Instrumental Analytical Chemistry (ACAC4101) Chapter 2 Introduction to Chromatography PDF

Instrumental Analytical Chemistry (ACAC4101) 2 – Introduction to Chromatography Reference Book: Fundamentals of Analytical Chemistry, 9th edition, D. A. Skoog et. al. Prepared By: Analytical Chemis...

Instrumental Analytical Chemistry (ACAC4101) 2 – Introduction to Chromatography Reference Book: Fundamentals of Analytical Chemistry, 9th edition, D. A. Skoog et. al. Prepared By: Analytical Chemistry Team- Dr.Elham (EFR)  Chapter 2. Introduction to Chromatography Outline 2.1. Basic Chromatographic Terminologies (Page 861) 2.2. Classification of Chromatography (Page 861-862) 2.3. Elution in Chromatograph (Page 862-864) 2.4. Migration rates of solute (Page 865-868) 2.5. Theories of Band broadening and Column Efficiency (Page 868-872) 2.6. Factors affecting column efficiency (Page 872-877) 2.7. Column Resolution (Page 877-882) page 861 Reference Book: Fundamentals of Analytical Chemistry, 9th edition, D. A. Skoog et. al. Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 2  Introduction to Chromatography Introduction to Chromatography Chromatography: is a technique in which the components of a mixture are separated based on differences in the rates at which they are carried through a fixed or stationary phase by a gaseous or liquid mobile phase. Stationary phase: is a phase that is fixed in place either in a column or planar surface. Mobile phase: is a phase that moves over or through the stationary phase carrying with it the analyte mixture. The mobile phase may be a gas, a liquid, or a supercritical fluid. page 861 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 3  Introduction to Chromatography Chromatographic separation page 861 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 4  2.1. Basic Chromatographic Terminologies Basic Chromatographic Terminologies  Chromatograph: Instrument employed for a chromatography.  Chromatogram: A plot of some function of solute concentration versus elution time or elution volume.  Eluent: The solvent used to carry the components of a mixture through a stationary phase (Fluid entering a column).  Eluate: The mobile phase that exits the column is termed the eluate (Fluid exiting the column).  Elution: The process in which solutes are washed through a stationary phase by the movement of a mobile phase.  Flow rate: How much mobile phase passed / minute (ml/min).  Linear velocity: Distance passed by mobile phase per 1 min in the column (cm/min). page 861 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 5  2.2. Classification of Chromatography Classification of Chromatography Chromatography can be classified based on the type of mobile phase, page 861-862 stationary phase and support material Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 6  2.2. Classification of Chromatography Chromatography can be classified based on: type of mobile phase type of stationary type of support material page 861-862 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 7  2.2. Classification of Chromatography Types of Chromatography 1. based on the type of mobile phase used in the system: Type of Chromatography Type of Mobile Phase Gas chromatography (GC) gas Liquid chromatograph (LC) liquid page 861-862 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 8  2.2. Classification of Chromatography 2. based on the type of stationary phase used in the system: as Chromatography Name of GC Method Type of Stationary Phase Gas-solid chromatography solid, underivatized support Gas-liquid chromatography liquid-coated support LiquidBonded-phase Chromatography gas chromatography chemically- derivatized support Name of LC Method Type of Stationary Phase Adsorption chromatography solid, underivatized support Partition chromatography liquid-coated or derivatized support Ion-exchange chromatography support containing fixed charges Size exclusion chromatography porous support Prepared By: Analytical Chemistry Team- Affinity chromatography Dr.Elham (EFR) support with 9 immobilized ligand  2.2. Classification of Chromatography 3. based on the type of support material used in the system: Packed bed (column) chromatography Open tubular (capillary) chromatography Open bed (planar) chromatography page 861-862 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 10  2.2. Classification of Chromatography Thin Layer (and Paper) Chromatography The ratio of distance travelled by the component (from origin) compared with the distance travelled by the solvent front (from origin) is called the Rf value. x Solvent Solventfront front a Rf of = a/x b c Rf of = b/x Rf of = c/x  2.2. Classification of Chromatography Thin Layer (and Paper) Chromatography A solution of a mixture is applied as a spot/band at the bottom of the plate and allowed to travel with the solvent up the plate. Mixed Unknown + standards standards standards A B C A+B+C A+B+C ??  