Ion exchange chromatography

Ion exchange chromatography is a process of separation of molecules of like or same charges from a mixture by use of ion exchange resins. Principle: The separation occurs byreversible exchange of ionsbetween the ions present in the solution and those present in the ion exchange resin. Ion exchange resins like anion exchange for anions and cation exchange resins for cation separation are employed. The ion retained in the resin matrix are removed by use of similar strong charged mobile phase. Resin-H +M (in solution) ------------> Resin-M + H (in solution). Resin-OH +A (in solution) ------------> Resin-A + OH (in solution). Many drugs in pharmaceutical products are either acidic or basic in nature. Their mixture can be separated into pure compound by Ion exchange chromatography.
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Applications of ion exchange chromatography.

Ion exchange chromatography is prominently used as preparatory chromatography to isolate a desired compound from mixture. Hence the applications are meant to obtain pure compounds. a) For deionisation and softening of water. b) Purification of solution to keep them ion free. c) In biochemistry for separation of drugs and metabolites from blood, urine etc. d) Separation of organic mixture of acidic and basic compounds. e) For extraction of enzymes from tissues.

Gel filtration chromatography

Also called gel exclusion or size exclusion chromatography. Separates molecules on the basis of molecular weight, size and shape. Done in a column packed with porous gel beads (usually crosslinked polysaccharides). The pore size is chosen so that small molecules can enter the beads, but large ones cant.

Applications of gel filtration

A gel filtration column can be calibrated with known substances to allow a rough measurement of molecular size, estimated according to the point at which a protein emerges. This technique will remove low molecular weight contaminants from solutions of large molecules. The technique will separate different sized molecules of all kinds, not just proteins. It is possible to study the binding of proteins to a ligand by using gel filtration: protein bound to ligand will emerge from the column before protein on its own, or ligand on its own.
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Can be used to calculate binding coefficients.

Cholesterol (a sterol) and Phosphatidylcholine (a

glycerophospholipid) are two examples of amphipathic lipids. Looking at cholesterol you can see the four rings that are characteristic of a steroid and the aliphatic chain extending from the five membered D ring. The two methyl groups which are attached to the bridge carbons project toward you. Notice that since the rings are not aromatic they are not planar. The structural feature, that makes cholesterol not only a steroid but also a sterol, is the hydroxyl group on ring A. An amphipathic substance is one that is polar at one end of the molecule (hydrophilic) and nonpolar (hydrophobic) at the other. It is the oxygen of the hydroxyl that makes the A ring end of the molecule slightly polar, and the remainder of the molecule is hydrocarbon and therefore nonpolar. With only one oxygen atom cholesterol is not stronly amphipathic, but it is amphipathic enough to be a component of lipid bilayers. Phosphatidylcholine, as with all glycerophospholipids, contains a diacylglycerol, a phosphate, and a nitrogen containing component which in this case is choline. The two acyl groups can easily be identified by looking for the two hydrocarbon chains that are somewhat twisted around each other. These two chains are attached to a glycerol,the three carbon segment with three oxygens, by carboxylic esters. The third oxygen of glycerol is connected to the

phosphate (orange) which being being present as a diester is also bonded to the choline which is a two carbon unit containing a nitrogen (blue) bonded to three methyl groups. The nitrogen with its four bonds has a permanent positive charge, and the phosphate is negative since at physiological pH its third acidic hydrogen is ionized. This phosphate/choline end of the molecule being charged is polar (hydrophilic), but the opposite end consisting of hydrocarbon chains is nonpolar (hydrophobic).

Definition

Difference in absorbance of ultraviolet light between single stranded and double stranded DNA

Hypochromicity describes a materials decreasing ability to absorb light. Hyperchromicity is the materials increasing ability to absorb light. The Hypochromic Effect describes the decrease in the absorbance of ultraviolet light in a double stranded DNA compared to its single stranded counterpart. Compared to a single stranded DNA, a double stranded DNA consists of stacked bases that contribute to the stability and the hypochromicity of the DNA. When a double stranded DNA is denatured, the stacked bases break apart and thus becomes less stable. It also absorbs more ultraviolet light since the bases no longer forms hydrogens bonds and therefore are free to absorb light. Ways to denature DNA include high temperature, addition of denaturant, and increasing the pH level. [edit]Importance

