Library Simple Science
Click to enlarge Click to watch video Click to follow link

Progress in most natural science disciplines is directly related to the development of new analytical methods. This article will review some of the modern analytical techniques that are widely used in physics, chemistry and biology. Each of them forever changed our understanding of the surrounding world and ourselves. All of the methods described below are “Nobel Prize winners”. So let's get started.

X-Ray Structural Analysis
One of the most important and reliable experimental methods for determining the three-dimensional organization of substances (first of all, crystals) with atomic precision is X-ray diffraction analysis (XRD). Due to its versatility, simplicity and relatively low cost, today it is the most common method for determining the structure of a substance. The method is based on the phenomenon of X-ray diffraction (scattering) by the analyzed object (usually a crystal).

A group of German physicists headed by Max von Laue discovered this phenomenon in 1912. In 1914, Laue was awarded the Nobel Prize for this discovery. Any substance has the ability to scatter the incident radiation, including X-rays. The direction and intensity of the scattered X-rays depend on the structure of the scattering object, and correspond to the arrangement of atoms in its crystal. To study the atomic structure, radiation with a wavelength of about 0.1 nm (which is on the order of atomic size) is used. XRD requires a small crystal (0.5-1 mm3) of the substance. It is positioned in the path of the narrow X-ray and a scattered X-ray detector is placed behind the sample. According to Laue, the technique "has extended the power of observing minute structure ten thousand times beyond that of the (optical) microscope". It can be said that X-ray diffraction analysis is similar to a microscope with atomic-level resolution (1, 2).


Simplified diagram of the setup for X-ray structural analysis.

According to Max von Laue's idea, X-rays, passing through the crystal, will be scattered by its atoms, and then interfere with each other like waves passing through a breakwater gap. In some places, the waves will amplify, and in others, they will cancel each other out. The resulting diffraction pattern can be used to reverse-calculate the location of the atoms scattering the original X-rays. Laue and his colleagues proved their theory in 1912 using a copper sulfate sample, laying the foundation for X-ray structural analysis (2).

X-ray structural analysis plays a fundamental role in many scientific fields, since it allows studying the structure of not only crystals of salts and minerals, but also metals, semiconductors, inorganic and organic compounds and, which is especially important, biological molecules (1, 2). In 1952 using X-ray crystallography Rosalind Franklin obtained a photographic image of DNA, which helped James Watson and Francis Crick to create their famous model of the double helix structure of DNA (2).


X-ray diffraction picture of crystallized DNA taken by Rosalind Franklin, known as Photo 51, helped James Watson and Francis Crick create their famous double helix model. The atomic resolution image of the structure, proposed in 1953, was obtained only in 1980 (2).

Thanks to improvements in the method, even large viruses and proteins can be “seen” (2). The importance of XRD for science reflects the fact that 25 Nobel Prizes have been awarded for work in this area since the discovery of X-rays in 1901.

Mass spectrometry
Mass spectrometry (MS) is the most powerful technique for the qualitative and quantitative analysis of various compounds. Mass spectrometry is like "weighing" of molecules in a sample. However, how this could be done if the weight of even such a large molecule as the protein hemoglobin is only about 10-20 g? This is where the principle discovered by Sir Joseph J Thomson,
Nobel Prize winner in physics , comes to the rescue. Scientists have shown that charged particles in the presence of a magnetic or electric field establish their trajectory according to their mass-to-charge ratio (3).

MS process includes three main stages. At the first stage, samples are converted to the gas phase (vaporized) and passed into ionization chamber where molecules are bombarded by a stream of electrons to generate positively charged ions (ionization). At the second stage, the ions are sorted according to their mass. They are accelerated to form a beam from which they are deflected by a magnetic field according to the mass-to-charge ratio. At the third stage, the deflected ions pass through a detector and generate electric current that is proportional to the abundance of the ion. At the same time, by varying the strength of the magnetic field a mass spectrum is generated. The resulting distribution of masses and charges is characteristic for a given molecule (3). The seemingly simple physical concept of MS requires a number of non-trivial solutions for practical implementation. For example, the process of ionization differs between inorganic and organic compounds. Also, biological molecules (proteins, DNA) and many organic compounds (polymers) are difficult to vaporize without decomposition. Therefore, to solve specific problems, mass spectrometers of various design have been developed.

Mass spectrometry is used in many different fields. For example:
• in pharmaceutical field - in the development of drugs, pharmacokinetics studies and production control;
• in scientific research - for the analysis of proteins and nucleic acids;
• in medical diagnostics - for the detection of infectious agents and doping control in sports;
• to test food and water quality; detect toxins;
• in forensic science - to trace illicit drugs and explosives; in the oil and gas industry and even in nuclear power (3).

Mass spectrometers travel into space as an integral part of the equipment on spacecraft and probes. They were sent to the Moon, Mars, and even Saturn's satellite Titan (aboard the Huygens probe, the device that has made the most distant landing to date) (4-6). The use of MS in real time during surgical operations is innovative. The method makes it possible to distinguish healthy tissue from, for example, a tumor, which enables the surgeon to act with extraordinary precision (3, 7).

