How Dorothy Hodgkin Established the Structure of Penicillin

By the early 1940s, penicillin could treat bacterial infections that had previously been fatal. Yet chemists still disagreed about the molecule’s exact structure. Several proposed arrangements could explain some of the available chemical evidence, but only one could be correct.

This created an important problem. If scientists wanted to understand why penicillin worked, improve its properties, or develop related antibiotics, they needed to know exactly how its atoms were connected. How could they establish a molecular structure when they couldn’t simply look at the molecule?

One of the scientists who helped answer this question was British chemist Dorothy Crowfoot Hodgkin. Her work on penicillin between 1941 and 1945 became an important demonstration of how X-ray crystallography could establish the three-dimensional structures of complex organic molecules.

01The problem: knowing which structure was correct

Penicillin G, also called benzylpenicillin, has the molecular formula:

\[
\mathrm{C}_{16}\mathrm{H}_{18}\mathrm{N}_{2}\mathrm{O}_{4}\mathrm{S}
\]

This tells us that each molecule contains 16 carbon atoms, 18 hydrogen atoms, two nitrogen atoms, four oxygen atoms, and one sulfur atom.

But does knowing the molecular formula tell us how these atoms are connected?

No. Different structural isomers can have exactly the same molecular formula while having different arrangements of atoms and different chemical properties.

In penicillin’s case, scientists were particularly interested in the arrangement of its nitrogen, sulfur, and carbonyl-containing groups. Several proposed structures could account for aspects of its observed chemical behaviour.

The correct structure included an unusual four-membered ring called a β-lactam ring. This is a cyclic amide, meaning that it contains a carbonyl group connected to a nitrogen atom within a ring.

Why was the β-lactam structure controversial?

The β-lactam structure was first proposed in October 1943, but it remained controversial until 1945.

One alternative, associated with prominent organic chemist Robert Robinson, was a thiazolidine-oxazolone structure. This proposed a different arrangement of atoms and rings.

The four-membered β-lactam ring also seemed chemically surprising. Small rings experience considerable angle strain because their bond angles are forced away from their preferred values.

A chemist might therefore predict that such a ring would be unstable. However, an unusual structure is not automatically an incorrect structure.

The important question was whether experimental evidence supported it.

The historical debate is discussed in the review Selective Reminiscences of Beta-Lactam Antibiotics.

02How X-ray crystallography revealed molecular structures

Hodgkin used a technique called X-ray crystallography.

The central idea is straightforward: a crystal contains an ordered, repeating arrangement of molecules. When X-rays interact with this arrangement, they produce a diffraction pattern that contains information about the positions of atoms.

To understand why this works, start with the apparatus.

What did the experiment look like?

Imagine a narrow beam of X-rays travelling from an X-ray source towards a tiny crystal mounted in the centre of an experimental apparatus.

The crystal is carefully positioned so that the X-rays interact with its regularly repeating atomic arrangement.

The scattered X-rays travel in different directions. At certain angles, the scattered waves reinforce one another through constructive interference.

A photographic plate positioned to record the scattered radiation captures a pattern of bright spots.

The experimental sequence is:

1. An X-ray source produces radiation with a known wavelength.
2. The beam is directed towards a small, mounted crystal.
3. The crystal scatters the X-rays.
4. Constructive interference produces diffraction maxima at particular angles.
5. A photographic plate records the positions and intensities of the diffraction spots.
6. Scientists analyse the pattern to determine the electron-density distribution within the crystal.

The diffraction pattern is not a photograph of the molecule.

Instead, it is a collection of measurements that scientists must interpret mathematically.

Think of a crystal as a three-dimensional diffraction grating. Like the closely spaced tracks on a CD, its regular structure causes waves to reinforce at particular angles.

The analogy has limits, though. A molecular crystal contains a repeating three-dimensional arrangement of electron density, rather than a simple two-dimensional pattern of grooves.

