Department of Chemistry Instrumental Analysis I Introduction By Dawit Alemu 1. Analytical Separation Techniques and Classical Method of Analysis Analytical Methods This can be divided in to two broad classes as classical and instrumental method of analysis. Classical Methods Separation of analytes by precipitation, extraction, or distillation. Qualitative analysis by reaction of analytes with reagents that yielded products that could be recognized by their colors, boiling or melting points, solubilities, optical activities, or refractive indexes. Quantitative analysis by gravimetric or by titrimetric techniques Cont… Instrumental Methods Measurement of physical properties of analytes - such as conductivity, electrode potential, light absorption or emission, mass-to-charge ratio, and fluorescencebegan to be employed for quantitative analysis of inorganic, organic, and biochemical analytes. Efficient chromatographic separation techniques are used for the separation of components of complex mixtures. Instrumental Methods of analysis (collective name for newer methods for separation and determination of chemical species.) Cont … Table 1.1. Comparisons between the two methods Classical Methods Instrumental methods Simple to use Somewhat complex Cheap equipment requirement Coasty and require continuous calibration Based on absolute (direct) measurement Are not absolute (indirect way) in measurement Special training not required to operate the Special training will require to operate the instruments instruments Consume large sample volume Small sample can be used Time consuming Save time since it is automatic Separation techniques Brainstorming: How do you define separation? What is analytical separation? Why separation is extremely important in chemical analysis? Separation techniques cont… Separation is the act of differentiating pure compound from interferent or separating mixture to its components. Separations are extremely important in synthesis, industrial chemistry, biomedical sciences, and chemical analysis. The goals of an analytical separation are usually : to eliminate or reduce interferences and to obtain pure components of a mixture so that quantitative analytical information can be obtained about complex mixtures. Cont … Table 1.2 Separation Methods Method Basis of Methods Mechanical phase separation Precipitation and filtration Difference in solubility of compounds formed Distillation Difference in volatility of compounds Extraction Difference in solubility in two immiscible liquids Ion exchange Difference in interaction of reactants with ion-exchange resin Chromatography Difference in rate of movement of a solute through a stationary phase Electrophoresis Difference in migration rate of charged species in an electric field Field-flow fractionation Difference in interaction with a field or gradient applied perpendicular to transport direction 2. Introduction to Chromatographic Separation 2.1. Historical Background Mikhail Tswett, Russian Botanst, 1872-1919 In 1906 Tswett used to chromatography to separate plant pigments He called the new technique chromatography because the result of the analysis was 'written in color' along the length of the adsorbent column Chroma means “color” and graphein means to “write” Mikhail Tswett Original Chromatography Experiment of Tswett Start: A glass column is filled with powdered limestone (CaCO3). An EtOH extract of leaf pigments is applied to the top of the column. EtOH is used to flush the pigments down the column. Later End: A series of colored bands is seen to form, corresponding to the pigments in the original plant extract. These bands were later determined to be chlorophylls, xanthophylls and carotenoids. Definition of Chromatography Chromatography is 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 through it in a definite direction. Chromatography is the term used to describe a separation technique in which a mobile phase carrying a mixture is caused to move in contact with a selectively absorbent stationary phase. • The chromatographic process occurs due to differences in the distribution constant of the individual sample components. Cont … Components or terms of chromatography mobile phase: a solvent that flows through the supporting medium. • Q? Which kind of phases can be used as mobile phase? stationary phase: a layer or coating on the supporting medium that interacts with the analytes. • Q? Which kinds of phases can be used for stationary phase? supporting medium: a solid surface on which the stationary phase is bound or coated. Cont … Note: In a chromatographic separation the mobile and stationary phase must have low interaction. Always when we select a mobile phase we should consider the following: The mobile phase must be the least eluted species. The mobile phase could not wash the stationary phase. a chromatogram: is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture. 2.2. Types (Classification) of Chromatography There are a number of different kinds of chromatography, which differ in the mobile and the stationary phase used. Chromatography can be classified based on three criteria. 1. the physical means by which the stationary phase and mobile phase are brought together. 