of Hypochromic Effect

Nucleic acid melting curve showing hyperchromicity as a function of temperature

The measurement of absorption of light is important in monitoring the melting and annealing of DNA. At Tm, the DNA is half denatured and half double stranded. By lowering the temperature below the Tm, the denatured DNA strands would anneal back into a double stranded DNA. When temperature is above the Tm, the DNA is denatured. Because the melting temperature (Tm), occurs almost instantly at a certain temperature, monitoring the absorbance of the DNA at various temperature would indicate the melting temperature. By being able to find the temperature at which DNA

melted and annealed, scientists are able to separate DNA strands and anneal them with other DNA strands. This is important in creating hybrid DNAs, which consists of two DNA strands from different sources. Since DNA strands can only anneal if they are similar, the creation of hybrid DNAs can indicate similarities between genomes of different organisms.

Hyperchromicity
From Wikipedia, the free encyclopedia

Nucleic acid melting curve showing hyperchromicity as a function of temperature

Hyperchromicity is the increase of absorbance (optical density) of a material. The most famous example is the hyperchromicity of DNA that occurs when the DNA duplex is denatured. The UV absorption is increased when the two single DNA strands are being separated, either by heat or by addition of denaturant or by increasing the pH level. The opposite, a decrease of absorbance is called hypochromicity. Heat denaturation of DNA, also called melting, causes the double helix structure to unwind to form single stranded DNA. When DNA in solution is heated above its melting temperature (usually more than 80 C), the double-stranded DNA unwinds to form single-stranded DNA. The bases become unstacked and can thus absorb more light. In their native state, the bases of DNA absorb light in the 260-nm wavelength region. When the bases become unstacked, the wavelength of maximum absorbance does not change, but the amount absorbed increases by 37%. A double strand DNA dissociating to single strands produces a sharp cooperative transition. Hyperchromicity can be used to track the condition of DNA as temperature changes. The transition/melting temperature (Tm) is the temperature where the absorbance of UV light is 50% between the maximum and minimum, i.e. where 50% of the DNA is denatured. The hyperchromic effect is the striking increase in absorbance of DNA upon denaturation. The two strands of DNA are bound together mainly by the stacking interactions, hydrogen bonds and hydrophobic effect between the complementary bases. The hydrogen bond limits the resonance of the aromatic ring so the absorbance of the sample is limited as well. When the DNA double helix is treated with denatured agents, the interaction force holding the double helical structure is disrupted. The double helix then separates into two single strands which

are in the random coiled conformation. At this time, the base-base interaction will be reduced, increasing the UVabsorbance of DNA solution because many bases are in free form and do not form hydrogen bonds with complementary bases. As a result, the absorbance for single-stranded DNA will be 37% higher than that for double stranded DNA at the same concentration.

proteolytic enzyme, also called Proteinase, any of a group of enzymes that break the long chainlike molecules of proteins into shorter fragments (peptides) and eventually into their components, amino acids. Proteolytic enzymes are present in bacteria and plants but are most abundant in animals. In the stomach, protein materials are attacked initially by the gastric enzyme pepsin. When the protein material is passed to the small intestine, proteins, which are only partially digested in the stomach, are further attacked by proteolytic enzymes secreted by the pancreas. These enzymes are liberated in the small intestine from inactive precursors produced by the acinar cells in the pancreas. The precursors are called trypsinogen, chymotrypsinogen, proelastase, and procarboxypeptidase. When the pancreatic enzymes become activated in the intestine, they convert proteins into free amino acids, which are easily absorbed by the cells of the intestinal wall. Trypsinogen is transformed to trypsin by an enzyme (enterokinase) secreted from the walls of the small intestine. Trypsin then activates the precursors of chymotrypsin, elastase, and carboxypeptidase. The pancreas produces a protein that inhibits trypsin. It is thought that in this manner the pancreas protects itself from autodigestion.

DNA Super Coiling:

Whether the DNA is single stranded or double stranded, inside the cell it is always subjected to bending, folding, over winding and under winding. Such changes make stable DNA into unstable or vice versa, because energy constraints. Thermodynamically complaint B- DNA contains 10.6 bp per turn, and such DNA without any constraints or stress or torsion, when placed on a flat surface it lies flat as a circular molecule or a linear molecule. Such DNA, which is free from stress, is called Relaxed DNA.