Bragg’s law: relating diffraction angles to atomic spacing

The relationship between the X-ray wavelength, the spacing between crystal planes, and the diffraction angle is described by Bragg’s law:

\[
n\lambda = 2d\sin\theta
\]

Where:

• \(n\) = order of diffraction, a positive integer.
• \(\lambda\) = wavelength of the incident X-rays, measured in metres or nanometres.
• \(d\) = perpendicular distance between adjacent crystal planes.
• \(\theta\) = angle between the incident X-ray beam and the crystal planes.

The detector commonly records the angle between the incident and diffracted beams, which is \(2\theta\) in the usual symmetric Bragg geometry.

The important idea is that diffraction occurs strongly when the extra distance travelled by the scattered waves is an integer multiple of their wavelength.

From diffraction spots to an electron-density map

Knowing the spacing between crystal planes is only the beginning.

Scientists need to work out where the individual atoms are located.

The positions and intensities of diffraction spots provide information about the distribution of electrons in the crystal. More precisely, the measured intensities are related to the amplitudes of the scattered waves.

However, there is a complication.

To reconstruct an electron-density map using Fourier analysis, scientists also need the phase information of the waves. Phase describes the position of a wave within its cycle.

While diffraction intensities can be measured, the phase information cannot be directly measured in the same way.

This is known as the phase problem in X-ray crystallography.

Scientists developed mathematical methods to work around it. One important technique was the Patterson method, which could help identify the positions of heavier atoms within a crystal.

Once enough information was available, Fourier calculations could be used to construct an electron-density map.

Peaks in this map indicated regions where electrons were concentrated, helping scientists determine where atoms were located.

This is how an apparently random collection of diffraction spots could eventually become a three-dimensional molecular model.

The International Union of Crystallography provides further explanation of the relationship between diffraction data and electron-density maps.

03Dorothy Crowfoot Hodgkin’s investigation of penicillin

Hodgkin began investigating penicillin during the Second World War. Her work extended from approximately 1941 to 1945.

The investigation was not straightforward.

The first problem: obtaining suitable crystals

Initially, penicillin itself was difficult to crystallise.

Without a suitable crystal, X-ray crystallography could not be used effectively.

Hodgkin therefore investigated breakdown products of penicillin, working with fellow Oxford crystallographer Barbara Low.

This allowed the researchers to gather useful structural information while other scientists continued working on the purification and crystallisation of the antibiotic.

Eventually, a small quantity of crystalline sodium benzylpenicillin became available.

In her 1964 Nobel Lecture, Hodgkin recalled growing crystals for X-ray analysis from just 3 mg of the sodium salt, which had been transported from the United States during the war.

This was an extremely small sample, but it was sufficient to begin the investigation.

The second problem: identifying the correct atomic arrangement

Hodgkin and her collaborators investigated different crystalline salts of benzylpenicillin.

These included:

• Sodium benzylpenicillin.
• Potassium benzylpenicillin.
• Rubidium benzylpenicillin.

Why use different salts?

The answer involves the way different atoms scatter X-rays.

Rubidium has a higher atomic number than potassium, meaning it contains more electrons and generally produces a stronger X-ray scattering contribution.

A heavier atom can therefore act as a useful reference point when determining the positions of other atoms.

This is related to the heavy-atom methods used in crystallography.

In an ideal case, scientists can replace an atom with a heavier atom while maintaining essentially the same crystal arrangement. This is called isomorphous replacement.

The stronger scattering contribution helps resolve the phase problem and makes it easier to calculate the electron-density map.

However, Hodgkin’s actual experiment was more complicated.

The sodium salt did not form an isomorphous series with the potassium and rubidium salts. The potassium and rubidium ions were also positioned in ways that meant they did not contribute strongly to many of the measured reflections.

Consequently, the researchers could not simply substitute one perfect map for another.

Instead, they compared imperfect maps from the different crystal forms and used the combined information to establish the molecular arrangement.

