Methods Used in the Bio-Medical Industry

In this essay we will be exploring three methods by which materials are either separated, analysed, or both and their relevance and application in the biomedical industry. We will be looking at Electrophoresis, Nuclear Magnetic Resonance (NMR), and Gas Liquid Chromatography-Mass Spectrometry (GLC-MS). Electrophoresis is a technique used to separate DNA material based on their size which has applications in DNA forensics. Nuclear Magnetic Resonance is a technique used to visually determine what the composition of a live tissue is which has applications in medical science. Gas Liquid Chromatography-Mass Spectrometry is a technique used to determine the chemical composition of the substance that is being tested which has applications in blood doping in sports where the blood needs to be chemically analysed for its composition whether it contains banned substances. Chemistry is a very broad subject which has influence in almost every industry. This essay will attempt to cover these three methods knowing that it has only skimmed the surface.

The first separation technique that we will discuss is electrophoresis. Electrophoresis is used extensively in biochemical analysis. In particular, it is used in DNA fingerprinting and profiling in the field of forensic science. It can be used to separate, identify and purify proteins and nucleic acids. It can be used with amino acids and peptides obtained when a protein is hydrolysed. This basis for how this method works is that it depends on the fact that all DNA molecules are polar. Thus it is known to be impossible for there to have a compound with the same polarities. Another issue that might be raised is would the mass of the sample affect this separation technique? The answer is yes and no. It will affect it by making the DNA fingerprinting band hard to form. Thus chemists have developed different agarose medium gels for different compounds that have different properties such as pH and mass. The agarose gel can differ in density and pH, for example, to accommodate the different types of sample that is being tested. In that way, the effect that mass or even pH might on the result is negated and an accurate result is produced.

The DNA of each person is basically similar in its chemical structure. The two strands in the double helix of DNA are held in place via hydrogen bonds between base pairs. The DNA stores the information – call the genes – that provide the genetic blueprints for making proteins. However, there are segments along the DNA molecules which do not seem to carry the instructions needed to make proteins. These bits of DNA are repeated along the DNA molecule. They are called ‘minisatellites’. The number and sequence of these is unique to each person.

DNA fingerprinting is based on matching these minisatellite regions of DNA. We inherit half from our mother and the other half from our father.

How does it work? Firstly, DNA would be extracted from a sample such as a murder weapon. Next, Restriction enzymes are used to ‘cut’ the DNA molecule at specific places where the same sequences occurs, making smaller fragments for analysis. Because DNA fragments are all negatively charged because of the phosphate groups present them will all move towards the positive electrode in gel electrophoresis. When they move towards the positive electrode in gel electrophoresis, the fragments move at different rates because they have different sizes. And this creates bands. The bands are then made visible by radioactive labelling of the bands with the phosphorus-32 isotope, which causes photographic film to fog. Thus the result is a film that can reveal the positions of the bands and by inference, the identity of the person whose DNA is being tested upon.

The analytical technique of electrophoresis is based on separating ions placed in an electric field. If a sample is placed between two electrodes, positively charged ions will move towards a negatively charged electrode. Negatively charged ions will move towards a positively charged electrode.

The sample is placed on absorbent paper or on a gel supported on a solid base such as a glass plate. A buffer solution carries the ions along. A buffer solution or medium is used in this method. This is to not only provide a means for the electricity to separate the ions but also as a means to stabilize the pH level because it will affect the movement of ions during electrophoresis. The rate at which the ions move towards the oppositely charged electrode depends, amongst other things, on the size and charge on the ions: larger ions will move more slowly; highly charged ions will move more quickly. Therefore the ions are separated as the electric field is applied. A series of lines or bands on the paper or gel appears once a chemical is applied. Sometimes ultraviolet light is used to show the bands up. The series of bands is called an electropherogram. The bands form a sort of fingerprint as every DNA will show up a different series of bands. In the same way that a thumbprint is unique to a person, these bands made by DNA is unique to every person.

A particular limitation is that this experiment does require electricity, an agarose gel medium, a container to store the gel, and it requires a lab free from impurities as it has a high intolerance for contaminants. This may limit the reach of DNA fingerprinting in rural areas or places in third-world countries where access to a biologically clean lab may be difficult.

The transport equation for electrophoresis is – .