2.2. Classification of Chromatography Column Chromatography A mixture is applied to a solid support in a chromatography column, and eluted by a solvent. Elute with solvent 1 2 3 4 Absorbent medium tap Cotton wool plug  Theory of Chromatography Parameters used in Chromatography : 1. Retention time tR 2. Retention volume VR 3. Partition Coefficient K 4. Efficiency (Number of Theoretical Plates) N 5. Height Equivalent to a Theoretical Plate (HETP) 6. Selectivity factor α 7. Capacity factor K' 8. Resolution Rs Prepared By: Analytical Chemistry Team- Dr.Elham (EFR)  2.3. Elution in Chromatography Elution in Chromatography Elution is the process in which solutes are washed through a stationary phase by the movement of a mobile phase. Processes during elution: Solutes move at different rates based on the interaction with the stationary phase Solutes spread from very short to much longer length of column – band broadening page 862-864 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 15  2.3. Elution in Chromatography Theory of Chromatography A chromatogram is a typical response obtained by chromatography Where: tR = retention time tM = void time Wb = baseline width of the peak in time units Wh = half-height width of the peak in time units page 862-864 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 16  2.4. Migration rates of solute Migration rates of solute inear Rate of Migration The retention time or retention volume of a solute in chromatography is directly related to the strength of the solute interaction with the mobile and the stationary phases. Retention on a given column relate to the particulars of that system: - size of the column particles - flow rate of the mobile phase Average migration rate , Where: tR = retention time page 865-868 Prepared By: Analytical Chemistry Team- L= length of the Dr.Elham (EFR) column 17  Retention factor (Capacity factor) 2.4. Migration rates of solute (k’):  is the time spent by the analyte in the stationary phase in relation to the mobile phase  = time required to elute analyte peak – time required for mobile phase to pass through the column. k’ = (tR –tM)/tM or k’ = (VR –VM)/VM  capacity factor is useful for comparing results obtained on different systems since it is independent on column length and flow-rate. page 865-868 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 18  Retention factor (Capacity factor) 2.4. Migration rates of solute (k’):  The value of the retention factor is useful in understanding the retention mechanisms for a solute: moles A stationary phase k’ = moles A mobile phase  k’ is directly related to the strength of the interaction between a solute with the stationary and mobile phases.  The amount of solute present in each phase at equilibrium is represented by moles A stationary phase and moles Amobile phase represents.  Equilibrium is approached at the center of a chromatographic peak. When k' is < 1.0, separation is poor When k' is 20- 30, separation is slow When k' is = 1-5, separation is optimum page 865-868 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 19  2.4. Migration rates of solute Partition factor (distribution constant )(K):  The ratio of concentrations of solute in the stationary and mobile phase = k’  As K increases, interaction of the solute with the stationary phase becomes more favorable and the solute’s retention (k’) increases  Separation between two solutes requires different Ks for their interactions with the mobile and stationary phases page 865-868 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 20  2.4. Migration rates of solute Selectivity factor (distribution constant )(K): The selectivity factor, , where B has stronger partitioning than A.  Provides a measure of how well the column will separate the two. page 865-868 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 21  2.5. Theories of Band broadening and Column Efficiency Theories of Band broadening and Column Efficiency page 868-872 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 22  2.5. Theories of Band broadening and Theoretical Plates Column Efficiency Separation on a column can be thought of as a series of extractions/distillations – In the “early” days multiple distillations took place with multiple glass interactions, called plates – In measuring the efficiency of a column, we still refer to plates page 868-872 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 23  2.5. Theories of Band broadening and Column Efficiency Number of theoretical plates (N): Compare efficiencies of a system for solutes that have different retention times 2 𝜎 𝐻= 𝐿 N is for a column, the better the column separation of two compounds. - the better the ability to resolve solutes that have small differences in retention page 868-872 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 24  2.5. Theories of Band broadening and Column Efficiency Rate Theory of Chromatography It describes the shapes and breadth of elution bands quantitatively based on a random-walk mechanism for the migration of analytes through a column. page 868-872 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 25  2.5. Theories of Band broadening and Band Broadening (Asymmetric Band Column Efficiency shapes) Theoretically, the band coming off a column should be Gaussian but this is not always the case This usually occurs when the partition coefficient, K (Cs/Cm) changes during the run  K : either bigger or smaller  K becomes bigger when stationary phase is overloaded by too much solute so band emerges gradually in front (fronting)  K becomes smaller due to tailing (trailing part is elongated) - this is when the solute binds strongly to some sites on the column page 868-872 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 26  2.5. Theories of Band broadening and Column Efficiency Band Broadening and Van Deemter Equation 𝑩 𝑯= 𝑨+ + 𝑪 𝒔 𝒖+ 𝑪 𝑴 𝒖 𝒖 Eddy Diffusion/ Longitudinal Equilibration Multiple flow paths/ Diffusion Time/ mass transfer page 868-872 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 27  2.5. Theories of Band broadening and Column Efficiency Band Broadening due to Eddy Diffusion, A: a.) Eddy diffusion A– random multiple flow paths through a packed column. As solute molecules travel through the column, some