Hodgkin later acknowledged the importance of her collaborators, particularly Barbara Low and Charles Bunn, in developing the methods used to interpret these difficult results.

The investigation was a substantial computational challenge.

Before modern computers, Hodgkin and her collaborators performed many calculations using specialised printed mathematical strips called Beevers-Lipson strips. They gradually constructed electron-density contours and assembled these into three-dimensional models.

The Oxford Museum of the History of Science describes how Hodgkin’s team developed contour maps and a physical Perspex model of penicillin from this work.

By spring 1945, Hodgkin and her collaborators had worked out the complete structure. The detailed research was published later, with Hodgkin’s X-ray analysis appearing in 1949.

You can read her own retrospective account in her 1964 Nobel Lecture, as well as the Oxford museum’s historical account of the investigation.

Why was this evidence convincing?

The X-ray results did not stand alone.

Other researchers had investigated penicillin using chemical reactions, degradation products, and different physical measurements.

These experiments provided independent information about the atoms, functional groups, and chemical behaviour of the molecule.

The crystallographic evidence added an important missing piece: the three-dimensional arrangement of those atoms.

This is an important principle in structural chemistry.

A molecular model becomes convincing when several independent observations support the same arrangement and competing structures fail to explain the combined evidence.

A later historical review, Drugs That Changed Society: History and Current Status of the Early Antibiotics, discusses the combined role of chemical reactions and X-ray crystallography in establishing penicillin’s structure.

04What did the established structure reveal?

The accepted structure of benzylpenicillin contains two fused rings:

1. A four-membered β-lactam ring.
2. A five-membered sulfur-containing thiazolidine ring.

It also contains an amide-containing side chain, a carboxylic acid group, and a benzene ring.

The four-membered β-lactam ring was particularly important.

Its geometry creates considerable ring strain. The unusual geometry also affects the resonance within the cyclic amide, making its carbonyl group more susceptible to nucleophilic attack than that of an ordinary, relatively unstrained amide.

This helps explain its chemical reactivity.

Connecting molecular structure to medicinal action

Today, we understand that penicillin works by interfering with the formation of bacterial cell walls.

Bacteria build their cell walls using a strong polymer called peptidoglycan. During bacterial growth, enzymes called transpeptidases help cross-link the peptide chains in this structure.

Penicillin binds to these enzymes, known as penicillin-binding proteins, and inhibits their activity.

Its β-lactam ring reacts with an active-site serine residue in the enzyme, forming a covalent interaction that prevents normal cell-wall cross-linking.

Without properly formed cell walls, susceptible bacteria become vulnerable to damage and death.

There is an important distinction here.

Hodgkin’s work established the molecular structure. It did not, by itself, establish the complete biological mechanism of penicillin.

That mechanism was developed through additional biochemical and biological investigations.

Nevertheless, knowing the structure helped scientists understand which parts of the molecule were chemically important and provided a foundation for developing related antibiotics.

The NCBI Bookshelf discussion of penicillin explains its interaction with bacterial cell-wall enzymes.

Structure determination was not the same as synthesis

It is tempting to assume that once scientists knew the structure of penicillin, they could simply manufacture it using chemical reactions.

That did not happen immediately.

Hodgkin’s contribution was to establish the molecular structure using crystallographic evidence. She did not discover penicillin, develop its original clinical use, or synthesise the antibiotic.

These were different scientific achievements.

The distinction matters because penicillin was already being used medically before Hodgkin completed her structural investigation.

Its widespread production relied primarily on growing Penicillium mould through fermentation, rather than manufacturing every molecule through a sequence of chemical reactions.

Although John Sheehan and Kenneth Henery-Logan later achieved the total synthesis of penicillin V, the process was difficult and did not replace fermentation as the main method of mass production.

The original 1957 report can be found in the Journal of the American Chemical Society: The Total Synthesis of Penicillin V.

