General Information: Chromatography is a method very widely used for the separation of mixtures of chemicals and biochemicals. The basic principle is that a mixture of compounds is applied to a stationary phase, which may be a solid or a liquid. A mobile phase is then passed over the stationary phase; the mobile phase may be a liquid or a gas. In paper chromatography the paper acts as a support for the aqueous stationary phase and the mobile phase is a solvent.
The mixtures of compounds to be separated are taken up into solution and a spot of this is applied to the paper. This area of application is termed the origin. The origin and the final position of the solvent front should be marked by pencil lines. One edge of the paper is dipped into a reservoir of the solvent, which begins to soak into the paper. More and more of the solvent is absorbed by the paper and the solvent front advances.
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While the chromatogram is running any compound at the origin which does not dissolve in the solvent will remain at the origin it self. Any compound which dissolves very readily will move along as the solvent front advances and will remain very close to the solvent front. Other compounds, which dissolve less readily, will move along the paper at a slower rate and will fall behind the solvent front. When paper is removed from the solvent or the solvent front stops moving, each compound remains in the paper at the site where it happens to be at that time.
Experimental details, procedure and calculations:
Three dyes are supplied: Methyl red, neutralot and Light green. Take three beakers one paper strip and three of the sample applicators. Place 5 drops of methyl red in one beaker, 5 drops of netralot in another and 5 drops od Light green in the third. Each will form a puddle in the bottom of the beaker. Stand an applicator in each puddle and leave for a minute so that the dye can soak into the wood. Now gently place the tip of the applicator on the paper strip to deposit a small spot of dye. Place small spots of the three dyes side by side as shown. At the other end of the strip, place three larger spots side by side. Now fold the paper in half and stand for about 5 minutes to allow the spots to dry. Pour some of solvent (80% methanol, 20% water) into the chromatography tank so that the solvent just covers the glass beads, but do not overfill. Place the paper strip in the tank and lay the lid on the top of the tank. The solvent will run up the paper to reach the top in 5-10 minutes. Take the paper out, using forceps, and allow to dry. Note the positions of the spots . You will probably find that some spots will not give a very noticeable colour. This is because the spots diffuse as they travel in the solvent stream and can become spread out too thinly to see. On the other hand, the larger spots may spread out over a sizeable area and trail backwards towards the origin. One of the difficulties of all chromatographic separations is the spotting of the right amount of sample.
Take another paper strip and spot the amounts of dyes which you now think will give good spots. Also, mix the three dyes together in one beaker and spot this mixture at the other end of the strip but use three times the amount you think necessary for any one dye, since they are now diluted with each other. Allow the spots to dry and develop the chromatogram as before (check that there is enough solvent in the tank). If you are not satisfied with the separation because you think the spots were too small or too big, use another paper strip to produce another chromatogram, using different amount of samples for spotting.
Take the different developed chromatograms and ring the spots in pencil. Make a mark where you think the colour is densest. Measure the distance from the origin to position where the solvent front stopped and call this A. Measure the distance from the origin to the centre of s spot and cal this B. This ratio B/A for any one dye should be the same in all chromatograms. This ratio is the Rf value, which is characteristic for any compound. If a different solvent is used, the compounds may well have a different Rf value, but the Rf value for any compound in any one solvent is constant. Wash out the beakers and the chromatography tank and dry. Note that both ends of an applicator can be used for spotting, but each end should be used for one sample liquid only. Keep your chromatograms for reference and label fully.
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1sthalf of chromatography paper.
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2nd half of chromatography paper.
Chromatography: Chromatography is the collective term for a set ofÂ laboratory techniquesÂ for theÂ separation of mixtures. It involves passing a mixture dissolved in a "mobile phase" through aÂ stationary phase, which separates theÂ analyteÂ to be measured from other molecules in the mixture based on differential partitioning between the mobile and stationary phases. Subtle differences in compoundsÂ partition coefficientÂ results in differential retention on the stationary phase and thusÂ changingÂ the separation.
Stationary phase: ChromatographyÂ is the collective term for a set ofÂ laboratory techniquesÂ for theÂ separation of mixtures. It involves passing a mixture dissolved in a "mobile phase" through aÂ stationary phase, which separates theÂ analyzeÂ to be measured from other molecules in the mixture based on differential partitioning between the mobile and stationary phases. Subtle differences in compoundsÂ partition coefficientÂ results in differential retention on the stationary phase and thusÂ changingÂ the separation.
Mobile phase: TheÂ mobile phaseÂ is the part of the chromatographic system which carries the solutes through the stationary phase. The mobile phases are either liquids or gases. The liquid mobile phases are used to adjust the chromatographic separation and retention in liquid chromatography and the temperature of the gas mobile phase is used to adjust the retention in gas chromatography.
