Chromatography PDF

CHEM 40.1 Sometimes, suitable solvents are composed of Basic Organic Chemistry Laboratory more than one liquid. For example, separation of amino acids tends to be more...

CHEM 40.1 Sometimes, suitable solvents are composed of Basic Organic Chemistry Laboratory more than one liquid. For example, separation of amino acids tends to be more EXERCISE 4: effective when a mixture of butanol, acetic Chromatography acid, and water of different proportions is used DEFINITION/S: A guide in choosing solvents as mobile phase is CONCEPT DEFINITION/S summarized by the diagram below: Chromatography a physical method of separation in which the components to be separated are distributed between TWO phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction (IUPAC Definition, 1993) MOBILE PHASE a liquid or a gas that When the stationary phase is polar, the chosen carries analytes through mobile phase should be relatively less polar and a liquid or solid vice versa. stationary phase (Skoog et al. 2014) The MOBILE PHASE moves through the STATIONARY PHASE a solid or an STATIONARY PHASE by capillary action (see immobilized liquid on figure below). This is a consequence of the which analyte species adhesive and cohesive forces interacting with the are partitioned during liquid and the surface passage of a mobile phase (Skoog et al., 2014) PRINCIPLE OF CHROMATOGRAPHY The components of a mixture move at different rates due to the differences in their physical properties (solubility, volatility, etc.), and different interactions (adsorption, solute partitioning, etc.) with the stationary phase MOBILE PHASE can be a liquid, gas, or supercritical fluid In an analysis, the choice of mobile phase will depend on: § possible identities of the sample components § polarity of the stationary phase § specific type of chromatography to be performed Prepared by MADArturo, 1st semester, A.Y. 2024-2025 UPLB  STATIONARY PHASE ANALYSIS OF SPOTS a liquid or solid adsorbent or a porous material, The bands or spots after the analysis are shown by which may or may not need solid support, that the CHROMATOGRAM (i.e. the plate which shows can retain solutes into its surface, as illustrated where layers of separated spots or bands can be below seen due to the separation of components of a mixture by chromatography) The intensity of the spots or bands indicates § relative amounts of components § concentrations of components However, some compounds appear as colorless spots since they cannot absorb light in the visible region and need to be visualized through several techniques. The STATIONARY PHASE may be composed of either The spots and bands generated from INORGANIC or ORGANIC materials: chromatography can be quantitatively characterized by measuring their Rf. INORGANIC ORGANIC paper, silica gel, cellulose, various RETENTION FACTOR / RETARDATION FACTOR kieselguhr, alumina substituted celluloses, (acid, neutral, or basic), impregnated celluloses, (Rf) magnesium silicate, microcrystalline the distance traversed by a compound calcium sulfate, celluloses, polyamides, from the origin divided by the distance zirconium phosphate, and powdered ion- traversed by the solvent from the origin hydroxylapatite exchanged resins 𝑅𝑓 = !"#$%&'( $*%+(,,(! -. '/01/2&! 3*/0 0 /*"4"& MECHANISM OF SEPARATION The Rf measured can either be close to 1 or to In general, the mobile phase carries the sample 0, and these imply different things: and separates the components as bands or spots in the stationary phase as shown by the figure below, CLOSE TO ZERO CLOSE TO ONE indicates that means the spot is closer to the the spot is closer to the origin, which implies solvent front, which that indicates that the component is the component is less strongly attracted to retained in the the stationary phase stationary phase but is instead of the mobile more affinitive to the phase mobile phase Rf can also differentiate or compare the identities of the solutes in a chromatogram, such as in identifying the identity of an (sample components are carried by the unknown. If the Rf value of the unknown is MOBILE PHASE through the solid STATIONARY similar or very close to the Rf value of a PHASE, and get separated by virtue of their standard compound, the unknown identity is solubility, polarity, among others) probably the said standard. Prepared by MADArturo, 1st semester, A.Y. 2024-2025 UPLB  TYPES OF CHROMATOGRAPHY CONSIDERATION OF CHOOSING AN STATIONARY MOBILE ADSORBENT PHASE PHASE Components best separated by adsorption NORMAL- polar relatively less chromatography are nonpolar and non-ionic PHASE polar than the organic compounds with low molecular mobile phase weights highly active adsorbents may give rise to REVERSED- relatively less polar irreversible solute adsorption PHASE polar than the Silica gel, which is slightly acidic, may firmly mobile phase retain basic compounds, while alumina (also an adsorbent) is basic and should not be used for the chromatography of base- ADSORPTION CHROMATOGRAPHY sensitive compounds solutes are separated based on their different abilities PARTITION CHROMATOGRAPHY to adsorb to the support’s surface or stationary solutes are separated based on their different abilities phase to partition or dissolve between the stationary and mobile phases In adsorption chromatography, the more strongly the solutes are adsorbed, the more strongly The stationary phases of adsorption and partition they are retained in the stationary phase. chromatography are different in terms of their physical states. This difference is summarized