If you take any medicine or wear makeup or are interested in identifying targeted therapies for currently incurable diseases, bioseparation is for you.
What is bioseparation?
Bioseparation is a technique to separate small scale molecules. It is very useful in the biopharmaceutical industry when purifying proteins and other chemical substances. The separations can also include: protein, DNA, antibiotics, vitamins, etc. which are useful in large scale productions of drugs, etc. There are two primary parts to bioseparation: separation and detection.
Bioseparation is very important because of its applications in the pharmaceutical field with purifying drugs along with in the cosmetic industry for purifying different types of makeup. It is also very important on a biological level, as bioseparation techniques are currently being improved in order to separate live tissue which can help us identify targeted therapies for certain diseases.
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Separation methods include filtration, distillation, centrifugation, and chromatography. Detection methods often are UV spec or mass spec which are used to identify whether a compound has fully been purified.
The problem: The primary issue currently is that bioseparation techniques are not the most efficient or effective. Improvements are currently being made, but different bioseparation methods serve different purposes and have a variety of applications.
Different bioseparation methods:
Background: Filtration is one type of bioseparation technique which separates compounds based on size. There are two types of filtration: conventional/dead end in which the fluid flows perpendicular to the medium and crossflow filtration where the fluid flows parallel to the medium. Filtration is primarily used in the earlier stages of bioproduct purification because it is able to filter out the bigger impurities first.
You can think of filtration in a different way – it uses the same principles that coffee filters do in weeding out the bigger impurities. Filtration is useful and is effective in volume reduction.
Applications: Filtration has many applications. Primarily, it is used when a product has been secreted from cells. It is also used to purify antibiotics and steroids, separate cells from a product that has been secreted, sterile fertilization in biopharmaecutical production, removal of cell debris from cells that have been lysed, removal of viruses from protein solutions, and more.
Liquid: Liquid chromatography is based on the affinity of soluble molecules for specific types of solids. There are multiple solutes, and liquid chromatography consists of a mobile phase and a stationary phase, where the mobile phase is a liquid phase and the stationary phase is a solid phase.
Elution Chromatography: Elution chromatography is basically where there is a liquid with a mixture of solutes and goes into an inlet of a column which already contains the stationary phase.
Gas Chromatography: Gas chromatography is used for the quantitative and qualitative analysis of mixtures, specifically used for the purification of compounds. There are many applications for gas chromatography including: determining thermochemical constants and monitoring industrial processes.
Countercurrent Chromatography/Liquid-Liquid Chromatography:
Countercurrent chromatography is a little more complicated, but essentially both the mobile phase and the stationary phase are liquid. The solute separation partitions both of the liquid phases. Centrifugal fields hold the stationary phase inside the column while the mobile phase is then passed through using a pump. As the liquid phase runs through the stationary phase (which the mixture has been injected into), the mixture separates. The stationary phase in CCC takes up approximately 90% of the column. Below, there is a quick video that demonstrates the principles behind countercurrent chromatography.
There are a number of advantages to using CCC.
- There is a high loading capacity
- Simple solute retention mechanism
- Either phase can be used as the mobile phase
- No irreversible solute adsorption
- No pH problem
- Less biological solute denaturation
- High stationary phase retention – more stationary phase remains within the column which is good because you want the stationary phase to remain stationary.
- Less loss of analyte and sample mixture
- Columns are less likely to break
HPLC: HPLC (High performance liquid chromatography) pumps mixtures at high pressures through a column. The sample is carried by moving carrier gas streams of either helium or nitrogen. HPLC is used to separate and identify compounds present in any sample.
Applications of chromatography:
There are a number of applications of chromatography in the real world.
- Purifies a wide variety of chemical substances
- Separations can include: protein, DNA, antibiotics, vitamins, natural products, enantiomers, etc.
- Lots of applications in the pharmaceutical industry for purifying drugs
- HPLC and CCC can separate from milligram quantities to kilogram quantities – this means they can separate small scale molecules as well as large scale molecules which makes it easier to manufacture on a larger scale.
