Impact of Chromatography on Life and Society

Tony Edge, Vice-President, The Chromatographic Society, UK, and Agilent Technologies, Stockport, Cheshire, UK.

A theme for The Chromatographic Society Diamond Jubilee Year is to increase public awareness of chromatography. Often the general public, and even chromatographers themselves, fail to appreciate the importance of chromatography. Here Tony Edge, The Chromatographic Society Vice-President, discusses just how much chromatography underpins everyday life.

From the development of separation science the world of analytical chemistry has been radically transformed in the type of analyses that can be performed. It affects many aspects of our lives, from the food that we eat and the medication that we use, to the better understanding of life outside of our planet. Without chromatography the world would be a less wealthy, less clean, and a less healthy place. So how is chromatography actually incorporated into these many varied aspects of our lives? In this article, examples of separation science are discussed alongside how the use of this critical science affects our standard of living and also our understanding of our place within the Universe.

Space Exploration

The Universe is one of the more esoteric areas where separation science has been employed. Chromatography has been used extensively in space to help look for the building blocks of life. As far back as the Viking missions to Mars in 1976, gas chromatography (GC) was being used to help scientists gain a better understanding of the solar system and the possibility of life on other planets. This analytical procedure was chosen because it is a robust technology that can separate relatively complex samples. One of the challenges that faces many separation scientists is accessing the level of technical support that is required to ensure that the chromatographic instrumentation is working in an appropriate fashion. The challenge of finding an appropriate service engineer is amplified substantially when dealing with extraterrestrial installations.

Another one of the challenges that faces extraterrestrial installations is ensuring that the analytical technology used is appropriate for the sample. Selection of the correct column, temperature gradient, and detector is critical for ensuring that the analysis is successful, and this is very dependent on knowledge of what the sample is. Thus, perversely, having a good indication of what the sample is will actually ensure that the best separation is achieved. This presents an obvious challenge in space where ordering a different column is simply not an option.

In general, when considering the selection of a detector, the more universal its applicability the better, because it will allow for a greater number of analytes to be detected. The first mission to Mars used a thermal conductivity detector (TCD), with the robustness and general applicability of this type of detector being a major consideration. However, there are limitations on the type of detectors that can be used; thus, flame ionization detectors (FIDs) are not typically used in space because of the consideration of transportation of the air and hydrogen gas as well as the more obvious risk of operating a flame at distance.

Sample pretreatment is also something that needs to be considered. In most laboratories, weighing out a sample and mixing with a solvent, sieving, and analyzing the resultant supernatant is considered routine: doing this remotely presents severe challenges. The application of more complicated pretreatments presents even more challenging issues. However, there have been a range of different sample matrices that have been analyzed in space including soil, ice, and dust.

Another consideration for space analysis is that the size and weight are absolutely critical parameters. Thus the electrical supply and capabilities of the instrumentation have to be very carefully considered. More complex instrumentation will require larger batteries, which will require a heavier fuel payload. For Mars, solar panels are effective at generating electricity, but for distances further away from the sun, this approach is not viable: examples here include the Rosetta and Cassini-Huygens missions.

As experience grew with extraterrestrial missions so the complexity of analyses developed. The use of GC coupled to mass spectrometry (MS) was demonstrated with the second Viking mission to Mars, and designs for using liquid chromatography (LC)–MS are very advanced. The use of LC in space is an exciting development, but also presents many challenges associated with the storage of the liquid and what to do with the waste.

It is quite often that a news reporter will state that NASA has identified chemical reagents that hint at life, but what is it that they are actually looking for? In many cases it is simple organic species and an understanding of the gas composition. All of this information helps scientists to determine if there is life or the potential for life on distant planets. However, one of the challenges that scientists face is contamination and there have been too many false hopes of detecting life, which subsequently have been found to be a contamination issue.

To date chromatographic systems have been sent to our moon (Apollo), Saturn (Cassini-Huygens), comets, (Rosseta), and to Mars (Viking, Curiosity).

Popular Culture

Chromatography has been seen in many forms of popular culture in particular on radio, television, and also in films. On the radio, the UK’s BBC Radio 4 recently devoted an hour to the discussion of what chromatography is and the applications of chromatography, with an expert panel from industry representing separation scientists, which included Dr Leon Barron, Professor Apryll Stalcup, and Professor Andrea Sella. Beyond the radio and moving into television, separation science has also been seen in The Simpsons on several occasions, including when Martin Prince gave a presentation on Archer Martin. High performance liquid chromatography (HPLC) was also seen in Professor Jonathan Frink’s laboratory in another episode. Beyond the cartoon characters, chromatography can also be seen in the popular crime scene investigation (CSI) programmes. The creators of these programmes do use technical help from a range of analytical and forensic scientists to ensure that the commentary associated with the text on these programmes is factually correct, although there have been examples where this has not always been the case. For example, trying to analyze the residuals of liquid nitrogen may prove challenging. Some of the laboratories shown are actual working laboratories, with one of them based on the R&D centre of Thermo Fisher Scientific in San Jose, USA.