Rf value : Â The distance travelled by a given component divided by the distance travelled by the solvent front. For a given system at a known temperature, it is a characteristic of the component and can be used to identify components. For example, the photosynthetic pigments of an organism and the metabolites of a drug excreted in the urine can be identified by theirÂ RFvalues in paper or thin-layer chromatography.
HPLC : High Pressure Liquid Chromatography.
High performance liquid chromatographyÂ (orÂ high pressure liquid chromatography,Â HPLC) is a form ofÂ column chromatographyÂ used frequently inÂ biochemistryÂ andÂ analytical chemistryÂ to separate, identify, and quantify compounds based on their idiosyncratic polarities and interactions with the column's stationary phase. HPLC utilizes different types of stationary phase (typically,Â hydrophobicÂ saturated carbon chains), a pump that moves the mobile phase(s) and analyte through the column, and a detector that provides a characteristic retention time for the analyte. The detector may also provide other characteristic information (i.e. UV/Vis spectroscopic data for analyte if so equipped). Analyte retention time varies depending on the strength of its interactions with the stationary phase, the ratio/composition of solvent(s) used, and the flow rate of the mobile phase.
With HPLC, a pump (rather than gravity) provides the higher pressure required to propel the mobile phase and analyte through the densely packed column. The increased density arises from smaller particle sizes. This allows for a better separation on columns of shorter length when compared to ordinary column chromatography.
The sample to be analyzed is introduced in small volume to the stream of mobile phase. The analyte's motion through the column is slowed by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. How much the analyte is slowed depends on the nature of the analyte and on the compositions of the stationary and mobile phases. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time; the retention time under particular conditions is considered a reasonably unique identifying characteristic of a given analyte. The use of smaller particle size column packing (which creates higher backpressure) increases the linearÂ velocitygiving the components less time toÂ diffuseÂ within the column, leading to improved resolution in the resultingÂ chromatogram. CommonÂ solventsÂ used include any miscible combination ofÂ waterÂ or various organic liquids (the most common aremethanolÂ andÂ acetonitrile). Water may contain buffers or salts to assist in the separation of the analyte components, or compounds such asÂ trifluoroacetic acidÂ which acts as an ion pairing agent.
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A further refinement to HPLC has been to vary the mobile phase composition during the analysis; this is known asÂ gradient elution. A normal gradient for reversed phase chromatography might start at 5% methanol and progress linearly to 50% methanol over 25 minutes; the gradient chosen depends on howÂ hydrophobicÂ the analyte is. The gradient separates the analyte mixtures as a function of the affinity of the analyte for the current mobile phase composition relative to the stationary phase. This partitioning process is similar to that which occurs during aÂ liquid-liquid extractionÂ but is continuous, not step-wise. In this example, using a water/methanol gradient, the more hydrophobic components willÂ eluteÂ (come off the column) when the mobile phase consists mostly of methanol (giving a relatively hydrophobic mobile phase). The more hydrophilic compounds will elute under conditions of relatively low methanol/high water.
The choice of solvents, additives and gradient depend on the nature of the stationary phase and the analyte. Often a series of tests are performed on the analyte and a number of trial runs may be processed in order to find the HPLC method which gives the best separation of peaks.
Partition chromatography was the first kind of chromatography that chemists developed. TheÂ partition coefficientÂ principle has been applied inÂ paper chromatography,Â thin layer chromatography,Â gas phaseÂ and liquid-liquid applications. The 1952Â Nobel PrizeÂ in chemistry was earned byÂ Archer John Porter MartinÂ andÂ Richard Laurence Millington SyngeÂ for their development of the technique, which was used for their separation ofÂ amino acids. Partition chromatography uses a retained solvent, on the surface or within the grains or fibres of an "inert" solid supporting matrix as withÂ paper chromatography; or takes advantage of some additionalÂ coulombicÂ and/orÂ hydrogen donorÂ interaction with the solid support. Molecules equilibrate (partition) between a liquid stationary phase and the eluent. Known as Hydrophilic Interaction Chromatography (HILIC) in HPLC, this method separates analytes based on polar differences. HILIC most often uses a bonded polarÂ stationary phaseÂ and a non-polar, waterÂ miscible, mobile phase. Partition HPLC has been used historically on unbonded silica or alumina supports. Each works effectively for separating analytes by relative polar differences, however, HILIC has the advantage of separatingÂ acidic,Â basicÂ and neutral solutes in a single chromatogram.
The polar analytes diffuse into a stationary water layer associated with the polar stationary phase and are thus retained. Retention strengths increase with increased analyte polarity, and the interaction between the polar analyte and the polar stationary phase (relative to the mobile phase) increases the elution time. The interaction strength depends on the functional groups in the analyte molecule which promote partitioning but can also includeÂ coulombicÂ (electrostatic) interaction and hydrogen donor capability.