2. the type of mobile phase and stationary phases 3. the type of interaction occur between the sample and stationary phase Cont… 1. Based on the physical means by which the stationary phase and mobile phase are brought together. Column chromatography Packed Open tubular Planar chromatography Paper TLC Cont … Column chromatography Column chromatography is a separation technique in which the stationary phase is within a tube. Planar chromatography Paper Chromatography Paper chromatography is a technique that involves placing a small dot or line of sample solution onto a strip of chromatography paper. Cont… Thin Layer Chromatography (TLC) TLC is a widely employed laboratory technique and is similar to paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. Cont … Rf = Distance the solute move Distance the solvent front move • Q? How you can identify if invisible spots could occur when you do TLC? • Q? Which types of mobile phases are used for column and planar chromatography? • Q? Which types of stationary phases can be used for column and planar chromatography? 2. Based on the type of mobile phase and stationary phases Gas chromatography (GC) includes all chromatographic methods in which gas is used as mobile phase. It uses only stationary phases either coated or packed on a column. Liquid chromatography (LC) includes all chromatographic techniques in which liquid is used as mobile phase. For LC a stationary phase is either packed on a column or coated on a column or a planar plate can be used. Supercritical fluid chromatography (SFC) includes chromatographic techniques which uses supercritical fluid as mobile phase. For this technique the stationary phase could be organic species bonded on a solid surface. Cont … Table 2.1. Classification of chromatography based on the type of mobile phase and stationary phases Chromatography technique Mobile phase Stationary phase Liquid chromatography (LC) a, Liquid-liquid chromatography Liquid Liquid immobilized on a solid surface b, Liquid-solid chromatography Liquid solid a, Gas-liquid chromatography Gas Liquid immobilized on a solid surface b, Gas-solid chromatography Gas solid Gas chromatography (GC) Supercritical fluid chromatography Supercritical fluid Organic species bonded on a solid (SFC) surface 3. based on the type of interaction occur between the sample and stationary phase Table 2.2. Types of chromatography based on interaction between sample and stationary phase Type of chromatography Type of interaction occur a. Adsorption chromatography Adsorption of a sample on a solid surface b. Partition chromatography Partition of a sample between two immiscible liquids c. Ion-exchange chromatography Ion exchange d. Size exclusion chromatography Partitioning/ Sieving Elution • Note: Separation in chromatography is based on the elution of the sample across the stationary phase by the mobile phase. Across the movement the components of the sample interacts with the stationary phase differently. The solute which interacts strongly with the stationary phase will be strongly retained. So the speed of this solute in the column or plane is very slow and takes more time to be eluted. The solute, which interacts with the stationary phase weakly, will be weakly retained. So the solute will be eluted easily. Cont …. A -is a solute which has a weak interaction with the stationary phase and weakly retained. B –is a solute which has a strong interaction with the stationary phase and strongly retained. Resolution Z Rs WA / 2 WB / 2 2Z Rs WA WB 2[(t R ) B (t R ) A ] Rs WA WB 2.4. Migration Rates of Solutes • The effectiveness of a chromatographic column in separating two solutes depends in part on the relative rates at which the two species are eluted. • These rates in turn are determined by the ratios of the solute concentrations in each of the two phases. • The separation is enhanced by altering the relative flow rate of solutes. i.e. o by increasing the flow rate of weakly retained species and o decreasing the flow rate of strongly retained species. Factors affecting Migration Rates of Solutes • Distribution Constant, K or D Ain mobile phase Ain stationary phase • The equilibrium constant K; for this reaction is called a distribution constant, which is defined as 𝐾= 𝐶𝑠 𝐶𝑚 Where: 𝐶𝑠 - Concentration in molar of solute A in the stationary phase 𝐶𝑚 - Concentration in molar of solute A in the mobile phase Note: • Large K value is obtained when the solute concentration in the stationary phase is large. This indicates the solute interacts with stationary phase strongly. Cont… • Retention time o The retention time (tR) is the time between injection of a sample and the appearance of a solute peak at the detector of a chromatographic column. o The dead time (void time) (tM) is the time it takes for an unretained species to pass through a chromatographic column. o All components spend this (tM) amount of time in the mobile phase. o Separations are based on the different times tS that components spend in the stationary phase. Velocities: Linear rate of solute migration! Velocity = distance/time length of column/ retention times Velocity of solute: v L tR Velocity of mobile phase: L tM The Relationship between Migration Rate and Distribution Constant To relate the migration rate of a solute to its distribution constant, we express the rate as a fraction of the velocity of the mobile phase. v fraction of solute spends in mobile phase moles of solute in mobile phase v total moles of solute cM VM v cM VM cSVS Cont… cM VM v cM VM cSVS 1 v 1 cSVS / cM VM cS K cM Distribution Constant 1 v 1 K VS / VM Cont… Retention Factor : It is the amount of time a solute spends in the stationary phase relative to the time it spends in the mobile phase. 