Chromatography is a highly effective and widely used technique for separation of individual components of a mixture. Translated from Greek, chromatography means "color writing"; this name was given to the method at the very beginning of the 20th century by its inventor, the Russian botanist Mikhail Semenovich Tsvet, who studied brightly colored plant pigments (8, 9). In 1918, Tsvet was nominated for the Nobel Prize (8), and the English biochemists Archer J.P. Martin and Richard L.M. Synge received the award in 1952 for the development of this method.

The method is based on the distribution of the components of the analyzed sample between two phases - stationary and mobile. The stationary phase is a porous solid material (adsorbent or matrix) or a thin liquid film on a solid support. Typically, the stationary phase is packed in a glass or metal tube called a column. A mobile phase is a liquid (solvent) or gas flowing through a stationary phase, sometimes under pressure. The components of the analyzed mixture move with the solvent along the stationary phase where they interact with the matrix surface with different strengths due to their individual properties and the nature of the interaction (adsorption or other mechanism) and, therefore, will pass through the column at different speeds. This makes it possible to separate the components of the mixture (8, 9). An analogy can be drawn with a marathon. If you look from a great height at the starting line when everyone is in the same crowd - it is not easy to distinguish the individual athletes. However, when the race proceeds along the course, the crowd of runners appears to thin out and it becomes possible to identify each athlete. So in chromatography, separation of complex mixtures occurs - some components will remain in the upper layer of the sorbent, others, to a lesser extent interacting with the sorbent, will reach the lower part of the column, and some will completely leave the column together with the mobile phase (9).

There are several different types of chromatography. They can be classified according to the physical state of the phases (gas, liquid), the retention mechanism (adsorption, ion exchange, affinity, etc.) and the geometry (columnar, planar) (9). In general, the following advantages of chromatographic techniques can be highlighted:
• Efficiency of separation due to the possibility of multiple repetition of adsorption and desorption;
• The ability to use completely different types of interaction of the analyte with the stationary phase: from purely physical and chemical to biospecific. This allows a very wide range of substances to be separated;
• Various fields (gravitational, electric, magnetic, etc.) can influence the separation conditions.

Chromatography is a hybrid method that allows to simultaneously separate, identify, purify, isolate and concentrate various compounds, and these tasks can be achieved simultaneously by performing them in real time (9). Hardly any other technique can compete with chromatography in versatility (from the analysis of the contents of a living cell to the atmospheres of the planets of the Solar System) and the efficiency of separation of the most complex multicomponent mixtures (isolation of more than a thousand of individual components in one analysis) (8). The method is indispensable in forensics, environmental monitoring, determination of water quality, medical diagnostics, pharmaceutical, chemical and petrochemical, gas, food and many other industries. Chromatography is used in archeology to study ancient dyes, varnishes, and materials (8). Thanks to the combination of chromatography with other methods, it became possible to decipher DNA sequence and complete the Human Genome project (8). Chromatography can be used to date organic material going back 1 million years and well beyond the range of the radiocarbon dating (8).

Chromatography–mass spectrometry
Extremely high accuracy and information content of the analysis was achieved by combining chromatography and mass spectrometry; this is one of the most effective methods for analyzing complex mixtures (9). The analytical capabilities of these methods complement each other perfectly. For example, the use of a gas chromatograph mass spectrometer (GC/MS) found no evidence of life on Mars (the detection limit for organic substances was 10-7%) (9). GC/MS is widely used for the determination of volatile organic compounds in urban and industrial air. GC/MS is currently practically the only acceptable method for the determination of some pollutants (for example, organometallic compounds). It is a key method of soil and water analysis, allowing the identification of most pollutants and their determination at extremely low levels. The sensitivity of GC/MS for some substances is 0.1 μg / L, and the detection limit for substances such as, for example, phenols and chlorophenols is up to 5-20 ng / L (9).

NMR spectroscopy
Nuclear magnetic resonance (NMR) is an interdisciplinary method for visualizing and studying chemical and biological objects. The phenomenon of NMR was discovered by the American scientist Isidor I Rabi in 1938, for which in 1944 he
received the Nobel Prize in Physics. NMR is widely used for spectroscopic analysis in physics and chemistry. The method made its way into medical diagnostics as well. For the development of the technique of NMR tomography (or as it is now called - magnetic resonance imaging, MRI) in the 70s of the last century, the American chemist Paul C. Lauterbur and the English physicist Sir Peter Mansfield were awarded the Nobel Prize in Physiology or Medicine in 2003. However, this award caused controversy, as an American physician and inventor, Raymond Damadian, one of the first researchers of the principles of MRI and the creator of the first commercial MRI scanner, publicly announced that he should also share the award. “Had I never been born, there would be no MRI today” Damadian said after the announcement of the award. Interestingly, Soviet scientist Vladislav Ivanov was the first to develop this method back in 1960, but his patent application was rejected. However, unlike Damadian, Ivanov never claimed the prize (10).