Evaluating the social impact

The impact of penicillin was substantial.

Before effective antibiotics became available, bacterial infections could turn relatively minor injuries into life-threatening medical emergencies.

Penicillin helped transform the treatment of bacterial infections, saved thousands of lives during the Second World War, and contributed to the development of modern medical practices.

However, its social impact should not be attributed to Hodgkin’s crystallographic work alone.

Fleming’s discovery, the Oxford team’s medical development, improved fermentation and manufacturing methods, and the work of many other researchers were all essential.

Structural determination made a different contribution. It provided scientists with a more precise understanding of the molecule and supported subsequent research into new antibiotics.

Penicillin also illustrates a continuing problem in medicine: antibiotic resistance. Some bacteria produce enzymes called β-lactamases, which hydrolyse the β-lactam ring and deactivate susceptible antibiotics.

Understanding the structure and reactivity of this ring is therefore relevant to both antibiotic development and resistance.

05Connecting the historical investigation to modern spectroscopy

Hodgkin’s work provides a useful way to understand how different analytical techniques contribute to molecular structure determination.

Modern chemists have access to several techniques that provide complementary information.

These techniques answer different questions.

For example, suppose two compounds have the same molecular formula and molecular mass.

Mass spectrometry may establish that their masses are consistent with the same formula, but this does not necessarily distinguish their structures.

IR spectroscopy may reveal that one contains a different functional group.

NMR spectroscopy can provide evidence about the number and arrangement of different atomic environments.

X-ray crystallography can then provide direct structural information about the arrangement of atoms within a crystal.

The strength comes from combining the results.

This is exactly the broader lesson from penicillin: scientists should not accept a molecular structure simply because it explains one experimental observation. They should test whether it explains several independent observations.

For another explanation of how molecular mass and fragmentation patterns help identify compounds, see tutorgum’s resource on Mass Spectrometry: Identifying and Measuring Water Pollutants.

One final historical distinction is worth remembering. Modern NMR and mass spectrometry were not available as the routine structural-analysis tools they are today. The penicillin investigation relied heavily on chemical transformations, crystallography, and the physical methods available at the time.

06Questions and solutions

Question 1

An X-ray beam with a wavelength of 0.154 nm produces a first-order diffraction maximum from a crystal with parallel planes separated by 0.308 nm.

Calculate:

(a) The Bragg angle, \(\theta\).

(b) The angle between the incident and diffracted beams, assuming symmetric Bragg geometry.

Solution 1

(a) The Bragg angle is approximately \(14.5^\circ\), and (b) the angle between the incident and diffracted beams is approximately \(29.0^\circ\).

Bragg’s law is:

\[
n\lambda=2d\sin\theta
\]

Step 1: Identify the known quantities.

\[
n=1
\]

\[
\lambda=0.154\ \mathrm{nm}
\]

\[
d=0.308\ \mathrm{nm}
\]

Step 2: Rearrange the equation to find \(\theta\).

\[
\sin\theta=\frac{n\lambda}{2d}
\]

Step 3: Substitute the values.

\[
\sin\theta=
\frac{1(0.154)}{2(0.308)}
\]

\[
\sin\theta=\frac{0.154}{0.616}
\]

\[
\sin\theta=0.250
\]

Taking the inverse sine:

\[
\theta=\sin^{-1}(0.250)
\]

\[
\boxed{\theta=14.5^\circ}
\]

Step 4: Calculate the angle between the incident and diffracted beams.

For symmetric Bragg geometry, this angle is twice the Bragg angle.

\[
2\theta=2(14.5^\circ)
\]

\[
\boxed{2\theta=29.0^\circ}
\]

The detector records the diffracted beam at an angle of approximately \(29.0^\circ\) relative to the incident beam.

Question 2

A chemist obtains a suitable crystal of an unknown organic compound and records an X-ray diffraction pattern.