1 v 1 K VS / VM k ' A K AVS / VM (Retention Factor) 1 v 1 k 'A L L 1 tR tM 1 k ' A tR tM k 'A tM Adjusted retention time Cont… Selectivity Factor: The selectivity factor for solutes A and B is defined as the ratio of the distribution constant of the more strongly retained solute (B) to the distribution constant for the less strongly held solute (A). B retained more than A >1 KB KA k 'B k 'A Distribution Constant Retention factor (t R ) A t M (t R ) B t M k 'A and k ' B tM tM (t R ) B t M (t R ) A t M Retention time Band Broadening and Column Efficiency Theoretical Plates and Plate Theories H plate height N number of plates L N H H 2 L L = length of column packing standard deviation 2 L variance per unit length. Cont… Relation between column distance and retention times L column length (distance) standard deviation in distance t R retention time standard deviation in time L tR L / tR Cont… Relation between column distance and retention times L tR L tR W 4 Tangent at Inflection point ~96% 2 WL 4 tR 2 W2 L H L 16 t R2 Cont… Determining the Number of Theoretical Plates N number of pates t N 16 R W W1/2 2 t N 5.54 R W1/ 2 2 2.5. Optimization of Resolution Effect of Retention Factor and Selectivity Factor on Resolution A useful equation that relates the resolution of a column to the number of plates also contains the retention and selectivity factors of pair of solutes on the column. 𝑅𝑠 = 𝑁 𝛼−1 𝐾′ 𝐵 ( )( ) 4 𝛼 1+ 𝐾′ 𝐵 Where: 𝐾 ′ 𝐵 is the retention factor of the strongly retained solute. 𝛼 is selectivity factor Rearrange to solve for the number of theoretical plates, N: 𝑁= 2 1+ 𝐾′ 2 𝛼 𝐵 16𝑅𝑠2 𝛼−1 𝐾′ 𝐵 Effect of Resolution on Retention Time The time required to obtain the resolution Rs is given by; (𝑡𝑅 )𝐵 = 3 16𝑅𝑠2 𝐻 𝛼 2 1+ 𝐾′ 𝐵 µ 𝛼−1 𝐾′ 𝐵 2 Factors affecting solutes separation in CC ( Factors affecting column efficiency) Factor Effect Particle size of solid stationary Decrease of size improves separation (but very small phase (or of support) particles need high pressure). Column dimensions Efficiency increases as ratio length / width increases. Uniformity of packing Non uniform packing results in irregular movement of solutes through column & less uniform zone formation, (i.e. band broadning or tailing). Column temperature Increase in column temperature results in speed of elution but does not improve separation (tailing). Eluting solvent Solvents should be of low viscosity (to give efficient resolution) & h igh volatility (to get rapid recovery of the substances). Solvent flow rate Uniform & low flow rate gives better resolution. Continuity of flow Discontinuous flow disturbs resolution Condition of adsorbent Deactivation of adsorbent decreases separation. Concentration of solutes Substances of high concentration move slowly. Column Efficiency Kinetic variables Zone Broadening Flow Rate of Mobile Phase Liquid chromatography Gas chromatography Note the differences in flowrate and plates height scales Zone Broadening Kinetic Processes Van - Deemter Equation λ and γ are constants that depend on quality of the packing. B is coefficient of longitudinal diffusion. Cs and Cm are coefficients of mass transfer in stationary and mobile phase, respectively. H A B / (CS CM ) Zone Broadening Multiple Pathways Eddy Diffusion: band broadening process results from different path lengths passed by solutes. 1. Directly proportional to the diameters of packing 2. Lower mobile-phase velocity, smaller eddy diffusion Zone Broadening Longitudinal Diffusion Column Diffusion • The higher the , the smaller the H • Much smaller in LC than in GC 1. the physical means by which the stationary phase and mobile phase are brought together. Paper Chromatography In paper chromatography, the mobile phase is a solvent, and the stationary phase is water held in the fibers of chromatography paper. A solution of the mixture to be separated is spotted onto a strip of chromatography paper (or filter paper) with a dropper. The chromatogram is developed by placing the bottom of the paper (but not the sample spot) in a tank containing a suitable solvent. The solvent is drawn up the paper by capillary action. The components of the mixture move up the paper with the solvent at different rates due to their differing interactions with the stationary and mobile phases. Cont … Rf = Distance the solute moves/Distance the solvent front moves Column Chromatography In column chromatography, the mobile phase is again a solvent, and the stationary phase is a finely divided solid, such as silica gel or alumina. Chromatography columns vary in size and polarity. There is an element of trial and error involved in selecting a suitable solvent and column for the separation of the constituents of a particular mixture. A small volume of the sample whose constituents are to be separated is placed on top of the column. The choice of the eluting solvent should ensure that the sample is soluble. However, if the sample was too soluble the mobile phase (solvent) would move the solutes too quickly, resulting in the nonseparation of the different constituents. Cont … • Thin Layer Chromatography • In thin layer chromatography, the mobile phase is also a solvent, and the stationary phaseis a thin layer of finely divided solid, such as silica gel or alumina, supported on glass or aluminium.Thin layer chromatography is similar to paper chromatography in that it involves spotting the mixture on the plate and the solvent (mobile phase) rises up the plate in the chromatography tank. • It has an advantage over paper chromatography in that its separations are very efficient because of the much smaller size of the particles in the stationary phase. • Thin layer chromatography is particularly useful in forensic work, for example in the separation of dyes fromfibres. Gas chromatography and high performance liquid chromatography are more sophisticated chromatographic techniques.