Analysis of Chemical Composition: What Is Inside a Cell?
Imagine you have a handful of living tissue — a piece of liver, a leaf, a bacterial pellet. You know it contains thousands of different molecules, but how do you begin to ask: what is actually in there? You cannot just look at it. You need a way to separate the big, structural stuff from the small, mobile stuff, and then figure out what each group is made of.
That is the core of analysis of chemical composition: a set of techniques to identify and quantify the chemical constituents of a biological sample. The goal is not just a list of names, but a map of how the material is organised — what is bound up in large structures and what is freely dissolved.
The Intuition: Big vs. Small
Think of a cell as a crowded room. The macromolecular fraction is like the furniture — the tables, chairs, shelves. These are large, insoluble, structural molecules: proteins, nucleic acids (DNA, RNA), polysaccharides (like starch or cellulose), and lipids in membranes. They do not dissolve easily in water; they stay put.
The micromolecular fraction is like the people moving around the room, the air, the dust motes. These are small, soluble molecules: amino acids, sugars, nucleotides, ions, vitamins, metabolic intermediates. They dissolve freely and can be extracted with water or mild solvents.
The trick is to separate these two pools cleanly, so you can study each one separately.
The Precise Statement: Trichloroacetic Acid (TCA) Fractionation
The classic method to achieve this separation uses trichloroacetic acid (TCA). Here is how it works, step by step:
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Homogenise the tissue in cold TCA (typically 5–10% w/v). TCA is a strong acid that denatures proteins and precipitates them, along with other large molecules like nucleic acids and polysaccharides.
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Centrifuge the mixture. You get two fractions:
- Pellet (insoluble): the acid-insoluble macromolecular pool — proteins, DNA, RNA, large polysaccharides, membrane lipids.
- Supernatant (soluble): the acid-soluble micromolecular pool — free amino acids, small peptides, nucleotides, sugars, organic acids, inorganic ions.
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Analyse each fraction separately using chemical tests, chromatography, spectrophotometry, or other methods.
The key distinction is solubility in cold TCA, not size alone. Some small molecules (like certain lipids) may also precipitate, and some large molecules (like glycogen) may remain partly soluble. But as a first approximation, TCA fractionation cleanly separates the structural/bound from the free/metabolic pools.
Why This Matters
This technique is foundational because it reveals the dynamic state of a cell. For example:
- If you measure total protein in the pellet, you know how much structural or enzymatic machinery is present.
- If you measure free amino acids in the supernatant, you know the pool available for new protein synthesis.
- In a pulse-chase experiment (feeding cells a radioactive amino acid), you can track radioactivity moving from the supernatant (free pool) into the pellet (newly made protein) — that is how you measure protein synthesis rates. …