Chromatography: Introduction to Chromatographic Separations PDF

INTRODUCTION TO CHROMATOGRAPHIC SEPARATIONS Chromatography - an analytical technique used to separate, identify, and quantify individual components in a sample mixture - invented by the Russian botanist Mikhail Tswett; he employed the technique to separate various plant pigments such as...

INTRODUCTION TO CHROMATOGRAPHIC SEPARATIONS Chromatography - an analytical technique used to separate, identify, and quantify individual components in a sample mixture - invented by the Russian botanist Mikhail Tswett; he employed the technique to separate various plant pigments such as chlorophylls and xanthophylls by passing the solutions through glass columns packed with finely divided calcium carbonate; the separated species appeared as colored bands on the column which accounts for the name he chose for the method (Greek chroma meaning “color” and graphein meaning “to write”) Figure 1. Separation of components of mixture by column chromatography (Source: Google.com) The separation requires 2 phases: Stationary (fixed) phase – may be solid or liquid Mobile (moving) phase – may be liquid (Liquid Chromatography) or gas (Gas Chromatography) In Normal phase chromatography, the stationary phase is polar and the mobile phase is nonpolar (for separation of polar compounds) In Reversed phase chromatography, the stationary phase in nonpolar and the mobile phase is polar (for separation of nonpolar compounds) Elution – a process in which solutes are washed through a stationary phase by the movement of a mobile phase Eluent – a solvent used to carry the components of a mixture through a stationary phase Table 1. Classification of Column Chromatographic Methods General Classification Specific Method Stationary Phase Type of Equilibrium Gas Chromatography Gas-Liquid Liquid adsorbed or Partition between gas (Mobile Phase: Gas) Chromatography (GLC) bonded and liquid to a solid surface Gas-Solid Solid Adsorption Liquid Chromatography Liquid-Liquid, or Liquid adsorbed or Partition between (Mobile Phase: Liquid) Partition bonded immiscible liquids to a solid surface Liquid-Solid, or Solid Adsorption Adsorption Ion Exchange Ion-exchange resin Ion exchange Size Exclusion Liquid in interstices of a Partition/sieving polymeric solid Affinity Group specific liquid Partition between bonded to a solid surface liquid and surface mobile liquid Supercritical Fluid Organic species bonded Partition between Chromatography (SFC) to a solid surface supercritical and (Mobile Phase: bonded surface Supercritical Fluid) The Chromatogram  - a plot of detector signal as a function of volume of eluate or elution time - the number of peaks correlates with the number of components in the sample Figure 2. Chromatogram (Source: Google.com) Migration Rates of Solutes The Partition Ratio All chromatographic separations are based upon differences in the extent to which solutes are partition between the mobile and stationary phase. For the solute species A, the equilibrium involved is described by the equation Amobile → Astationary The equilibrium constant for this reaction is called a partion ratio or partion coefficient, K, and is defined as K = cS / cM where cS = molar analytical concentration of a solute in the stationary phase CM = molar analytical concentration of a solute in the mobile phase The Capacity Factor, k’ - an important experimental parameter that is widely used to describe the migration rates of solutes on columns - for solute A, k’A = KAVS / VM where KA = partition coefficient VS = volume of stationary phase VM = volume of mobile phase k’A = (tR – tM) / tM where tR = retention time (time required for solute to reach the detector after sample injection) tM = dead time (time required for the unretained species to reach the detector; provides a measure of the average migration rate of the mobile phase) The Selectivity Factor, α - capability of chromatographic system to distinguish between two components - for solutes A and B, α = KB / KA where KB = partition coefficient of the more retained species B KA = partition coefficient of the less retained species A α = k’B / k’A where k’B = capacity factor for solute B k’A = capacity factor for solute A α = [(tR)B – tM] / [(tR)A – tM] When α = 1: separation is difficult α = 2: clean separation in minimal time is possible Band Broadening and Column Efficiency Chromatographic efficiency is affected by the amount of band broadening that occurs when a compound passes through the column. The Plate Theory of Chromatography - chromatographic column is envisioned as being composed of a series of discrete, narrow horizontal layers called plates - equilibration of solute between the stationary and mobile phases takes place at each step - the solute moves down the column by transfer of equilibrated mobile phase from one plate to the next - movement of solute and solvent is a series of stepwise transfers from one plate to the next - note: the plates do not really exist Column Efficiency - a measure of the chromatographic column’s ability to separate two solutes - if two similar solutes are separated in a minimum column length, the column has high efficiency - quantitative measures of column efficiency: (a) plate height, H (b) number of theoretical plates, N N=L/H where L = length of column packing - efficiency of a column increases as the number of theoretical plates increases - decreasing the plate height will lead to greater column efficiency - band broadening decreases column efficiency The Rate Theory of Chromatography - describes how various column parameters and operating conditions can be altered to increase column efficiency - the chromatographic peaks are generally broader than expected from the random nature of migration process alone due to 3 kinetically controlled processes: eddy diffusion, longitudinal diffusion, and mass transfer - the magnitudes of these effects are determined by controllable variables such as flow rate, particle size of packing, and thickness of stationary layer on the packing Eddy Diffusion (A) - arises from the multitude of pathways by which a molecule can find its way through a packed column - involves the packing effect in the column and the particle diameter - band broadening due to eddy diffusion is minimized by careful packing of column with small spherical particles possessing a limited range of sizes Longitudinal Diffusion (B) - results from the tendency of molecules to migrate from the concentrated center part of a band toward more dilute regions on either side - can occur in both phases - the amount of diffusion increases with time so extent of broadening increases as the flow rate decreases Nonequilibrium Mass Transfer (C) - due to the nonequilibrium conditions that results from rapid flow rates - equilibrium is not instantly achieved leading to broadening at both ends of the solute band - effects become smaller as flow rate is decreased since there is more time for attainment of equilibrium The van Deemter Equation - the effect of the column variables is expressed in the van Deemter equation: H = A + B/u + Cu where H = height equivalent to a theoretical plate (cm) u = flow rate of the mobile phase (cm2/s) A = constant that accounts for the effects of eddy diffusion B = constant that accounts for the effects of longitudinal diffusion C = constant that accounts for the effects of nonequilibrium mass transfer - the modified van Deemter Equation is H = A + B/u + CSu + CMu where CSu = mass transfer coefficient for the stationary phase CMu = mass transfer coefficient for the mobile phase The van Deemter Plots - a plot of plate height vs. average linear velocity of the mobile phase - such plots are of considerable use in determining the optimum mobile phase flow rate Column Resolution - the ability of a column to resolve two solutes - determined by the difference in elution times between the peaks and the peak widths - the further apart the peaks and the narrower the peaks, the better their separation Rs = ΔtR / wave = 2 [(tR)B – (tR)A] / (wA + wB) where w = peak width of the base √𝑵 𝜶 − 𝟏 𝒌′𝐵 𝑹𝑺 = ( )( )( ) 𝟒 𝜶 𝟏 + 𝒌′𝑩 Rearranging equation above gives - RS values of 0.50 and 0.75 gives poor resolution and two peaks overlap - RS of 1.50 gives an essentially complete separation of two peaks Remember! The goal in chromatography is to have the highest possible resolution in the shortest possible time. Other useful equations: N = 16 (tR / w) 2 (See derivation from Skoog)  N = 5.55 (tR / w1/2) 2 where w1/2 = width at half peak height The time required, tR, to elute the species with a resolution of Rs is given by where H = plate height u = flow rate of the mobile phase α = selectivity factor k’ = capacity factor Example Substances A and B have retention times of 16.40 and 17.63, respectively, on a 30.0-cm column. An unretatined species passes through the column in 1.30 min. The peak widths at base for A and B are 1.11 and 1.21 min, respectively. Calculate (a) column resolution, (b) average number of plates in the column, (c) plate height, (d) length of column required to achieve a resolution of 1.5, and (e) time to elute substance B on the longer column. Answer: (𝐭𝐑)𝐁 – (𝐭𝐑)𝐀 𝟏𝟕.