In the real world, in addition to their laboratory role, forensic scientists will also testify as expert witnesses in both criminal and civil cases, and can work for either the prosecution or the defence. While any field could technically be forensic, certain sections have developed over time to encompass the majority of forensically related cases. So what exactly does chromatography have to do with forensics though? Chromatography is used wherever it is necessary to determine an organic chemical, such as a poison in the body, or residues of gunpowder, or indeed to try and identify the type of rubber used in a car tyre, as was noted in the film My Cousin Vinny. Samples taken from the scene of a crime may require some form of sample preparation to convert the raw sample into a form that is suitable for chromatographic analysis, or sometimes it is possible to analyze the sample directly (for example if it is suspected that the contents of a glass of water have been contaminated with a deadly poison). In general the chromatographic technique is coupled to MS to aid identification and quantification.

Within the film industry, chromatography has also been incorporated successfully to ensure a viable plot. This was particularly true in the much acclaimed film, Medicine Man, starring Sean Connery. Connery, who plays the lead character, is seen as a loner employed by a pharmaceutical company to discover new drugs from the Amazonian rainforest where he has been sent. Connery’s predecessor is a local medicine man who has the cure for cancer, which he demonstrates when a boy is brought to him with malignant neoplasms. Connery, along with another colleague from the pharmaceutical company, then goes in search of the elusive component that can cure one of man’s deadliest enemies. This is initially focused on the flowers the medicine man is seen using to treat the boy. Using a gas chromatograph, Connery is able to confirm that the flowers are not in fact the source of the cure, but that the source comes from an endangered species of ants, who, with a degree of irony, get wiped out by a subsequent fire after the cure for cancer is found using the GC–MS. There are a few inaccuracies in the film, relating to the use of suitable gas cylinders and identification of an unknown compound, but in general the film portrays an accurate reflection of an analytical laboratory.

However, it is not just the arts where chromatography has found a new home. In the world of finance chromatography has also appeared, with an old-style HPLC system on the back of the Bank of Scotland £20 note. Many suggestions have been put forward as to which system is being used is but to date none have been officially verified.

The pumping system is undoubtedly a gradient system using two Waters M6000A pumps, but the identity of the detector is less certain The chromatographer in the image is Janet Mullen, who took over from Betty Trafford as Wolfson Unit technician in around 1977. Other Wolfson members at that time, other than Knox himself, were Dick Wall, who played a principal role in the development of a would-be Shandon LC system, and post-docs John Done and Jadwiga Jurand. Others who worked with Knox outside the Wolfson Unit were post-doc Andrew Pryde and PhD student Forbes Mclennan, who were succeeded by Mary Gilbert and Harry Ritchie, respectively.

Food Analysis

The largest industry in the world is the food industry, which is worth about 10% of the global GDP, or about 10 trillion dollars. Food is an essential requirement for life and, as such, it is critical that appropriate measures are put in place to ensure that the quality of the food is at a high level. Unfortunately, because of the value of this industry, it is often targeted by criminals and cheaper substitutes can be introduced to improve profit margins. However, in some cases these substitutes can be harmful to humans, such as when melamine was substituted for milk powder in China. In some cases the substitutes are deemed to be legal and in general these will affect the quality of the food. For example, the use of monosodium glutamate, which is used to enhance the taste of food but has little nutritional value.

Drug Testing

Many of the drugs that the pharmaceutical industry have developed have a variety of side effects, some of which at higher concentrations can have a stimulating effect on the user. However, these drugs can be very addictive and long-term use will invariably result in adverse effects for the user, and ultimately this could result in death. Substance abuse is widespread, with an estimated 120 million users of hard drugs such as cocaine, heroin, and other synthetic drugs. In 2013, drug use disorders resulted in 127,000 deaths, up from 53,000 in 1990. The highest number of deaths are from opioid use disorders at 51,000. Cocaine use disorder resulted in 4300 deaths and amphetamine use disorder resulted in 3800 deaths. Alcohol use disorders resulted in an additional 139,000 deaths. Governments around the world have taken steps to address the high death rates associated with the abuse of drugs, but in order to determine what an individual is using the world of separation science is required.

