What are the molecule separation techniques based upon size

The techniques based on size and weight of the molecules are Gel Filtration, Osmotic Pressure & Centrifugation

Gel-filtration Chromatography:
Gel-filtration chromatography is a separation based on size. It is also called molecular exclusion or gel permeation chromatography. In gel filtration chromatography, the stationary phase consists of porous beads with a well-defined range of pore sizes. The stationary phase for gel filtration is said to have a fractionation range, meaning that molecules within that molecular weight range can be separated.

Proteins that are small enough can fit inside all the pores in the beads and are said to be included. These small proteins have access to the mobile phase inside the beads as well as the mobile phase between beads and elute last in a gel-filtration separation. Proteins that are too large to fit inside any of the pores are said to be excluded. They have access only to the mobile phase between the beads and, therefore, elute first. Proteins of intermediate size are partially included-meaning they can fit inside some but not all of the pores in the beads. These proteins will then elute between the large ("excluded") and small ("totally included") proteins.

Consider the separation of a mixture of glutamate dehydrogenase (molecular weight 290,000), lactate dehydrogenase (molecular weight 140,000), serum albumin (MW 67,000), ovalbumin (MW 43,000), and cytochrome c (MW 12,400) on a gel-filtration column packed with Bio-Gel P-150 (fractionation range 15,000 to 150,000). When the protein mixture is applied to the column, glutamate dehydrogenase would elute first because it is above the upper fractionation limit. Therefore, it is totally excluded from the inside of the porous stationary phase and would elute with the void volume (VO). Cytochrome c is below the lower fractionation limit and would be completely included, eluting last. The other proteins would be partially included and elute in order of decreasing molecular weight.

These separations can be described with this equation

Vr, = Vo + KVi

where Vr is the retention volume of the protein, Vo is the volume of mobile phase between the beads (outside the beads) of the stationary phase inside the column (sometimes called the void volume), Vi is the volume of mobile phase inside the porous beads (also called the included volume), and K is the partition coefficient (the extent to which the protein can penetrate the pores in the stationary phase, with values ranging between 0 and 1). In the mixture of proteins listed above, the partition coefficient (K) for glutamate dehydrogenase would be 0 (totally excluded), K = 1 for cytochrome c (totally included), and K would be between 0 and 1 for the other proteins, which are within the fractionation range for the column.

In practice, gel-filtration can be used to separate proteins by molecular weight at any point in purification of a protein. It can also be used for buffer exchange; a protein dissolved in a sodium acetate buffer, pH 4.8, can be applied to a gel-filtration column that has been equilibrated with Tris buffer pH 8.0. Using the Tris buffer as the mobile phase, the protein moves into the Tris mobile phase as it travels down the column, while the much smaller sodium acetate buffer molecules are totally included in the porous beads and travel much more slowly than the protein. Similarly, it can be used for the separation of salts and other small molecules from a protein sample.

Osmotic Pressure:
Molecules always move from the region of their higher concentration to the region of their lower concentration. It is applicable to both solvent and solute molecules in the case of a solution. When a solution is separated from a pure solvent by a membrane that is permeable to the solvent alone, the molecules of the solvent will move into the solution. The flow of the solvent molecules into the solution can be prevented by applying some amount of pressure that is equal to the pressure exerted by the solvent molecules to enter into the solution compartment through the membrane partition. The pressure that is needed to prevent the entry of the solvent molecule into the solution is called osmotic pressure.

The osmotic pressure of a solution depends on the concentration of a solute and the temperature of the solution. It can be used for the calculation of the molecular weight of the solute.

#V=nRT

where #is the osmotic pressure, V is the volume of the solution, n is the number of moles of solute, R is gas constant, and T is the absolute temperature.

#= n/V x RT (n/V= M, molarity of the solution)

Therefore, # = MRT

But in this equation, n-the number of moles = weight of the solute in grams/molecular weight.

i.e.#V = Wt 9 /MW x RT Therefore,

MW = Wt 9 /#V x RT. (Wt 9 /Volume = Concentration, C)
i.e. MW = CRT/#.