C would represent the concentration of the substance undergoing electrophoresis and t wold represent the transport after progressing for a time. This equation explains how time actually affects the concentration of the substance. (Jordan and Mills, 1966)

The next technique that we will look at is an analytical technique called the nuclear magnetic resonance (NMR). NMR is mainly used to diagnose medical problems. The technique of MRI (Magnetic Resonance Imaging) scanning has been adapted from NMR spectroscopy. The patient is placed inside a body scanner which generates a powerful magnetic field. A computer analyses the radiowaves absorbed by ¹H nuclei in successive ‘slices’ of the body, combining these to make a 3-D image of organs inside the body. The reason that a 3D picture of an organ can be produced just by flipping protons in different magnetic environments can be explained as such. By flipping the protons, a magnetic wave is produced. This wave contains energy that can be measured. When scanning the body, the strength and pattern of this wave is hugely affected by the type, density, and weight of the body that is being measured. Different parts of the body will give off a different wave because not all parts of the body are the same. Some parts of the body contain more muscle or bone than other parts. Thus after scanning the body, data is collected from scanning the different parts of the body that yield different results. Computer imaging software then processes the data that has been collected by the MRI machine and produces a 3D image based on the type of data that it receives. In this way NMR can be explained as such. MRI is much safer than high-energy X-ray imaging. As an example of its use, MRI can monitor the success of cancer treatment in reducing the size of tumours.

Nuclear magnetic resonance (NMR) spectroscopy is a widely used analytical technique for organic compounds. NMR is based on the fact that the nucleus of each hydrogen atom in an organic molecule behaves like a tiny magnet. The nucleus of a hydrogen atom consists of a single proton. The proton can spin. This movement of the positively charged proton causes a very small magnetic field to be set up.

In NMR the sample is goes to be analysed in a magnetic field. The hydrogen nuclei (protons) either line up with the field or, by spinning in the opposite direction, line up against it.

There is a tiny difference in energy between the oppositely spinning ¹H nuclei. This difference corresponds to the energy carried by waves in the radiowave range of the electromagnetic radiation spectrum. In NMR spectroscopy the nuclei ‘flip’ between the two energy levels. Only atoms whose mass number is an odd number, e.g. ¹H or ¹³C, absorb energy in the range of frequencies that are analysed.

The size of the gap between the nuclear energy levels varies slightly, depending on the other atoms in the molecule (the molecular environment). Therefore, NMR can be used to identify ¹H atoms in different parts of a molecule. In NMR spectroscopy, we vary the magnetic field as that is easier than varying the wavelength of radiowaves. As the magnetic field is varied, the ¹H nuclei in different molecular environments flip at different field strengths. The different field strengths are measured relative to a reference compound which is given a value of zero. The standard compound chosen is tetramethylsilane (TMS). TMS was chosen because it is an inert, volatile liquid which mixes well with most organic compounds. Its formula is Si (CH₃)₄, so all its H atoms are equivalent (i.e. they are all in the same molecular environment). TMS only gives one, sharp absorption, called a peak, and this peak is at a higher frequency than most other protons. All other absorptions are measured by their shift away from the TMS line on the NMR spectrum. This is called the chemical shift (δ), and is measured in units of parts per million (ppm).

The spins within the MRI possess a natural frequency that is proportional to the magnetic field. This is called the Larmor relationship equation. This equation explains the method behind the MRI.

Larmor relationship equation – ω = γB

Some limitations that can be inferred from data would be that portability, the need for a large amount of electricity, the exclusion of people with tattoos that has ink mixed with metal, people with pacemakers, morbidly obese people, or people who are claustrophobic.

Lastly we will look at a separation and analytical technique called the gas-liquid chromatography/mass spectrometer technique. To identify the components in a mixture, it is possible to link a gas-liquid chromatography (GLC) apparatus directly to a mass spectrometer.

This combined technique is very sensitive, and any two solutes that can be separated with a time gap of 1 second on a GLC column can be identified almost instantly by the mass spectrometer without the need to be collected. Identification is by comparing the mass spectrum of each solute with the mass spectra of known compounds, using a computer’s spectral database. The generated is complex. There can be many components in a mixture, each with a peak at its particular retention time on the chromatogram, and each peak will generate its own characteristic series of lines in the mass spectrometer. It is possible to combine the chromatogram and the mass spectra to display the data on a 3-D graph.