by ADSORBENT the table below. may be packed in a column or spread on a plate; high surface area, active solid; finely divided silica TYPE PHYSICAL STATES and alumina are two of the stationary phases used Stationary Mobile phase for adsorption chromatography phase ADSORPTION SOLID LIQUID/GAS In the experiment, SILICA (silicon dioxide) was used PARTITION Liquid LIQUID/GAS to create the TLC plate due to its higher sample supported on capacity. The silica powder was made into a slurry by the surface of a mixing it with water to create silica gel which was solid then placed on the surface of the TLC plate. Using the table above, we can say that the paper chromatography performed in the experiment is After making the plate, the surface of the TLC plate is under partition chromatography (the said to be composed of mainly Si-OH groups as STATIONARY PHASE being water molecules are shown below, adsorbed on the surface of cellulose present in filter paper), while the thin-layer chromatography is under adsorption chromatography. THIN-LAYER CHROMATOGRAPHY A type of solid-liquid adsorption chromatography where the sample is spotted near one end of a sheet of paper or plate coated with a thin layer of an adsorbent as shown below (there exists an irregular tridimensional framework of alternating silicon and oxygen atoms; additionally, Si-O-H bonds are found at the surface of the silica gel which makes the plate to be HIGHLY POLAR Prepared by MADArturo, 1st semester, A.Y. 2024-2025 UPLB  IDENTITY OF SPOTS STATIONARY PHASE OF TLC All types of leaves contain chloroplast silica gel, alumina, kieselguhr, and cellulose pigments, namely, chlorophylls and powder in ‘TLC grade’ carotenes. incorporation of fluorescent compound such as zinc sulfide to facilitate the detection 1. The chlorophylls are the major pigments of the resolved components of the mixture by present in leaves and are present both as viewing the plates under UV light chlorophyll a and chlorophyll b A binder is sometimes mixed with the slurry (structures shown below) to improve the adhesion of the solid particles to each other and the surface. The plates used in TLC can be conventional or high-performance 1. Conventional plates - thicker layers (200 to 250 mm) with ≥20 mm particle sizes 2. High-performance plates - thickness of 100 mm and particle diameters of 5 mm or less CHLOROPHYLL A TLC GENERAL PROTOCOLS: 1. SPOTTING - Applied 1-2 cm from edge using capillary tubes, toothpicks, or other pointed materials that allow for ample and steady application of the sample 2. Equilibration of chamber by placing filter paper inside first - To ensure that the container is saturated with solvent vapors for a fair resolution and rapid separation CHLOROPHYLL B SEPARATION OF MALUNGGAY PIGMENTS BY TLC As shown, both chlorophylls contain polar C- STATIONARY PHASE SILICA GEL O and C-N bonds and magnesium bonded MOBILE PHASE 8:2 (v/v) petroleum to nitrogen, forming a bond so polar that it ether:ethyl acetate is almost ionic. AND 1:1 (v/v) petroleum ether:ethyl acetate Chlorophyll a and chlorophyll b almost have similar structures except for chlorophyll a NORMAL OR REVERSED NORMAL PHASE having a methyl group (-CH3) in a position PHASE where chlorophyll b has an aldehyde group (-CHO), making the chlorophyll b Choosing the most appropriate solvent system for slightly more polar than chlorophyll a. the separation will depend on MOBILE PHASE which: Chlorophyll a is blue-green and is more § separates the highest number of intense than chlorophyll b. On the other components, hand, chlorophyll b is green to yellow- § has the least degree of overlaps, and green. § has the spots traveling not very close to the origin Prepared by MADArturo, 1st semester, A.Y. 2024-2025 UPLB  2. Carotenes, meanwhile, have eight isoprene PAPER CHROM. GENERAL PROTOCOLS: units or 40 carbon atoms, one example of § similar to TLC which is β-carotene (structure shown § With the movement of the mobile phase, the below). They are classified as components of the mixture will be brought tetraterpenes. along at various speeds. § As the mobile phase reaches the solvent front, the paper is removed, and the separated discrete zones (spots or bands) are located and observed as illustrated below, When consumed, β-carotene is broken to create two vitamin A molecules and is the primary source of this vitamin in the diet. When carotenes are oxidized, they produce the oxygen-containing xanthophylls, which are yellow pigments present in spinach leaves. In green leaves, the most abundant xanthophylls are lutein, zeaxanthin, violaxanthin, and neoxanthin In the performed TLC analysis, the compounds that are more polar or are containing more polar groups are more retained in the stationary SEPARATION OF FOOD DYE COMPONENTS BY PC phase. STATIONARY PHASE water adsorbed on the On the other hand, those that are less polar or pores of Whatman No. 1 are containing a few to no polar groups are held filter paper more strongly by the mobile phase, given that MOBILE PHASE 2% NaCl a normal-phase chromatography is employed. NORMAL OR REVERSED REVERSED-PHASE PHASE (Do note however that if reversed-phase chromatography is performed, the exact opposite Whatman No. 1 Filter Paper of the separation in normal-phase chromatography - “No. 1” indicates that it is of Grade 1 quality is true) - particle retention of 11 μm at 98% efficiency and has a thickness of 0.18 mm PAPER CHROMATOGRAPHY - most widely used filter paper for routine A type of liquid-liquid partition chromatography applications with medium retention and flow