Another student claims that the diffraction pattern is essentially a photograph of the molecule, with each bright spot representing an individual atom.

Explain why this interpretation is incorrect.

In your response, describe:

• Why a crystal is required for conventional single-crystal X-ray crystallography.
• What information the positions and intensities of diffraction spots provide.
• How electron-density maps are produced.
• Why the phase problem makes the interpretation more complicated.

Solution 2

The diffraction pattern is not a direct photograph of individual atoms. It is a pattern produced by constructive interference of X-rays scattered by the repeating electron density within a crystal.

Step 1: Explain the role of the crystal.

A crystal contains a regular, repeating three-dimensional arrangement of molecules.

This periodic structure allows scattered X-ray waves to reinforce one another at particular angles, producing sharp diffraction maxima.

Without this regular arrangement, the clear diffraction pattern required for conventional single-crystal crystallography would not be obtained.

Step 2: Explain what the diffraction pattern measures.

The positions of diffraction spots provide information about the spacing and symmetry of the crystal lattice.

Their intensities provide information about how electrons are distributed within the repeating structure.

Individual spots do not correspond directly to individual atoms.

Step 3: Explain how the molecular structure is reconstructed.

Scientists use mathematical methods, including Fourier calculations, to convert diffraction information into an electron-density map.

Regions of high electron density help identify atomic positions.

By analysing the resulting map, scientists can construct a model showing the three-dimensional arrangement of atoms.

Step 4: Explain the phase problem.

The intensity measurements provide information about the amplitudes of scattered waves, but their phases are not directly measured.

Both amplitude and phase information are required to reconstruct the electron-density distribution completely.

Scientists therefore use methods such as Patterson analysis and heavy-atom techniques to obtain additional structural information.

The student’s interpretation is incorrect because diffraction spots represent the behaviour of waves scattered by the entire repeating crystal structure, not separate photographs of individual atoms.

Question 3

During the investigation of benzylpenicillin, Hodgkin and her collaborators examined sodium, potassium, and rubidium salts.

Explain why comparing potassium and rubidium salts could provide useful structural information.

In your response:

(a) Explain why rubidium provides a stronger X-ray scattering contribution than potassium.

(b) Explain how replacing one counterion with a heavier atom can help resolve the phase problem.

(c) Explain why Hodgkin’s actual comparison was more complicated than an ideal isomorphous replacement experiment.

(d) Explain why the results needed to be considered alongside other chemical evidence.

Solution 3

Comparing the potassium and rubidium salts could provide useful structural information because rubidium scatters X-rays more strongly than potassium, potentially helping researchers identify atomic positions and improve their electron-density maps.

However, the comparison was not a straightforward example of perfect isomorphous replacement.

Step 1: Explain the difference in X-ray scattering.

Potassium has atomic number 19, whereas rubidium has atomic number 37.

Rubidium therefore contains more electrons and generally contributes more strongly to X-ray scattering.

This makes it a potentially useful reference point when interpreting diffraction data.

Importantly, the potassium and rubidium ions are counterions in the crystalline salts. They are not covalently bonded parts of the benzylpenicillin molecule.

Step 2: Explain how heavier atoms help.

In an ideal heavy-atom experiment, a heavier atom is introduced or substituted at a known position without significantly changing the crystal structure.

The stronger scattering contribution helps researchers establish phase information.

This allows more reliable electron-density maps to be calculated.

Step 3: Explain the historical complication.

Hodgkin’s sodium salt was not isomorphous with the potassium and rubidium salts.

Furthermore, the potassium and rubidium ions occupied positions that meant they did not contribute strongly to many diffraction reflections.

The researchers therefore had to compare imperfect maps rather than relying on a simple, clean heavy-atom substitution.

Hodgkin’s own Nobel Lecture describes these difficulties.

Step 4: Explain why independent evidence mattered.

Even a useful diffraction comparison should not be interpreted in isolation.