This is an EM of a Super coiled and one relaxed DNA

When a relaxed DNA is subjected to bends, or openings of DNA, over winding or unwinding, its base pairs per turn changes, and the DNA is subjected to stress and strain. In order to overcome such distortion, which has rendered the DNA unstable, the DNA twist around itself, like a circular rubber band undergoing twisting, such a twists on its own thread is called Super coiling; it is also referred to as tertiary structure. To explain super coiling phenomenon, if a 5300 bp long circular DNA, which contains 500 helical turns with 10.6bp per turn, is twisted 50 times by 360^0 each time, in right handed direction i.e. towards its own right handed coil, the DNA becomes over coiled or over winded and the helix becomes tight. It virtually means adding 50 more helical coils to the already existing 500 helical turns, making the total number of turns to 550. So the 5300 bp long DNA has to accommodate 50 more turns, which is torsionally a stressed condition. If 550 divide 5300 bp, the number of base pairs per turn will be 9.6; it is a drastic deviation from the normal and thermodynamically feasible and stable state of 10.6 per turn. This over winding is thermodynamically incompatible, so in order to accommodate the excess number of coils, the helical DNA twists on its own length in space in left handed direction, like a rubber band twist, in left handed way, such a twist is called super coil; in this case the super coiling is positive.

Such super coilings overcome stress energy and accommodate the extra 50 numbers of added coils to the existing 500 helical turns. It is because of super coiling, now the number of base pairs per turn would be 10.6 Negative super coiling: Similarly, if a 5300bp long circular DNA with 500 turn is twisted 50 times 360^0 each, in left handed way, the DNA gets under wounded, and likely the DNA strands can separate. In this case the number of turns lost are 50 and to adjust the number of coils, the number of base pairs per turn will be11.6, which is not thermodynamically feasible, hence the DNA takes a right handed twist on its own; the DNA helix is in the form of negatively super coiled DNA. This super coiling accommodates the stress energy and the DNA becomes stable with 10.6 bp per coil. Generally the density of the super coils a DNA is one super coil per 200bp or per ~20 turns of the helix. Negative super coils help in stabilizing certain DNA structures i.e. Z-DNA, cruciform DNA, triplex DNA. It also helps in unwinding of the DNA during replication as replication bubble or during transcriptional initiation as transcriptional bubble.

Detection of super Coils: Super coiled DNA can be distinguished from the normal DNA using gel electrophoresis. Super coiled DNA moves faster in an agarose gel than the relaxed DNA. If a purified plasmid DNA is run on the gel, and if one finds more than two bands, it means the one that moved faster is fully super coiled and the one that moves slower is nicked, so

it moves slow. By subjecting such super coiled DNA to Topoisomerases, an enzyme that nicks and relaxes the super coiled DNA, one cut per digestion and such digested DNA if ran on an agarose gel, one can observe a number of bands from the bottom. The bottom most is the most super coiled and uncut. The band found next to the uncut shows that it is relaxed by one super coil. The uppermost band is the one, which is totally relaxed. The number of bands found on the gel gives the total number of super coils in a given DNA. During replication, transcription, recombination, DNA repair and bending or folding of DNA or during gene expression or compacting of DNA as chromosomes, the normal relaxed DNA of 10.6 bp per turn, dramatically changes its base pairs per turn and the DNA is subjected to torsional stress; to accommodate such strain the DNA has to undergo super coiled states. Such topological changes in DNA can described by three parameters, they are

With respect to circular DNAs and supercoiling, students should note the following: 1. Circular DNAs have no free ends. 2. Enzymes called topoisomerases can take apart a circular DNA, introduce additional twists into it, and then reseal the structure (Figure 4.24). Adding twists to circular DNA introduces tension into the molecule. This kind of tension would not stay in linear DNA for very long because the force could be dissipated through the ends, and the DNA would "relax". The extra tension in circular DNA (or in linear DNA whose ends are anchored to prevent tension from being released) usually causes the molecule to writhe to alleviate the tension. Like an overwound rubber band, the circular DNA assumes a new shape, called a supercoil. 3. Supercoiling can be positive (additional twists added beyond the normal amount for linear DNA) or negative (reduced numbers of twists compared to linear DNA).