Thus, if the osmotic pressure and the concentration of the solution are available the molecular weight of the solute can be determined.

Tags: Bio Technology, Bio Genetics , Biochemical Techniques

What is Density Gradient Centrifugation

Density gradient centrifugation are of two types:

1) Rate zonal centrifugation

2) Isopycnic centrifugation

Rate zonal centrifugation:
In rate zonal centrifugation, the sample is applied in a thin zone at the top of the centrifuge tube on a density gradient. Under centrifugal force, the particles will begin sedimenting through the gradient in separate zones according to their size, shape, and density or the sedimentation coefficient(s). The run must be terminated before any of the separated particles reach the bottom of the tube. S is the sedimentation coefficient and is usually expressed in Svedbergs (S) units.


Isopycnic centrifugation:
In the isopycnic technique, the density gradient column encompasses the whole range of densities of the sample particles. The sample is uniformly mixed with the gradient material. Each of the particles will sediment only to the position in the centrifuge tube at which the gradient density is equal to its own density, and there it will remain.

The isopycnic technique, therefore, separates particles into zones solely on the basis of their buoyant density differences, independent of time. In many density gradient experiments, particles of both the rate zonal and the isopycnic principles may enter into the final separations. For example, the gradient may be of such a density range that one of the components sediments to its density in the tube and remains there, while another component sediments to the bottom of the tube. The self-generating gradient technique often requires long hours of centrifugation.

Isopynically banding DNA, for example, takes 36 to 48 hours in a self-generating cesium chloride gradient. It is important to note that the run time cannot be shortened by increasing the rotor speed; this only results in changing the position of the zones in the tube since the gradient material will redistribute further down the tube under greater centrifugal force.

Tags: Bio Technology, Bio Genetics , Biochemical Techniques

What is Centrifugation Process for isolation of molecules

Biomolecules can be isolated and purified by applying different techniques, which are based on various chemical and physical properties. The main physical and chemical properties that can be exploited for their separation and characterization of biomolecules are molecular weight and size, interaction with electromagnetic radiations or spectroscopic properties, solubility, molecular charge, and polarity.

Centrifugation is a technique based on size and weight of the molecules.

A centrifuge is a device for separating particles from a solution according to their sedimentation rate, which depends on factors like size, shape, density, viscosity of the medium, and centrifugal force (rotor speed). This process of separation of particles based on its sedimentation rate is called centrifugation. In biology, the particles are usually cells, sub-cellular organelles, viruses, and large molecules such as proteins and nucleic acids. The rate of sedimentation will be directly proportional to the molecular weight or size, if all other factors are constant. To simplify mathematical terminology we will refer to all biological material as spherical particles.

Following are the ways to classify centrifugation:

1) Analytical and Preparative Centrifugation:

The two most common types of centrifugation are analytical and preparative; the distinction between the two is based on the purpose of centrifugation. Analytical centrifugation involves measuring the physical properties of the sedimenting particles such as sedimentation coefficient or molecular weight. Optimal methods are used in analytical ultracentrifugation. Molecules are observed by an optical system during centrifugation, to allow observation of macromolecules in the solution as they move in the gravitational field. The samples are centrifuged in cells having windows that lie parallel to the plane of rotation of the rotor head. As the rotor turns, the images of the cell (proteins) are projected by an optical system onto film or a computer. The concentration of the solution at various points in the cell is determined by absorption of light of the appropriate wavelength (Beer's law is followed). This can be accomplished either by measuring the degree of blackening of a photographic film or by the pen deflection of the recorder of the scanning system, which is fed into a computer.

The other forms of centrifugations are preparative and the objective is to isolate specific particles, which can be reused. There are many types of preparative centrifugation such as rate zonal, differential, and isopycnic centrifugation.

