Fruits of Spin

It’s no secret. Buy a mango that looks perfectly ripe, especially off-season, and you’ll very likely be taking home not so much the king of fruits, but an impostor.

Carbide, the vendor might reluctantly admit, blaming it on his supplier.

What really happens when carbide is used to artificially ripen fruits?

Prof. Chandrakumar’s research group at the MRI/MRS Centre of Department of Chemistry, IIT Madras, set out to investigate this using a technique that relies on the same quantum-mechanical phenomenon as the brain scanners used in hospitals.

When a fruit ripens, it softens, changes colour, and smells different. And it’s sweeter. All this is due to the molecular changes happening inside. In some fruits the plant hormone ethylene, a gas, is produced naturally. It calls upon various other hormones which collectively lead to, among other things, a decrease in acidity and an increase in the sugar to acid ratio.

If fruits are harvested when ripe, they would have a short shelf life. Which is why artificial ripening appears attractive. Carbides are a class of compounds commonly used for this purpose because they generate acetylene, also a gas, which mimics the behaviour of ethylene. However, the most commonly used among them, calcium carbide, is often contaminated with hazardous elements like arsenic and phosphorus. For this reason, its use for artificial ripening is banned in India.

A ripening fruit can, of course, be studied by extracting its juice. But there’s a better, non-destructive way.

Nuclear magnetic resonance (NMR) is a phenomenon that magnetic resonance imaging (MRI) machines in hospitals use for non-invasive scanning. Spectroscopy – the study of matter by exploiting its interaction with electromagnetic radiation – based on NMR is widely used for probing molecular structure. Prof. Chandrakumar realized that a form of NMR spectroscopy, called volume-localized spectroscopy, offered a nondestructive and reliable method to study natural and artificial ripening at the molecular level.

The phenomenon of magnetic resonance arises from several fundamental principles of physics. One of them, angular momentum, is a property that rotating objects have. But elementary particles have an intrinsic spin angular momentum which is a purely quantum-mechanical property; it has no analogue in classical mechanics. For a given type of particle, the magnitude of spin is a constant.

If a nucleus has an even number of protons, they pair up to cancel each other’s spin; the same applies for an even number of neutrons. In such cases, the net spin of the nucleus becomes zero. If there’s an odd number of either, or both, of them, the nucleus will have a net spin. The direction of this nuclear spin, unlike its magnitude, can be manipulated using an external magnetic field, an effect which depends on the exact number of neutrons and protons.

To see what happens, consider the nucleus of a hydrogen atom, a single proton, which behaves as a tiny bar magnet of its own pointing in the direction associated with its spin. When an external magnetic field is applied, the direction of this proton magnet rotates about the field direction, tracing out a cone. Known as precession, the frequency of this rotation is called Larmor frequency, which depends on the strength of the external magnetic field as well as a quantity called the gyromagnetic ratio, which is a constant for a given nucleus

The result is that the net magnetism of the sample aligns either parallel or anti-parallel to the external field, being a resultant of the magnetism of the individual protons which have a component in one of these two directions. The energy difference between these two states is determined by the Larmor frequency. The lower-energy, parallel, state has slightly more nuclei, but they can jump to the higher-energy, anti-parallel, state if radiation at the Larmor frequency is applied, a phenomenon called resonance.

“When the frequency of the electromagnetic radiation nearly matches the energy level separation, you have the possibility to flip the spin. But the crucial point there is that the two energy levels are a quantum-mechanical consequence of the fact that spins can have only a certain finite number of allowed stable orientations in an external field. Unlike a classical compass needle,” says Prof. Chandrakumar.

The radiation used to excite the nuclei can be applied continuously, or can be in bursts, called pulses, each lasting between one microsecond to a few milliseconds, depending on the experiment. When a proton absorbs this energy, it enters a state which is neither the ground state nor the excited state – a superposition of the two states.

For this to happen though, the radiation has to have its magnetic field component oscillating perpendicular to the applied magnetic field. “If not, under standard NMR conditions, we cannot excite the system at all,” says Prof. Chandrakumar. The duration of the pulse matters too. The product of the amplitude of the magnetic field component of the radiation and the duration of the pulse determines the angle through which the bulk magnetization flips. In the present experiment, it’s 90 degrees.

Before the radiation is switched on, the nuclear spins have an equilibrium distribution – all the magnetization is parallel to the external field; none perpendicular to it. Applying radiation at resonant frequency, as a 90 degree pulse, results in the opposite situation. Then, the radiation is switched off.

“There is going to be a spontaneous return of the system to equilibrium non-radiatively,” says Prof. Chandrakumar. This process is called relaxation. And relaxation takes time. “If excitation needed, say, 1 microsecond to 10 milliseconds…relaxations would need hundreds of milliseconds to tens of seconds. So it’s a much slower process.”

