarrow Na_3C_6H_5O_7 (aq) + H_2O~~~~~~~~~~~~~~~~~~(1)$$ $$Na_3C_6H_5O_7 (aq) + CaCl_2 (aq) \rightarrow Ca_3(C_6H_5O_7)_2 (s) + NaCl (aq) ~~(2)$$

Alright, with a scheme to precipitate calcium citrate, all we need is a good source. We'll go with the original source—citrus fruit (Hmm...wonder why they have that name?). Oranges are nice, but too sweet. Grapefruit are large, but they still have a bit of sweetness (honest). Now which citrus fruit would you be most disinclined to simply peel and eat ... lemons. Yes, that's the most sour of the common citrus fruit (and probably the one with the most citric acid). The industry began with Italian lemons, but wars in Europe force innovation. Most production today uses microorganisms in culture, saving fresh lemons for kitchen decorations and garnishes.

1. Spin the lemon juice at 10,000 g for 10 minutes to pellet the remaining fruit pulp.
2. Pour off the liquid into a boiling flask.
3. Add the sodium hydroxide to the boiling flask 1 mL at a time with 60 seconds of swirling after addition. The pH will slowly climb as the citric acid is titrated. You'll know you are close to a high pH when what has looked like lemonade changes to the color of day old orange juice (note that as you add base, the local color will darken, but return on swirling—just like a pH indicator in a titration).
4. Precipitate the citric acid by adding the CaCl2 in 1 mL volumes. The results should be dramatic from the start.