2) Ultracentrifugation vs Low-speed Centrifugation:

Another system of classification is the rate or speed at which the centrifuge is turning. Ultracentrifugation is carried out at speed faster than 30,000 rpm. High-speed centrifugation is at speeds between 10,000 and 30,000 rpm. Low-speed centrifugation is at speeds below 10,000 rpm (mostly between 3,000 to 9,000 rpm).

3) Moving Boundary vs Zone Centrifugation:

A third method of defining centrifugation is by the way the samples are applied to the centrifuge tube. In moving boundary or differential centrifugation, the entire tube is filled with the sample and centrifuged. Through centrifugation, one obtains a separation of the mixture into two parts-a supernatant and a pellet. But any particle in the mixture may end up in the supernatant or in the pellet or it may be distributed in both fractions, depending upon its size, shape, density, and conditions of centrifugation. The pellet is a mixture of all of the sedimented components, and it is contaminated with whatever unsedimented particles were in the bottom of the tube initially. The only component that is purified is the slowest sedimenting one, but its yield is often very low. The two fractions are recovered by decanting the supernatant solution from the pellet. The supernatant can be recentrifuged at higher speeds to obtain further purification with the formation of a new pellet and supernatant.

Tags: Bio Technology, Bio Genetics , Biochemical Techniques

What is Biopiracy

Plants constitute a rich source of therapeutically important products. Only 10 percent of plant species have already been tested for pharmaceutical value. About 120 medicines commonly prescribed by doctors are based on plant extracts. Some of the most important medicines used by humans have a history that traces back to medicinal plants collected from wild flora, and others are just now beginning to be discovered.

Aspirin is one example of the health benefits provided by plants. This medicine is based on acetyl-salicylic acid, a very common analgesic. Its history began with the Greek doctor Hypocrites, who in the fifth century B.C. used a bitter powder to treat pain and lower fever. This mystical powder was collected from the cork of Salix a tree of the family Salicaceae. Although the mode of action of aspirin was only unraveled in the 1970s, this medicine has been used as a painkiller and to improve the elasticity of the circulatory system for millions of people. If this species had gone extinct before that discovery, man would have never known the valuable medicine. In fact, Americans annually consume about 80 billion aspirin tablets.

The history of biodiversity collection dates back to 1500 B.C., when Egyptian rulers gathered plant species from their military expeditions. Charles Darwin, the renowned naturalist of the 19th century, accomplished one of the most famous trips for biological collection. During his travels on the ship the HMS Beagle, he collected samples of everything that interested him, from which he elaborated the Theory of the Evolution, the foundation of modern biological research. More recently, Nicolai I. Vavilov, a Russian scientist in the beginning of the 20th century, also collected samples of plant species from five continents, with which he established the Theory of the Center of Origin of the Crop Species.

None of those famous expeditions were legally or morally questioned. Today, the paradigms and laws have changed, and biopiracy is considered a crime. Biopiracy is the unauthorized appropriation of any biological resources. The extraction of aromatic, ornamental, or medicinal plants without the proper authorization is considered biopiracy. Natural resources primarily from Africa and South America are becoming increasingly valued in the international market. In the 1500s, Brazilian wood was prized for making red dyes; today, targeted Brazilian species number about 50,000 plant species, 534 mammals, 3,000 fishes, approximately 1,700 birds, 500 amphibians, and 470 reptiles. The wealth of Brazilian biodiversity makes the country a valuable source of genetic diversity. Every year, thousands of tourists, scientists, environmentalists, and biologists travel around the world under the umbrella of ecological tourism. Although this type of travel has improved research on biodiversity, it has also caused problems relating to biopiracy.

Scientists and pharmaceutical companies are obtaining several patents using plant extracts from different regions around the world. Recently, the English chemist Conrad Gorinsky received a worldwide patent for two pharmaceutical products: Rupununine, extracted from seeds of the Octotea rodioei, for birth control; and Cunaniol, a nervous system stimulant, extracted from Clibadium sylvestre. The use of the plants is part of the traditions of the native Wapixana Indians, who live in the Brazilian state of Roraima. Several other bioprospecting projects are underway in Africa and other places to identify plant extracts, animal toxins, and microorganisms for different purposes such as production of plastics or ore purification and fermentation processes.