Chemical degradation experiments and other physical measurements provided additional constraints on the possible arrangements of atoms.

The correct structure became convincing because the crystallographic evidence was consistent with the wider body of chemical evidence.

This is an important feature of scientific structural determination: several independent observations should support the same model.

Question 4

A historical article makes the following statement:

“Dorothy Crowfoot Hodgkin discovered penicillin, synthesised its molecular structure, and created the antibiotic that saved thousands of lives.”

Evaluate this statement.

Your response should:

(a) Identify the historical inaccuracies.

(b) Explain Hodgkin’s actual contribution.

(c) Distinguish structural determination from total chemical synthesis.

(d) Discuss how establishing the structure contributed to later medicinal chemistry.

Solution 4

The statement is historically inaccurate because it combines several separate scientific achievements and incorrectly attributes them to Hodgkin.

Step 1: Identify the discovery of penicillin.

Alexander Fleming discovered the antibacterial effects of Penicillium mould in 1928.

He observed that bacteria growing near the mould were inhibited.

However, Fleming did not develop the purification and manufacturing processes needed to make penicillin a widely available medicine.

Step 2: Identify the medical development of penicillin.

Howard Florey, Ernst Chain, Norman Heatley, and their collaborators at Oxford developed methods to isolate, purify, test, and produce penicillin.

Their work made the antibiotic medically useful.

Large-scale production and distribution during the Second World War helped transform the treatment of bacterial infections.

Step 3: Explain Hodgkin’s actual contribution.

Hodgkin investigated the molecular structure of penicillin using X-ray crystallography.

Working with other researchers, including Barbara Low and Charles Bunn, she helped establish the three-dimensional arrangement of its atoms.

Her work provided strong structural evidence supporting the unusual β-lactam-containing model.

She did not synthesise penicillin or discover its antibacterial activity.

Step 4: Distinguish structural determination from chemical synthesis.

Structural determination means establishing how atoms are connected and arranged in a molecule.

Chemical synthesis means producing that molecule through controlled chemical reactions involving other substances.

These are different scientific tasks.

Hodgkin established the structure, whereas John Sheehan and Kenneth Henery-Logan later achieved the total chemical synthesis of penicillin V in 1957.

Step 5: Explain the contribution to medicinal chemistry.

Knowing the molecular structure allowed chemists to understand the functional groups and structural features responsible for penicillin’s chemical behaviour.

It also supported research into related antibiotics with modified side chains and improved properties.

For example, penicillin V is more acid-stable than penicillin G.

The correct conclusion is that Hodgkin made an important contribution to structural chemistry and subsequent medicinal research, but penicillin’s discovery, medical development, production, and synthesis involved different scientists and research teams.

Question 5

A pharmaceutical research group investigates three samples of benzylpenicillin.

The following observations are recorded.

Additional information:

• Samples A and B have the same elemental composition.
• Both have been dissolved in the same solvent before IR and NMR measurements.
• Refinement of their X-ray diffraction data shows the same covalent connectivity and stereochemistry, but different unit-cell dimensions and arrangements of surrounding ions.
• The molecular mass measurements for sample C have been corrected for counterions, solvent, and instrumental adducts.
• Assume the mass increase represents a genuine change in the organic molecule.

A student makes two claims:

Claim 1: Samples A and B must contain different molecules because their X-ray diffraction patterns are different.

Claim 2: Samples A and B must have identical crystal structures because their IR and NMR spectra are the same.

A third student argues that sample C is simply another crystalline form of penicillin.

Evaluate all three claims.

Your answer must:

(a) Identify the most appropriate description of samples A and B.

(b) Explain why different X-ray diffraction patterns do not necessarily indicate different molecular structures.

(c) Explain why identical solution IR and NMR spectra do not establish identical crystal structures.

(d) Identify the most likely chemical change in sample C and explain how the observations support your conclusion.

(e) Suggest one additional experiment that could strengthen the identification of sample C.