wherein solutes are separated based on the rate differences in their solubility towards the stationary phase and mobile phase under the influence of pores in the paper STATIONARY PHASE OF PAPER CHROM a thin film of water adsorbed onto the filter paper, made up of pure cellulose, approximately 23% water by mass Prepared by MADArturo, 1st semester, A.Y. 2024-2025 UPLB  RESULTS OF PC A SOLVENT SYSTEM MUST ALSO: Since the type of chromatography employed (PC) is § not dissolve the adsorbent reversed-phase, the results imply that in terms of § not react irreversibly with the adsorbent or polarity: compounds to be separated. blue > yellow > red § have a large selectivity in desorbing or The possible compounds that compose the blue, dissolving the solutes yellow, and red pigments are Brilliant Blue FCF, § dissolve the compounds to be separated Tartrazine, and Allura Red AC, respectively. § be relatively non-toxic and inexpensive. § composition should not change with time In order of increasing separation (more retained in § be capable of being prepared as required by the stationary phase): simple mixing or of being prepared in bulk and stored till required. Allura Red AC > Tartrazine > Brilliant Blue FCF § Be capable of rapid, complete, and easy removal from the paper or plate after the Basis: polarity of the pigment, (number of -OH and chromatogram has been run. -NH groups) and ionic character SPOTTING CONSIDERATIONS: In general, the order of polarity from most to least When too much of the sample is applied, polar is: ionized/charged groups > -OH groups > - the spot gets too concentrated with the NH2 groups > -NHR groups > OR > NR2, where R solutes, causing spots to appear as long is an alkyl or aryl group. streaks or “blobby” spots on a filter paper or a TLC plate WHY PENCIL IS USED IN MARKING THE ORIGIN Pencil marks are composed of graphite, a Band broadening covalent network of carbon atoms, which is - may cause sample spots to be too close to insoluble in the solvent, hence, it will not each other, resulting in the bleeding be carried through the paper together or combining of solutes - resulting spots or bands will always be broader or larger than the sample spot's GENERAL CONSIDERATIONS original size. This is because the sample diffuses as it travels, which occurs in all directions CHOICE OF MOBILE AND STATIONARY PHASE - happens when TLC plate is placed in a slight A proper balance of intermolecular forces tilt in the developing chamber among the three participants in the - may be due to splashing the solvent onto separation process—the solutes or the plate or paper during placement in the compounds to be separated, the mobile container phase, and the stationary phase—is - can occur by jostling or bumping the needed for successful and effective chamber during the separation. chromatography In general, the polarities of common organic functional groups in increasing order are hydrocarbons < ethers < ketones < aldehydes < amides < alcohols Note however that water is more polar than compounds containing any of the preceding functional groups Also note that ionic solvents are the most polar and will associate to a great extent with charged and polar groups Prepared by MADArturo, 1st semester, A.Y. 2024-2025 UPLB CHROMATOGRAM DEVELOPMENT FACTORS AFFECTING Rf VALUES: CONSIDERATIONS: 1. The dimensions of the apparatus or For PC, it should be ensured that the filter developing chamber paper does not touch the sides of the 2. The grade of paper, or nature and thickness developing chamber to ensure that the of the adsorbent separation of components will occur in 3. Ascending or descending solvent flow and a single direction on a single lane. length of the flow for TLC, silica gel plate should only be held 4. The volume used and composition of the at the corners to prevent the chapping off mobile phase or breaking of the silica gel 5. Any other liquid or vapors incorporated for developing chamber or beaker should be special purposes, e.g., iodine vapors for covered tightly during the separation to visualization prevent errors caused by external factors: 6. Equilibration time (if any) 1. inclusion of foreign particles 7. Temperature 2. change in temperature 8. The nature of the mixture to be 3. irregularities in mobile phase movement chromatographed 4. evaporation of the solvent APPLICATION/S OF TLC AND PAPER CHROM.: VISUALIZATION TECHNIQUES FOR TLC identification of an unknown to establish the purity and authenticity of METHOD Procedure and examples starting materials and reagents DESTRUCTIVE performed when the solutes are not to monitor the reaction UV-active to optimize experimental conditions to check the isolation and purification in this method, the spots are stained procedures (spray or dip) and heated (heat gun to achieve the separation of product or oven) mixtures should this not be possible using Examples of stains used: p- distillation, recrystallization, or sublimation anisaldehyde, vanillin, procedures, permanganate, phosphomolybdic provide a further check on the authenticity acid, iron(III) chloride, and of the final product (supplemental bromocresol green information) SEMI- uses Iodine vapors; semi-destructive DESTRUCTIVE since the complexation of the compounds with iodine is reversible. NON- uses ultraviolet (UV) absorption of DESTRUCTIVE the spots long-wave UV – absorbents are included with a fluorescent powder that glows bright green short-wave UV - only the compounds will glow, with the TLC plates remaining dark Prepared by MADArturo, 1st semester, A.Y. 2024-2025 UPLB