The physical processes underlying NMR spectroscopy are quite complex. The method is based on the absorption of electromagnetic radiation by the nuclei of a sample placed in a magnetic field. The nuclei of atoms spin on an axis. Since nuclei are composed of positively charged protons, the spinning produces a magnetic field, the poles of which are located on the axis of rotation. Usually, the rotation axes of various molecules are randomly distributed, but when placed in an external magnetic field, their directions become aligned with or against the field (similar to how a compass needle is aligned in the Earth's magnetic field). Perturbation of this alignment leads to a resonance of atomic nuclei, and as a result, they emit electromagnetic radiation. The extent of excitation and decay of such resonant radiation are registered. The hydrogen nuclei in water molecules provide the highest resonance signal and this is exactly what is measured in medical diagnostics.

Polymerase chain reaction
Polymerase chain reaction (PCR) is a powerful molecular biology technique everybody has heard about. The discovery of PCR has become one of the most prominent developments in the field of molecular biology over the past half century. The method has gained enormous practical application in completely different fields, from medical diagnostics, to industry, agriculture, and even forensic science (11). The PCR method was invented in 1983 by an ordinary employee of the Cetus Corporation Kary B. Mullis. For this discovery, he was
awarded the Nobel Prize in Chemistry in 1993.

PCR technique can significantly increase small concentrations of a specific segment of DNA in a biological sample. If a sample (for example, blood) contains only very few DNA molecules of interest (for example, bacterial or viral DNA), PCR can generate enough copies to detect them. The method is based on making multiple copies (amplification) of a specific target region of DNA (template) by a special enzyme, DNA polymerase, during temperature cycles. At each cycle of amplification, the previously synthesized fragments are copied again. Thus, the number of specific DNA fragments increases exponentially. Depending on the initial quantity of template and the efficiency of the reaction, 20 to 50 PCR cycles are required. It is thought that even starting with a single copy of DNA molecule and using 40 cycles of highly efficient PCR, it is possible to obtain a sufficient amount of the reaction product for a subsequent detection and analysis (11). The reaction is run in a special device – a thermocycler that performs the heating and cooling cycles required for DNA amplification. At the end of the reaction, the presence of amplified DNA fragments is detected using various methods. It is important to note that bacteria living in hot springs have helped to significantly simplify PCR and make it a routine technique. The fact is that, unlike previously used enzymes, their DNA polymerase withstands high temperatures (necessary for the reaction) and retains its biological activity after heating.

However, the classical PCR technique is limited by the ability to accurately determine the amount of template present in a sample. The registration of the result at the endpoint of the reaction does not provide information about the effectiveness of the process. In terms of the amount of information content, this can be compared with a still photo of a figure skater, according to which the judges must give marks for the technique and artistry of the entire performance. Therefore, in the early 1990s, it was proposed to record the accumulation of amplification products during the entire PCR reaction. This approach was called "real-time" or quantitative PCR, and made it possible to accurately measure the initial number of templates in a sample. Thus, the researcher obtains much more information about the sample. Continuing the analogy, we can say that this is like a video footage of a skater's performance (11).

The range of applications for which PCR is used is constantly expanding. This includes:
• Scientific research and medical diagnostics (for example, determining the level of bacterial or viral load, cancer diagnostics);
• Paternity testing, forensic science (personal identification);
• Microbiological environmental monitoring;
• Food quality tests;
• Searching for elements of genetically engineered constructs.

Even the most knowledge-intensive analytical methods are simply tools to conduct research. However, it is not the methods themselves that are essential, but the correct formulation of a question they help to find an answer. In turn, the discovery of each method listed in this article became the correct answer to a good and, importantly, timely asked question. Or, as Honore de Balzac said: “The key to all sciences is unquestionably the question mark”.

1.    https://en.wikipedia.org/wiki/X-ray_crystallography.

2.    Jones N. (2014). Crystallography: Atomic secrets. Nature, 505(7485): 602–603.

3.    Maher S., Jjunju F.P.M., Taylor S. (2015). Colloquium: 100 years of mass spectrometry: Perspectives and future trends. Reviews of Modern Physics, 87(1), 113–135.

4.    Yokota S. (2018). Isotope Mass Spectrometry in the Solar System Exploration. Mass spectrometry (Tokyo, Japan), 7(2), S0076.

5.    The Connection between Mass Spectrometry and Space Exploration. 2016

6.    Arnaud C.H. (2018). Sending mass specs to Mars. Chemical & Engineering News, 96 (22).

7.    Woolman M., Qiu J., Fischer C., Zarrine-Afsar A. (2020). In situ tissue pathology from spatially encoded mass spectrometry classifiers visualized in real time through augmented reality. Chemical Science.

8.    Ettre LS, Zlatkis A, eds. (26 August 2011). 75 Years of Chromatography: A Historical Dialogue. Elsevier. ISBN 978-0-08-085817-3.

9.    https://en.wikipedia.org/wiki/Chromatography

10.    MacWilliams B. (2003). Russian claims first in magnetic imaging. Nature, 426: 375.

11.    https://en.wikipedia.org/wiki/Polymerase_chain_reaction