When a sample of a species is collected illegally and a new drug or an isolated gene from that sample is patented, the patent can be revoked. If there is proof that the active ingredient used in the new drug was in public use, even if restricted to an indigenous tribe, revocation of the patent is possible. The great dilemma in patenting a natural product is that pharmaceutical companies take advantage of the ethnobiological knowledge of indigenous populations, and later, the companies are the only ones to collect profits from the marketing and production of the drugs.

To prevent such exploitation, regulations are being made worldwide to govern the use of biological diversity. In 1992, the United Nations Conference on Environment and Development (ECO 92) met in Rio de Janeiro, Brazil, with representatives from 120 countries. This conference recognized the national sovereignty of the nations and the genetic resources within their borders. Beyond this international work, the national laws of each country further govern the conservation and development of biodiversity within their respective boundaries. The Brazilian Congress recently recognized the importance of the protection of its biodiversity. This came after an accord with some multinational companies relating to the development of medicines resulting from the exploration of plants and microorganisms from the Atlantic rainforests and the Amazon.

Only 20 years ago, legal aspects related to the collection of samples of plants, microbes, and animals were largely ignored. In most cases, researchers simply made a trip to the place where the species of interest could exist in nature, collected the samples, and returned to their laboratories. Clearly laws didn't exist to regulate that practice. Sometimes, researchers obtained informal authorization from the local authority or from the landlord where the samples were collected. The days of locating, collecting, and returning home are running out, at least legally in most countries. More often it is today considered biopiracy.

Tags: Bio Technology, Bio Genetics, Bio diversity

What is Genetic Erosion

Until the 1940s, the centers of origin of crop species and animals were considered limitless sources of genetic variability. After World War II, agriculture in developing countries suffered great changes. The expanded use of improved varieties resulted in the reduction of traditional varieties, a process called genetic erosion. The expansion of the agricultural frontiers also contributed to the risk of loss of the wild relatives of crop species.

According to a study carried out by the National Academy of Sciences in the United States, of approximately 3,000 possible plant species, only 20 to 30 constitute the basis of agriculture. For example, amaranth has high economic potential and has been recommended as a species that deserves more attention from plant breeders, with the objective of improving the plant to make it more valuable for commercial use. This requires the removal of undesirable traits and the improvement of other traits to allow for improved production.

The process of genetic erosion also occurs with many other species of flora, fauna, and microorganisms, and it is the first sign indicating possible species extinction. Environmental deterioration initially results in local extinction and later culminates with the global extinction of the species. For instance, well before species vanish, a small number of survivors could result in inbreeding of the population. Inbreeding results from intermating between related individuals that causes the generation of less fit individuals with a greater likelihood of genetic defects.

Biotechnology can help in the diagnosis of genetic erosion before any conventional techniques. This can be achieved by DNA analyses that quantify the remaining genetic diversity. However, as each organism has a different genome, these methods would have to be developed for each species. This technique has been used with success in the study of wolf species, fish, cattle, macaws, whales, and other animals. In many cases, the studies were used to justify the creation of new refuges where such species dwell.

Tags: Bio Technology, Bio Genetics, Bio diversity

Why Preserve Biodiversity

The preservation of genetic variation has become an important subject for many species. Various species of plants, animals, and microorganisms have been collected and stored, so the immense species variation might not be lost. The culture of cells and tissue, an area within biotechnology, is being used for the maintenance of live collections of the most varied types of plant species of economic importance or others at risk for extinction. For instance, the preservation of the genetic diversity of cassava is accomplished at tissue culture laboratories, where thousands of different varieties and species are maintained in small petri dishes. In the Frozen Zoological Garden in San Diego, California, there are live cellular lineages of species of several families of mammals, many close to extinction. It is expected that in the near future, cloning techniques will be used to regenerate whole animals from the cells. Had tissue culture technologies not been developed, the required space and costs to preserve rare species would be many times larger, limiting the number of species that could be preserved.