- Can separate and purify chemically complex samples
Distillation: Distillation is yet another type of bioseparation technique. Distillation separates based on differences in a mixture based on conditions that are required to change the phase components of the mixtures. The liquid can be heated based on the differing boiling points and then the gas can be condensed and then collected. There are three primary forms of distillation.
Simple distillation: Simple distillation is used when boiling points of the compounds in a mixture are different from one another. The mixture is heated to change the component from liquid to vapor. Fractions based on density values can separate themselves using simple distillation.
Fractional distillation: Fractional distillation is used when the boiling points of the components of the mixtures are close together. A fractioning column is used to separate the components. When the mixture is heated, the vapor rises and the vapor, as it cools, condenses on the column. The heat then vaporizes again and it moves it through the column, eventually purifying the sample.
Vacuum distillation: Vacuum distillation separates components that have very high boiling points. Lowering the pressure lowers the boiling points. Vacuum distillation is specifically used when the normal boiling point exceeds the decomposition temperature.
Applications: Distillation is primarily used in industrial processes like the production of gasoline, distilled water, alcohol etc. Gases can also be liquified and separated like nitrogen, oxygen, argon, etc.
Centrifugation: Centrifugation is used for larger scale molecules and uses principles of sedimentation.
Differential centrifugation uses principles of different densities to suspend sediments at different rates. The sedimentation rates can be increased by using centrifugal force. Particles of different densities will sediment at different rates where the largest particles will sediment the fastest. Applications of differential centrifugation are in harvesting cells or producing subcellular fractions.
After the separation has been completed, detection methods are used to see whether a compound has been purified or not.
There are two primary detection methods: Mass spectrometry and UV spectrometry.
Mass spec: Mass spec measures the mass to charge ratio of ions by identifying and quantifying the molecules in simple and complex mixtures. The analysis is usually done after sample preparation and liquid chromatography based separation. Mass spec has many applications in the field of proteomics with determining protein structures and identifying proteins from peptide fragments, in drug discovery (determining the structures of drugs), clinical testing for forensic analysis or detecting disease biomarkers, genomics in sequencing oligonucleotides, the environment (testing water quality, for example), geology, carbon dating, and more. The method involves using an ion source, mass analyzer, and an ion detector. Below, see the figure which shows the process used in mass spec.
The ions deflected by the mass analyzer hit the ion detector and the computer analyzes the ion detector data.
UV Spec: UV spec uses the principles of differentiating between compounds based on their colors. Component colors are separated by passing sunlight. UV Spec measures the beam of light after it passes through a sample. There are many applications for UV spec as well. It is often used for the detection of functional groups, can easily detect impurities, is used for both qualitative and quantitative analysis, and can show the relationship between different (functional) groups.
What this means on a global and local level:
On a both local and global level, what is very important in purifying compounds are these bioseparation methods. Currently, some of these methods like distillation and filtration and centrifugation can only separate larger scale molecules which makes small-scale purification, and therefore larger scale industrialization of those methods harder. Currently, in the bioseparation fields, countercurrent chromatography and HPLC have been very useful in separating small proteins and molecules, and can be easily applied on a larger scale based on the columns that are being used. This is very important in the pharmaceutical industry for the purification of drugs, the cosmetic industry for purifying makeups, the food industry for ensuring that foods are of good quality (the FDA actually uses HPLC and CCC currently in purifying foods and drugs). On a biological level, the separation of enantiomers and natural products and antibiotics, etc. are very helpful in identifying defects within the human body to therefore be able to identify/provide treatment to individuals. Finally, bioseparation techniques are critical as they are currently being expanded to separating and purifying live tissue, which can one day help us create more targeted therapies for diseases that currently do not have any cures or therapies. Overall, on both a global and local level, bioseparation techniques are critical to making progress in both the pharmaceutical world and the biological world. Improvements are currently still being made and work in this area is very important to attaining success in the biological areas.
On a Personal Level
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