GLC linked to a mass spectrometer is used for analysing complex mixtures. The combined technique is fast and gives reliable results that can identify trace quantities of pollutants, drugs, biochemical molecules and toxins. This means it is used in: forensics, environmental monitoring of pollutants, drug testing in sports, geological and archaeological dating, or even airport security.

Gas-liquid chromatography, referred to as GLC, uses partitioning to separate and identify the components in a mixture.

How does it work? First, an inert carrier gas such as nitrogen passes in the gas chromatograph to flush the mixture of vapours through the instrument. The mixture to be separated is injected into the instrument through a self-sealing rubber port. Next, an oven heats the injector to vaporise the contents of the mixture, to turn the mixture into a gas if it is not a gas yet. The sample passes through a snail like column oven. After which, the column oven keeps the mixture inside the column in the gaseous state and at a constant temperature. Within the long and thin column there will be a stationary phase, which is often a non-volatile liquid coated onto a solid support. Next, the components of the mixture interact with the stationary phase to different extents, so they move through the column at different rates. Then it passes the sample on to the mass spectrometer to be identified through a tube that is kept at a warm temperature.

The stationary phase is an inert carrier gas. This is packed tightly into a column. This has to be forced under pressure through the densely packed column where separation occurs. The tiny solid particles in the column have a very large surface area over which partitioning can occur, resulting in excellent separation. The more polar components in the mixture have a greater relative solubility in the polar solvent. Therefore they are carried through the column faster than components whose molecules are more non-polar (which dissolves better in the non-polar stationary phase in the column). The detector records retention times, i.e. how long it takes each component to pass through the column. The area under each peak recorded is proportional to the amount of solute emerging from the column.

For quantitative analysis, the component peaks are first identified and then the area of each is measured. The peaks are roughly triangular in shape so their area follows the area formula.

Area is

The sample would now go into the mass spectrometer where it will analyse the components of the mixture as they emerge from the column. In the mass spectrometer’s results you can determine the relative proportions of the components of the mixture (from the relative areas of the peaks obtained from the recorded current flow) and the identity of each substance (by matching their mass spectra against a computer database of know spectra (fingerprinting).

How does the mass spectrometer separate and identify the gas? First, The vacuum pump first removes unwanted previous sample and air which could interact with the sample by either colliding or reacting with the sample which would contaminate the sample and interfere with the final result. The sample then enters through the sample inlet to proceed to the inside of the mass spectrometer. Next, the sample would enter the vaporisation chamber where a heating coil in the vaporisation chamber converts the sample to a gas if it had not been a gas already. Following that, the sample would proceed into the ionisation chamber where an electron gun bombards electrons at the gaseous sample converting them to positive ions. The equation for this is – . The sample at this stage would be go to the accelerator which contains negatively charged electric plates where it accelerates the ions towards the magnetic field. The gaseous ions when approaching the magnetic field would be separated according to their mass to charge ratio (). The lighter ions are deflected more, and the heavier ions are deflected less. At the end of the sample’s route is an ion detector where the signal is converted to an electric one. The electric signal is sent to a recorder that interprets this data and plots a graph for analysis because the ions hit the recorder in different positions according to their mass to charge ratio. Lastly, the position where the ions hit the detector plate tells you their relative atomic mass.

A practical application for a gas-liquid chromatography/mass spectrometer is in the analysis of urine samples from athletes for banned substances such as steroids or stimulants or even in medical research to separate peptides and proteins. Some advantages of using this method in separating and analysing the sample is that it is possible to determine the percentages of dissolved oxygen, nitrogen, carbon dioxide and carbon monoxide in blood samples as small as 1.0 cm³. Some of the disadvantages are that similar compounds will have similar retention times and if a newly discovered compound is detected it will not have a match in the computer’s database of retention points.

As a conclusion, electrophoresis, nuclear magnetic resonance (NMR), and gas-liquid chromatography/mass spectrometer are techniques that affect everyday life. Electrophoresis has a useful function in helping to match the DNA from a crime scene to the criminal in cases where more evidence were need to convict the right person. The nuclear magnetic resonance machines have a the ability to form a 3D image of your inner body and that is useful in the case where detailed analysis of patients in a hospital is important. Other techniques for analysis are mostly either too invasive or too slow. Last but not least is the gas-liquid chromatography/mass spectrometer whose function is to separate compounds to analyse the composition.

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