Use of moreÂ polar solvents in the mobile phase will decrease the retention time of the analytes, whereas more hydrophobic solvents tend to increaseÂ retention times.
Partition and NP-HPLC had fallen out of favor in the 1970s with the development of reversed-phase HPLC because of a lack of reproducibility of retention times as water or protic organic solvents changed the hydration state of the silica or alumina chromatographic media. Recently it has become useful again with the development of HILIC bonded phases which improve reproducibility.
Also known as Normal phase HPLC (NP-HPLC), or adsorption chromatography, this method separates analytes based on adsorption to a stationary surface chemistry and by polarity. It was one of the first kinds of HPLC that chemists developed. NP-HPLC uses a polar stationary phase and a non-polar, non-aqueous mobile phase, and works effectively for separating analytes readily soluble in non-polar solvents. The analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity, and the interaction between the polar analyte and the polar stationary phase (relative to the mobile phase) increases the elution time. The interaction strength depends not only on the functional groups in the analyte molecule, but also on steric factors. The effect of sterics on interaction strength allows this method to resolve (separate)Â structural isomers.
The use of more polar solvents in the mobile phase will decrease the retention time of the analytes, whereas more hydrophobic solvents tend to increase retention times. Very polar solvents in a mixture tend to deactivate the stationary phase by creating a stationary bound water layer on the stationary phase surface. This behavior is somewhat peculiar to normal phase because it is most purely an adsorptive mechanism (the interactions are with a hard surface rather than a soft layer on a surface).
NP-HPLC fell out of favor in the 1970s with the development ofÂ reversed-phaseÂ HPLC because of a lack of reproducibility of retention times as water or protic organic solvents changed the hydration state of the silica orÂ aluminaÂ chromatographic media. Recently it has become useful again with the development ofÂ HILICÂ bonded phases which improve reproducibility.
The basic principle ofÂ displacement chromatographyÂ is: A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities.Â There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of the peaks can be achieved only with gradient elution and low column loadings. Thus, two drawbacks to elution mode chromatography, especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings. Displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than "peaks". Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentrations.
Reversed phase HPLC (RP-HPLC or RPC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is a silica which has been treated with RMe2SiCl, where R is a straight chain alkyl group such as C18H37Â or C8H17. With these stationary phases, retention time is longer for molecules which are more non-polar, while polar molecules elute more readily. An investigator can increase retention time by adding more water to the mobile phase; thereby making the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase. Similarly, an investigator can decrease retention time by adding more organic solvent to the eluent. RPC is so commonly used that it is often incorrectly referred to as "HPLC" without further specification. The pharmaceutical industry regularly employs RPC to qualify drugs before their release.
RPC operates on the principle of hydrophobic forces, which originate from the high symmetry in the dipolar water structure and play the most important role in all processes in life science. RPC is allowing the measurement of these interactive forces. The binding of the analyte to the stationary phase is proportional to the contact surface area around the non-polar segment of the analyte molecule upon association with the ligand in the aqueous eluent. ThisÂ solvophobicÂ effect is dominated by the force of water for "cavity-reduction" around the analyte and the C18-chain versus the complex of both. The energy released in this process is proportional to theÂ surface tensionÂ of the eluent (water: 7.3Â Ã-Â 10-6Â J/cmÂ², methanol: 2.2Â Ã-Â 10-6Â J/cmÂ²) and to the hydrophobic surface of the analyte and the ligand respectively. The retention can be decreased by adding a less polar solvent (methanol,Â acetonitrile) into the mobile phase to reduce the surface tension of water.Gradient elutionÂ uses this effect by automatically reducing the polarity and the surface tension of the aqueous mobile phase during the course of the analysis.
Structural properties of the analyte molecule play an important role in its retention characteristics. In general, an analyte with a larger hydrophobic surface area (C-H, C-C, and generally non-polar atomic bonds, such as S-S and others) results in a longer retention time because it increases the molecule's non-polar surface area, which is non-interacting with the water structure. On the other hand, polar groups, such as -OH, -NH2, COO-Â or -NH3+Â reduce retention as they are well integrated into water. Very large molecules, however, can result in an incomplete interaction between the large analyte surface and the ligand's alkyl chains and can have problems entering the pores of the stationary phase.
Retention time increases with hydrophobic (non-polar) surface area. Branched chain compounds elute more rapidly than their corresponding linear isomers because the overall surface area is decreased. Similarly organic compounds with single C-C-bonds elute later than those with a C=C or C-C-triple bond, as the double or triple bond is shorter than a single C-C-bond.
