Cell Fractionation and Ultracentrifugation Revision Notes | A Level AQA Biology

1. Why Separate a Cell's Contents?

A cell is extraordinarily crowded. Mitochondria, ribosomes, nuclei, and dozens of other structures all share the same cytoplasm — and if you want to study any one of them in isolation, you need a way to pull them apart without destroying them. That is exactly what cell fractionation and ultracentrifugation allow scientists to do.

These techniques transformed cell biology in the mid-twentieth century. Before them, researchers could see organelles under a microscope but could not isolate enough material to study their chemistry. Once fractionation was established, scientists could collect pure samples of mitochondria, for example, and directly measure their enzyme activity.

There is also a subtler story here: for decades, scientists argued fiercely about which structures in their preparations were real organelles and which were artefacts created by the preparation process itself. Understanding both the technique and that historical debate is required by the AQA specification.

2. Cell Fractionation — The Principles

Cell fractionation begins by breaking cells open — a step called homogenisation. Tissue is placed in a blender or ground with a pestle and mortar to rupture the plasma membranes and release the cell contents. The resulting mixture is called the homogenate.

Three conditions must be maintained throughout to keep organelles intact and functional. These are not arbitrary rules — each one has a precise biological reason.

• Ice-cold temperature: slows enzyme activity, preventing autolysis (self-digestion by lysosomal enzymes released during homogenisation).
• Isotonic solution: the buffer has the same water potential as the cell contents, so organelles neither gain nor lose water by osmosis and do not burst or shrivel.
• Buffered pH: maintains a stable pH so that enzyme structure and organelle membranes are not disrupted by acid or alkali conditions.

The homogenate is then filtered to remove any unbroken cells and large debris such as connective tissue. What remains is a suspension of organelles ready for the next stage.

3. Ultracentrifugation — Separating Organelles by Size and Density

Once the homogenate is filtered, it is placed in a centrifuge tube and spun. The principle is straightforward: larger, denser structures experience a greater centrifugal force relative to their drag, so they sediment to the bottom of the tube first, forming a pellet. The remaining liquid above the pellet is called the supernatant.

In differential centrifugation — the standard approach — the supernatant is decanted and spun again at a higher speed. Each successive spin brings down the next-largest fraction. This is repeated at progressively higher speeds until the desired organelle fraction has been isolated.

The order in which organelles pellet is predictable and worth committing to memory. Nuclei are the largest and densest organelles, so they sediment first at relatively low speeds. Mitochondria (and chloroplasts in plant cells) and lysosomes co-sediment at medium speeds. Endoplasmic reticulum fragments follow at higher speeds. Finally, ribosomes — the smallest organelles — require the highest speeds to pellet, not because of low density, but due to their small size.

The speeds given above are approximate and vary between protocols, but the order is fixed by the physical properties of the organelles. Examiners expect you to know that order, not the precise rpm values.

4. A Step-by-Step Walk Through the Procedure

Exam questions often ask you to describe or explain the fractionation procedure in sequence. The step-by-step below models the kind of methodical answer that earns full marks.

Worked example: Isolating Mitochondria by Differential Centrifugation

Describe how you would isolate a fraction enriched in mitochondria from liver tissue.

1. Grind liver tissue in ice-cold, isotonic, buffered solution using a blender or homogeniser. — Ice-cold prevents autolysis; isotonic prevents osmotic damage to organelles; buffered pH prevents enzyme denaturation. This produces the homogenate.
2. Pass the homogenate through muslin or a fine filter to remove unbroken cells and connective tissue debris. — Unbroken cells would contaminate later fractions with intact nuclei and all organelles mixed together.
3. Centrifuge the filtered homogenate at low speed. Discard the pellet (nuclei and cell debris). Retain the supernatant. — Nuclei are the largest organelles and sediment first. Discarding this pellet removes them from the preparation.
4. Centrifuge the supernatant at medium speed. Collect the pellet. — Mitochondria are now the largest remaining organelles and sediment into this pellet. The supernatant above still contains smaller organelles such as ribosomes.
5. Resuspend the medium-speed pellet in fresh isotonic buffer. — The resulting suspension is enriched in mitochondria. Purity can be checked using electron microscopy or enzyme assays specific to mitochondria (e.g., testing for succinate dehydrogenase activity).

Answer: The medium-speed pellet, resuspended in isotonic buffer, provides a fraction enriched in mitochondria.

5. Artefacts Versus Genuine Organelles — A Historical Debate

Here is where the science gets genuinely fascinating. When early cell biologists first used centrifugation and electron microscopy in the 1940s and 1950s, they saw structures they had never seen before. The immediate question was: are these real features of living cells, or are they artefacts — structures created by the preparation process itself?

An artefact can arise in several ways. Harsh homogenisation can shear membranes into vesicles that look like genuine organelles. Chemical fixatives used in electron microscopy can precipitate proteins into granule-like structures. Staining agents can bind non-specifically and create apparent boundaries where none existed in the living cell.

The scientific community therefore spent a considerable period debating each newly described structure. The debate was resolved only when multiple independent lines of evidence converged: consistent appearance across different preparation methods, biochemical activity specific to the structure, and eventually direct observation in living cells using fluorescence microscopy.

The ribosome is a good example. When ribosomes were first isolated in centrifuge pellets in the early 1950s, some researchers argued they were clumps of protein produced by the centrifugation process. It took biochemical evidence — showing that these particles contained RNA and were the site of protein synthesis — to confirm they were genuine organelles.

This history matters for your exam because AQA explicitly requires you to appreciate that distinguishing artefacts from real organelles was not straightforward. It reflects a broader principle of science: a single observation is rarely sufficient; reproducibility and convergent evidence are essential before a structure is accepted as real.

6. Exam Technique and Common Mistakes

Fractionation questions appear regularly in AQA papers and follow predictable patterns. The marks are often lost not through ignorance of the facts but through imprecise language or missing the logic of the question.

7. Pulling It Together — Exam-Style Application

The final check is whether you can apply these principles to an unfamiliar scenario — exactly what AQA 'suggest' and 'evaluate' questions demand. Work through the long-answer question below using the marking points as a guide.

Cell fractionation and ultracentrifugation together provide the methodological foundation for almost all biochemical studies of organelle function. Mastering the conditions, the order of sedimentation, and the historical context of artefact identification covers both specification requirements and equips you to handle novel application questions with confidence.