These approaches work to identify the original drug and to identify if a suspect has been using a drug. Unlike in the films and various TV situational dramas, it is not possible to determine by taste what a particular drug is. For seizures of drugs in their pure form, the analysis is relatively simple and requires dilution of the sample in an appropriate solvent before analysis can be performed. Over the years the approach to drug analysis has changed, with recent innovations such as GC–MS and LC–MS/MS becoming the mainstream approach for confirmation.

Urine or blood sampling is typically employed when testing a potential drug user and will require some form of sample preparation. This could be to remove the matrix in blood or plasma samples or indeed a simple dilution for urine analysis. Sample dilution is used ever increasingly when LC–MS/MS is used as the analytical technique because of the specificity and sensitivity of the technique. Even when mass spectrometry is involved, the separation is required to minimize ion suppression effects because it is essential that the data can be relied upon.

Newborn Screening

The first interaction that most people have with chromatography is the heel prick test, which was developed by Robert Guthrie for the analysis of phenylketonuria, a condition where the body cannot take in phenylanaline, resulting in neurological damage. The initial test was developed in the early 1960s shortly after Guthrie’s 15-month‑old niece had been diagnosed with phenylketonuria, and used a bacterial inhibition assay, but it was soon realized that the approach could be applied to the diagnosis of many other compounds. The test involves taking a spot of blood from a heel prick on the newborn onto a piece of what is essentially blotting paper. This original sample can then be analyzed in a laboratory.

Across the world many of the 134 million babies born each year are tested using the heel prick test. This type of analysis has developed and now many of the test compounds are analyzed using LC–MS/MS. In the UK the tests that use separation science
include:

• phenylanaline (phenylketonuria)

• octanylcarnitine/decanoylcarnitine (MCADD - Medium chain acyl‑CoA dehydrogenase deficiency)

• amino acid profile

• propionic acidemia, methylmalonic acidemia, and isovaleric acidemia

• disorders of the distal urea cycle, such as citrullinemia, argininosuccinic aciduria, and argininemia

• maple syrup urine disease (MSUD) - Leucine

• homocystinuria - methionine

• Isovalerylacidaemia (IVA) - C5 (Isovalerylcarnitine)

• glutaric acidaemia type 1 (GA1) - C5DC (glutarylcarnitine)

• LCHADD (Long chain 3-OH acyl co-A dehydrogenase deficiency) - C16OH (3-OH palmitoylcarnitine).

Other tests are being developed to allow greater use of LC–MS/MS technology. In this area the use of chromatography is key to ensuring our children are fit and healthy.

Sports Science

Sports science can be traced back to its origins in Ancient Greece. The noted Ancient Greek physician Galen (131–201) wrote 87 detailed essays about improving health (proper nutrition), aerobic fitness, and strengthening muscles. Assyrian Hunayn ibn Ishaq translated Galen’s work, along with that of Hippocrates, into Arabic, which led to the spread of Greek physiology throughout the Middle East and Europe. This was very much the beginning of sports science and the thirst to get a better understanding of how the human body works would eventually lead to the involvement of separation scientists.

New ideas upon the working and functioning of the human body emerged during the Renaissance as anatomists and physicians challenged the previously known theories. These new scholars went beyond the simplistic notions of the early Greek physicians and shed light upon the complexities of the circulatory and digestive systems. Today chromatography is an essential analytical tool to fully describe these processes and a better understanding allows athletes to train harder and to get more from their training sessions, with careful monitoring of metabolic markers.

2016 is not only the 60th anniversary of the ChromSoc, but is also marks another Olympic year. It is sad to note that separation science, as well as helping improve the performance of athletes in a legal manner, is also employed to allow illegal improvements in performances to be detected.The use of a range of drugs has been noted by many athletes and trainers to improve performance. However, these drugs are dangerous and the long-term effects are inevitably detrimental to the athlete concerned.

Conclusion

These are just a few of the areas that separation science plays a fundamental role. There are many other areas where separation scientists are employed, but in this brief snapshot it is not feasible to highlight them all. In a world that craves information, it is analytical scientists that feed that desire, and amongst analytical scientists, chromatographers are without doubt one of the leading resources. As the world develops, it is envisaged that separation scientists will continue to play a critical role in ensuring that the world is healthier, cleaner, and wealthier.

Tony Edge is R&D Manager, Polymeric Materials, Agilent CrossLab Group and Vice‑President of The Chromatographic Society.