Aside from mobile phase surface tension (organizational strength in eluent structure), other mobile phase modifiers can affect analyte retention. For example, the addition of inorganic salts causes a moderate linear increase in the surface tension of aqueous solutions (ca. 1.5Â Ã-Â 10-7Â J/cmÂ² per Mol for NaCl, 2.5Â Ã-Â 10-7Â J/cmÂ² per Mol for (NH4)2SO4), and because theÂ entropyÂ of the analyte-solvent interface is controlled by surface tension, the addition of salts tend to increase the retention time. This technique is used for mild separation and recovery of proteins and protection of their biological activity in protein analysis (hydrophobic interaction chromatography, HIC).
Another important component is the influence of theÂ pHÂ since this can change the hydrophobicity of the analyte. For this reason most methods use aÂ buffering agent, such asÂ sodium phosphate, to control the pH. The buffers serve multiple purposes: they control pH, neutralize the charge on any residual exposed silica on the stationary phase and act as ion pairing agents to neutralize charge on the analyte.Â Ammonium formateÂ is commonly added in mass spectrometry to improve detection of certain analytes by the formation of ammoniumÂ adducts. A volatile organic acid such asÂ acetic acid, or most commonlyÂ formic acid, is often added to the mobile phase if mass spectrometry is used to analyze the column eluent.Â Trifluoroacetic acidÂ is used infrequently in mass spectrometry applications due to its persistence in the detector and solvent delivery system, but can be effective in improving retention of analytes such asÂ carboxylic acidsÂ in applications utilizing other detectors, as it is one of the strongest organic acids. The effects of acids and buffers vary by application but generally improve the chromatography.
Reversed phase columns are quite difficult to damage compared with normal silica columns; however, many reversed phase columns consist of alkyl derivatized silica particles and shouldÂ neverÂ be used with aqueousÂ basesÂ as these will destroy the underlying silica particle. They can be used with aqueous acid, but the column should not be exposed to the acid for too long, as it can corrode the metal parts of the HPLC equipment. RP-HPLC columns should be flushed with clean solvent after use to remove residual acids or buffers, and stored in an appropriate composition of solvent. The metal content of HPLC columns must be kept low if the best possible ability to separate substances is to be retained. A good test for the metal content of a column is to inject a sample which is aÂ mixtureÂ of 2,2'- and 4,4'-Â bipyridine. Because the 2,2'-bipy canÂ chelateÂ the metal, the shape of the peak for the 2,2'-bipy will be distorted (tailed) whenÂ metalÂ ionsÂ are present on the surface of theÂ silica.[
Size exclusion chromatography (SEC), also known asÂ gel permeation chromatographyÂ orÂ gel filtration chromatography, separates particles on the basis of size. It is generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining theÂ tertiary structureÂ andÂ quaternary structureÂ of purified proteins. SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of a particle. The larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time.
This technique is widely used for the molecular weight determination of polysaccharides. SEC is the official technique (suggested by European pharmacopeia) for the molecular weight comparison of different commercially available low-molecular weightÂ heparins.
A separation in which the mobile phase composition remains constant throughout the procedure is termedÂ isocraticÂ (meaningÂ constant composition). The word was coined by Csaba Horvath from Yale University, who was one of the pioneers of HPLC.
The mobile phase composition does not have to remain constant. A separation in which the mobile phase composition is changed during the separation process is described as aÂ gradient elution.Â One example is a gradient starting at 10% methanol and ending at 90% methanol after 20 minutes. The two components of the mobile phase are typically termed "A" and "B";Â AÂ is the "weak" solvent which allows the solute to elute only slowly, whileÂ BÂ is the "strong" solvent which rapidly elutes the solutes from the column. SolventÂ AÂ is often water, whileÂ BÂ is an organic solvent miscible with water, such as acetonitrile, methanol,Â THF, or isopropanol.
In isocratic elution, peak width increases with retention time linearly according to the equation for N, the number of theoretical plates. This leads to the disadvantage that late-eluting peaks get very flat and broad. Their shape and width may keep them from being recognized as peaks.
Gradient elution decreases the retention of the later-eluting components so that they elute faster, giving narrower (and taller) peaks for most components. This also improves the peak shape for tailed peaks, as the increasing concentration of the organic eluent pushes the tailing part of a peak forward. This also increases the peak height (the peak looks "sharper"), which is important in trace analysis. The gradient program may include sudden "step" increases in the percentage of the organic component, or different slopes at different times - all according to the desire for optimum separation in minimum time.
In isocratic elution, the selectivity does not change if the column dimensions (length and inner diameter) change - that is, the peaks elute in the same order. In gradient elution, the elution order may change as the dimensions or flow rate change.
The driving force in reversed phase chromatography originates in the high order of the water structure. The role of theÂ organic component of the mobile phaseÂ is to reduce this high order and thusÂ reduce the retarding strength of the aqueous component.