The bulk magnetization, which is now perpendicular to the field direction, precesses around the latter, inducing a voltage in a coil surrounding the sample. This is how the NMR signal is detected.

“The NMR spectrum, therefore, is not an absorption or an emission spectrum like in other spectroscopic methods”

“It is an induction signal,” says Prof. Chandrakumar. “You have this dynamo on bicycles. So it’s exactly the dynamo effect as far as the detection is concerned.”

What makes NMR such a good probe of molecular structure is the fact that the Larmor frequency depends on the strength of the magnetic field at the position of the nucleus, which is, in general, different from the strength of the applied field. This difference arises because the negatively charged electron generates a small field opposing the applied field. This changes the magnetic field strength experienced by the nucleus and causes a shift in its Larmor frequency. Known as chemical shift, this effect depends on the distribution of electrons around the nuclei being probed as well as the strength of the applied field. “Chemical shifts indicate structural features in the molecule. If you’re looking at a proton NMR, then [you can see] if this proton NMR belongs to a methyl group or an aromatic ring or something else,” says Prof. Chandrakumar.

One could use NMR to study bulk objects, such as a fruit. The chosen one for this study, was sweet lime.

A set of three sweet limes was artificially ripened by keeping them in a desiccator containing calcium carbide, while another set of three was allowed to naturally ripen. Over the course of a month, NMR spectra of the two sets were taken to see how their molecular constituents changed. “When we pick a suitable region of the fruit where we know from the image there’s enough fleshy part, we can get a high-resolution NMR spectrum. We’re trying to get the molecular imprint of what is happening in a natural process, without interfering with it,” says Prof. Chandrakumar.

An obvious constituent of any fruit is water. So the NMR signal from the hydrogen nuclei – protons – in water molecules has to be suppressed selectively to prevent it from swamping the signals from protons in other compounds. Once this is done, the NMR spectrum of the naturally-ripened sweet lime shows several peaks that are characteristic of the molecules involved.

The horizontal axis of an NMR spectrum represents the resonance frequency of various nuclei, but not in the usual units of hertz. This is because the extent of the shielding effect of the electrons, and hence the resonance frequency of various nuclei, depends on the strength of the applied magnetic field. To remove this dependence, the difference between the resonance frequency of the nuclei and that of a standard is calculated relative to the standard. This is the number, called the chemical shift, which appears on the horizontal axis and has the dimensionless units of parts per million (ppm).

The most prominent peaks in the sweet lime NMR spectrum are from the hydrogen nuclei of the different sugars present, mainly sucrose, glucose and fructose. Tracking how these peaks change over time reveals the change in total sugar concentration as the fruit ripens.

The characteristic peaks from sucrose and glucose appear at one end of the spectrum. At the other end are the broadly spread smaller peaks from citric acid. These two regions of the NMR spectrum can be used to calculate the sugar to acid ratio, which provides a measure of the extent of ripening.

“We track the sugar to acid ratio, typically, as a function of ripening”

This ratio, for the most part, increases as the fruit ripens. Sucrose and glucose also have other peaks which lie to the right of their characteristic peaks (in the main sugar region), which can also be used to calculate this ratio. This gives specific information about the concentrations of these two sugars.

That was for the naturally-ripened sweet limes. For the ones artificially ripened using calcium carbide, the sugar to acid ratio increases, then decreases and ends up below the initial value. As in natural ripening, the skin of the fruit turns yellow, but this happens faster for the artificially-ripened ones. At the molecular level, this is manifested as a faster increase in the ethanol content, the peaks of which are more prominent than those in the NMR spectra of the naturally-ripened fruits. Acetylene is what does the trick.

These experiments reveal only the relative concentrations of what’s inside the fruit. Their absolute concentrations were determined by comparing the NMR spectra of the fruit with that of a model solution containing the same constituents whose concentrations – both relative and absolute – are known.

This study was done by Abhishek Banerjee, Christy George who were Ph.D. students working with Prof. Chandrakumar at the time along with Sathyamoorthy Bharathwaj, who was an M.Sc. student.

One of Prof. Chandrakumar’s current Ph.D. students, Amey Kirwai, is studying the guava’s ripening process, which is different from that of sweet lime, using NMR signals from phosphorus nuclei instead of hydrogen. “He’s looking at the phosphorus reservoir in terms of sugar phosphate, adenosine triphosphate and diphosphate and so on, and trying to track the ripening under various conditions – such as in the presence and absence of oxygen – of a fruit like guava, which is a fruit crop of great relevance in India and other tropical climes,” says Prof. Chandrakumar.

All this using a completely non-invasive and nondestructive method. What next?

“There have been some discussions on fruit quality control with a research institution and a major food retail chain. Though the technique is expensive, it gives you a quick non-invasive method for tracking, at a molecular level, anything that is to be learned about the food material. So, people who are interested in the shelf life of foodstuffs find this kind of approach would be valuable,” says Prof. Chandrakumar.