arrive at the end sooner than others simply due to the different path traveled around the support particles in the column that result in different travel distances. Longer path arrives at end of column after (1). page 868-872 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 28  2.5. Theories of Band broadening and Column Efficiency Band Broadening due to Longitudinal Diffusion, B/U: the diffusion of the solute away from the concentrated center of a band to the more dilute regions on either side The degree of band-broadening due to longitudinal diffusion depends on: 1) the diffusion of the solute 2) the flow-rate of the solute through the column Broadening is inversely proportion to the flow rate. To minimize the broadening of the peak use faster flow rate page 868-872 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 29  2.5. Theories of Band broadening and Band Broadening due to Mobile phase mass Column Efficiency transfer, CMu – a process of peak broadening caused by the presence of different flow profile within channels or between particles of the support in the column. A solute in the center of the channel moves more quickly than solute at the edges, it will tend to reach the end of the channel first leading to band-broadening The degree of band-broadening due to mobile phase mass transfer depends mainly on: 1) the size of the packing material 2) the diffusion rate of the solute page 868-872 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 30  2.5. Theories of Band broadening and Band Broadening due to Stationary phase mass Column Efficiency transfer, Csu band-broadening due to the movement of solute between the liquid phase and the stationary phase. Since different solute molecules spend different lengths of time in the stationary phase, they also spend different amounts of time on the column, giving rise to band-broadening The degree of band-broadening due to stationary phase mass transfer depends on: 1) the retention and diffusion of the solute 2) the flow-rate of the solute through the column 3) the kinetics of interaction between the solute and the stationary phase page 868-872 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 31  2.5. Theories of Band broadening and Van Deemter Equation Column Efficiency relates flow-rate or linear velocity to H - The plate height equation as a result of the four band broadening mechanisms 𝑩 𝑯= 𝑨+ + 𝑪 𝒔 𝒖+ 𝑪 𝑴 𝒖 𝒖 Eddy Diffusion/ Longitudinal Equilibration Multiple flow paths/ Diffusion Time/ mass transfer m optimum Van Deemter Equation represents the use of plate height (H) is to relate these kinetic process to band broadening to a parameter of the chromatographic system page 868-872 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 32  2.6. Factors affecting column efficiency Factors affecting column efficiency page 872-877 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 33  2.7. Column Resolution Column Resolution Resolution : is how far a part the two bands relative to their width or the quantitative measure of column ability to separate analytes A and B. Not separated Separated, 4% Rs = 1.5 is a complete separation but 0.75 resolution does not Well separated, 0.13% page 877-882 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 34  2.7. Column Resolution  k’ and a Effect on Rs kB is the retention factor of the slower-moving species a is the selectivity factor m is the linear velocity of the mobile phase  tR and Rs page 877-882 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 35  2.7. Column Resolution Factors affecting column Resolution: # plates Partition Capacity coefficients factor column length Ways to increase resolution: – Increase column length (Square root of N) – Change phase interaction – Increase capacity factor (Increase fraction of time solute spends in stationary phase) page 877-882 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 36  2.7. Column Resolution Factors affecting column Resolution: Effect of column length on the resolution. Chromatograms obtained with a GC instrument illustrating that by doubling the length of the capillary column, the resolution is multiplied by 1.41 or √2 (adapted from a document of SGE Int. Ltd). page 877-882 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 37  Example: The following data came from an HPLC separation, using length of packing of 10 cm, and a flow rate of 1 ml/minute: Material Retention time Width of peak (min) base (min) Non – retained 1.45 - A 3.00 0.25 B 3.50 0.29 C 5.00 0.42 D 6.00 0.48 Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 38  Calculate the number of plates for each peak? What is the average and standard deviation for N? What is the plate height for the column? Which peaks are better resolved, A&B or C&D? What is the selectivity factor for B relative to A? Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 39  Example: On a gas chromatography column L = 30 m and compounds elute in 10 min with w1/2 = 5 s. What's a number of theoretical plates ? And what's the plate height? = 79776 = 0.375 mm Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 40  Example: In liquid chromatography, columns are only L = 25 cm. Compounds elute in 10 min with a w1/2 = 5 sec. What's the number of theoretical plates and what's the plate height? 𝑁=5.54 ¿ Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 41  Example: A solute has a retention time of 5 min and 12 sec base width. A nearby peak eluted at 5.8 min with a base width of 16 sec. What is the resolution between these peaks? 𝑅 𝑠 =2 ¿ ¿ Prepared By: Analytical Chemistry Team- Dr.Elham (EFR) 42