𝟔𝟑 – 𝟏𝟔.𝟒𝟎 (a) 𝑹𝑺 = 𝟐 ( ) = 𝟐( ) = 𝟏. 𝟎𝟔 𝐰𝐀 + 𝐰𝐁 𝟏.𝟏𝟏 + 𝟏.𝟐𝟏 (b) N = 16 (tR/w)2 NA = 16 (16.40/1.11)2 = 3493 NB = 16 (17.63/1.21)2 = 3397 Nave = (3493 + 3397) / 2 = 3445 (c) N = L / H H = L / N = 30.0 cm / 3445 = 8.71 x 10 -3 cm (d) k’and α do not change greatly with increasing N and L thus substituting N 1 and N2 into the equation √𝑵 𝜶−𝟏 𝒌′ 𝑹𝑺 = ( 𝟒 ) ( ) (𝟏+𝒌′𝐵 ) and dividing one of the resulting equations by the 𝜶 𝑩 other give 𝑹𝒔𝟏 √𝑵𝟏 𝟏.𝟎𝟔 √𝟑𝟒𝟒𝟓 𝑹𝒔𝟐 = → 𝟏.𝟓 = √𝑵𝟐 √𝑵𝟐 N2 = 3445 (1.5)2/ (1.06)2 = 6899 = 6.9 x 10 3 Therefore, L = N (H) = 6.9 x 10 3 (8.71 x 10 -3 cm) = 60. cm (e) Again, k’and α do not change greatly with increasing N and L thus substituting Rs1 and Rs2 into the equation and dividing one of the resulting equations by the other give tR1 / tR2 = (Rs1)2 / (Rs2)2 17.63 / tR2 = (1.06)2 / (1.5)2 Thus, tR2 = 35 min Exercise The following data are for a liquid chromatographic column: Length of packing 24.7 cm Flow rate 0.313 mL/min VM 1.37 mL VS 0.164 mL A chromatogram of a mixture of species A, B, C, and D provided the following data: Retention Time, min Width of Peak Base, min Unretained 3.1 --- A 5.4 0.41 B 13.3 1.07 C 14.1 1.16 D 21.6 1.72 Calculate (a) the number of plates from each peak (b) the mean and the standard deviation for N (c) the plate height for the column (d) the capacity factor for A, B, C, and D (e) the partition coefficient for A, B, C, and D (f) the resolution for species B and C (g) the selectivity factor for species B and C (h) the length of column necessary to separate species B and C with a resolution of 1.5 (i) the time required to separate species B and C on the column in (h). Optimizing k’ to Improve Resolution - the easiest way to improve resolution is by optimizing k’ - the optimal value of k’B lies in the range from 1 to 5; k’ B values of greater than about 10 should be avoided because they provide little increase in resolution but markedly increase the time required for separations - for gaseous mobile phases, k’ can be improved by temperature changes (temperature programming) - for liquid mobile phases, changes in the solvent composition (gradient elution or solvent programming) often permit manipulations of k’ to yield better separations - other means of optimizing k’: changing composition of stationary phase and using special chemical effects such as incorporating a species which complexes with one of the solutes into the stationary phase Applications of Chromatography Qualitative Analysis - chromatography is widely used for recognizing the presence or absence of components in mixtures that contain a limited number of species whose identities are known - based on retention time - confirmation of identity would require spectral or chemical investigation of isolated components - the chromatogram may not lead to positive identification of the species in the sample but it often provides sure evidence of the absence of species - failure of a sample to produce peak at the same retention time as a standard obtained under identical conditions is strong evidence that the compound in question is absent (or present at a concentration below the detection limit of the procedure) Quantitative Analysis - based upon a comparison of either the peak height or the peak area of the analyte with that of one or more standards - analysis based on peak height can be used provided variations in column conditions do not alter peak widths during recording of chromatograms; peaks should not be distorted or the column overloaded - analysis based on peak area is less sensitive to small changes in operating conditions; independent of broadening effects Calibration with Standards External Standard Method - involves the preparation of a series of standard solutions that approximate the composition of the unknown - the calibration is a plot of peak height (or peak area) vs. concentration of standard Internal Standard Method - minimizes uncertainty in sample injection and variability due to instrument conditions - a carefully measured quantity of an internal standard is introduced into each standard and sample, and the ratio of analyte peak area (or peak height) to internal standard peak area (or peak height) is obtained; the calibration curve is a plot of this ratio vs. concentration - requirements for internal standard: must be completely resolved; must elute near peaks of interest; similar in concentration to peaks of interest; chemically inert and absent in the sample