The preservation of microbial diversity has also been made possible by biotechnological techniques. If the bacteria, fungi, and viruses had to be maintained in their traditional hosts, only a small fraction of the biodiversity of microorganisms could be preserved. The germplasm banks of bacteria and fungi require a relatively small and rudimentary laboratory for preservation. The main objectives of microorganism gene banks are related to the preservation of species for subsequent laboratory studies.

Legal mechanisms should be developed to protect the biodiversity of the world. The mechanisms should promote the conservation of biodiversity and its use for the well-being of mankind. Developed countries and many others have taken the lead to ensure germplasm preservation for years to come. Extensive efforts are being made to characterize, catalog, and store the germplasm resources collected over many years from all over the world. Further steps are being taken to guarantee the preservation of the biodiversity of ecosystems and centers of origin for future research. Biotechnology will benefit from the world's biodiversity, while creating a means of preservation and continuation of the diversity of life found around the world.

There is an incredible amount of biodiversity worldwide, and much of it is relatively unknown. Despite the actions to explore this diversity, steps are being taken to characterize and preserve this valuable resource.

Tags: Bio Technology, Bio Genetics, Bio diversity

Understanding Biodiversity

Biodiversity, or biological diversity, refers to every form of life within an area or ecosystem. This includes the genetic variability within the populations and species; the different species of flora, fauna, and microorganisms; the variety of functions and ecological interactions carried out by the organisms in the ecosystems; and the various communities, habitats, and ecosystems formed by the organisms. Biodiversity is the fruit of the great laboratory, which is the planet Earth, with its more than 30 million different species resulting from 4 to 5 billion years of evolution.

The importance of preserving biodiversity is also referred to in sacred books, such as the Bible, which relates that Noah saved domestic and wild animals from the great flood. Biodiversity is one of the fundamental properties of nature responsible for the balance and stability of ecosystems. It is also of great economic value. This diversity is the basis of farming and food production, and it is essential for biotechnology. The ecological functions carried out by various organisms are still poorly understood, but biodiversity is thought to be responsible for the natural processes and products supplied by ecosystems. It accounts for the species that sustain other life forms and also modifies the biosphere, making it suitable and safe for life. Biological diversity possesses, besides an intrinsic worth, a value of ecological, genetic, social, economical, scientific, educational, cultural, recreational, and aesthetic importance.

A reduction in biological diversity is hazardous to sustainable development. Genetic erosion (the loss of species variability) and the extinction of species can influence us to develop strategies that contribute to the preservation of the remaining biodiversity on the planet, at a level that is already smaller than it was a century ago. The preservation of biodiversity is also essential for human well-being. However, recent studies have indicated that extinction rates are 1,000 times faster than those expected naturally, with 50,000 species extinguished every year. Currently, about 34,000 plant species and 5,200 animal species are at risk of becoming extinct.

Biotechnology can be understood as a technology that explores biological systems instead of individual living organisms. Therefore, the preservation of the biological systems with all of their diversity can be considered a priority as well as a challenge to mankind.

Microbes, such as bacteria, are the most diverse of all living organisms. Some estimates indicate that there exist more than 1 million different species of bacteria in the world. Recent reports suggest that an extremely large number of bacteria exist in the biosphere awaiting the development of appropriate techniques needed to grow them, so that they can be characterized.
This is one example of one key part of the greater picture of biodiversity. Plants, animals, and even fungi are also important aspects of the world's biodiversity. This idea of biodiversity is an important part of biotechnology, as useful traits and chemicals are becoming part of important new biotechnology applications. Biotechnology brings, simultaneously, promises of biodiversity preservation and also the fear of genetic erosion and biopiracy.

Tags: Bio Technology, Bio Genetics, Bio diversity