Solution 5

Samples A and B are different crystalline forms of the same benzylpenicillin sodium salt, while sample C has undergone a chemical transformation most consistent with hydrolysis of the β-lactam ring.

The key distinction is between a change in the arrangement of molecules within a crystal and a change in the covalent structure of the molecules themselves.

Step 1: Classify samples A and B.

Samples A and B are best described as different polymorphic forms of the same compound.

Polymorphism occurs when the same chemical substance can form crystals with different arrangements of molecules or ions.

The molecular connectivity and stereochemistry remain unchanged, but the way the molecules pack together in the crystal differs.

This explains why their unit-cell dimensions and diffraction patterns are different.

Step 2: Explain why their X-ray patterns differ.

X-ray diffraction depends on the repeating arrangement of electron density throughout the crystal.

Changing the positions and orientations of molecules or ions changes the crystal lattice.

This alters the angles and intensities of the diffraction maxima.

Therefore, two crystals containing the same organic molecule can produce different X-ray diffraction patterns.

The first student’s mistake is assuming that a diffraction pattern describes only the isolated molecular structure.

It also contains information about how molecules are arranged throughout the crystal.

Step 3: Explain why identical solution spectra do not prove identical crystals.

The IR and NMR measurements were performed after dissolving the samples.

Dissolving the crystals destroys their original long-range crystal packing.

The dissolved molecules can therefore produce identical solution spectra even when their original solid-state arrangements were different.

IR and NMR provide information about molecular functional groups and atomic environments, but these measurements do not automatically reveal the original crystal lattice.

The second student’s mistake is confusing molecular identity with crystal identity.

Step 4: Identify the chemical change in sample C.

Sample C most likely underwent hydrolysis of the β-lactam ring.

Hydrolysis is a reaction in which water breaks a chemical bond.

In this case, water can open the strained β-lactam ring, producing a ring-opened penicilloic-acid-type product.

For the free benzylpenicillin molecule, the overall change can be represented as:

\[
\ce{C16H18N2O4S + H2O ->
C16H20N2O5S}
\]

The molecular mass increases by approximately:

\[
\Delta M=18.0\ \mathrm{g\,mol^{-1}}
\]

This corresponds to the addition of water.

The changed carbonyl environment is also consistent with the opening of the cyclic amide.

Importantly, the loss of antibacterial activity supports this explanation because the intact β-lactam ring is essential for penicillin’s antibacterial action.

This is not simply another polymorphic form. The covalent molecular structure has changed.

Step 5: Suggest an additional experiment.

A useful additional experiment would be to perform NMR analysis of sample C and compare its signals with those of the original benzylpenicillin.

The new chemical environments could provide further evidence that the β-lactam ring has opened.

Alternatively, mass spectrometry with fragmentation analysis, followed by comparison with an authentic ring-opened product, could strengthen the identification.

The combined evidence is much stronger than relying on the changed X-ray pattern or molecular mass alone.

The central lesson is that scientists must distinguish between changes in crystal packing and changes in molecular connectivity. Different experimental techniques measure different aspects of a chemical substance, and their results must be interpreted together.

07Where the investigation leads next

Once scientists establish a molecule’s structure, the next useful question is how changing that structure affects its properties and behaviour.

Penicillin provides a good example.

Penicillin G and penicillin V share the same essential β-lactam-containing molecular framework, but their side chains differ. This changes properties such as acid stability, influencing how the medicines can be used.

This is the foundation of understanding structure-property relationships and structure-activity relationships in medicinal chemistry.

For HSC Chemistry, the useful skill is to connect the evidence: use molecular formulae to constrain possible structures, spectroscopy to identify functional groups and atomic environments, and chemical reactions to test whether a proposed structure behaves as expected.

Hodgkin’s investigation demonstrates why no single measurement needs to provide the entire answer. A reliable molecular structure emerges when independent evidence points towards the same arrangement of atoms.