Introduction to Separations - Advanced Analytical Chemistry - Lecture Slides, Slides of Analytical Chemistry

Download Introduction to Separations - Advanced Analytical Chemistry - Lecture Slides and more Slides Analytical Chemistry in PDF only on Docsity! Introduction to Separations Science  What is separations science? – A collection of techniques for separating complex mixtures of analytes – Most separations are not an analytical technique in their own right, until combined with an analytical detector (often a type of spectrometer)  Key analytical branches discussed in this class: chromatography, electrophoresis, extraction docsity.com What is a Separation? ( a + b + c + d + ……) (a) + (b) + ( c ) + (d) +…… COMPLETE SEPARATION ( a + b + c + d + ……) (a) + ( b + c + d+ …..) PARTIAL SEPARATION ( a + b + c + d + ……) ( a + b ) + ( b + a) + …….. ENRICHMENT D ET EC TI O N  Separations are key aspects of many modern analytical methods. Real world samples contain many analytes, many analytical methods do not offer sufficient selectivity to be able to speciate all the analytes that might be present.  Most separation methods involve separation of the analytes into distinct chemical species, followed by detection: docsity.com The 100-Year History of Separations Russian chemist and botanist Michael Tswett coined the term “chromatography” Chromatography was the first major “separation science” Tswett worked on the separation of plant pigments, published the first paper about it in 1903, and tested >100 stationary phases  Separated chlorophyll pigments by their color using CaCO3 (chalk), a polar “stationary phase”, and petroleum ethers/ethanol/CS2 Mikhail Tswett , Physical chemical studies on chlorophyll adsorptions Berichte der Deutschen botanischen Gesellschaft 24, 316-23 (1906) Tswett’s original adsorption chromatography apparatus docsity.com History of Analytical Chromatography  Chromatography was “rediscovered” by Kuhn in 1931, when its analytical significance was appreciated  Chromatography very rapidly gained interest: Kuhn (Nobel prize in Chemistry 1937) separates caretenoids and vitamins  1938 and 1939: Karrier and Ruzicka, Nobel prizes in Chemistry  1940: established analytical technique  1948: A. Tiselius, Nobel prize for electrophoresis and adsorption  1952: A. J. P. Martin and R. L. M. Synge, Nobel prize for partition chromatography, develop plate theory  1950-1960: Golay and Van Deemter establish theory of GC and LC  1965: Instrumental HPLC developed Photographs from www.nobelprize.org A. Tiselius R. Kuhn A. J. P. Martin R. L. M. Synge docsity.com Introduction to Chromatography: Terminology  IUPAC Definition: chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary while the other moves in a definite direction  Stationary phase (SP): common name for the column packing material in any type of chromatography  Mobile phase (MP): liquid media that continuously flows through the column and carries the analytes  Analyte: the chemical species being investigated (detected and quantitatively measured) by an analytical method  docsity.com  A way to characterize chromatographic retention is to measure the time between injection and the maximum of the detector response for the analyte. This parameter, which is usually called the retention time tR, is inversely proportional to the eluent flow rate.  Retention time is dictated by physics and chemistry: – Chemistry (factors that influence distribution)  stationary phase: type and properties  mobile phase: composition and properties  intermolecular forces  temperature – Physics (flow, hydrodynamics)  mobile phase velocity  column dimensions Retention Time docsity.com Retention Volume  The product of the retention time and the eluent flow rate (F) is called the retention volume VR and represents the volume of the eluent passed through the column while eluting a particular analyte  Component retention volume VR can be divided into two parts: – Reduced retention volume, which is the volume of the eluent that passed through the column while the component was retained. – Dead volume, which is the volume of the eluent that passed through the column while the component was moving with the liquid phase. FtV RR  docsity.com Chromatograms and Electropherograms  Dead time (volume): the “mobile phase holdup time”, or the time it takes for an unretained analyte to reach the detector  A chromatogram or electropherogram shows detector response to analyte presence/concentration tM (tR)A wB tM = dead time (a.k.a. t0) tR = retention time wB = peak width at base docsity.com Retention and Differential Migration in Chromatography  Note: the arrows represent “approximate” equilibration Distribution constant (partition ratio, partition coefficient), where c is concentration: KB KA A M A SA ccK / B M B SB ccK / docsity.com Mobile Phase Velocity and Flow Rate  The average linear velocity of analyte migration (in cm/s) through a column is obtained by dividing the length of the packed column (L) by the analyte’s retention time: Rt L   The average linear velocity of the mobile phase is just: Mt Lu   Flow rate (mL/min) (F) is commonly used as an experimental parameter, it is related to the cross sectional area of the column and its porosity:  0 2 urF  L = length of column tR = retention time of analyte tM = retention time of mobile phase (“dead time”) u0 = linear velocity at column outlet  = fraction of column volume accessible to liquid docsity.com Relationship Between Retention Time and Distribution Constant solute ofmolestotal phase mobilein solute of moles  u MMSSSSMM MM VcVc u VcVc Vcu /1 1     MS VKV u /1 1   M S V KVk   We need to convert distribution constant (K) for an analyte into something measurable. Here’s how: average linear velocity of analyte migration average linear velocity of MP  Define k: Substitute in definition of K k u   1 1 Then substitute in definitions of u and  kt L t L MR   1 1 docsity.com Band Broadening (Column Efficiency)  After injection, a narrow chromatographic band is broadened during its movement through the column.  The higher the column band broadening, the smaller the number of components that can be separated in a given time.  The sharpness of the peak is an indication of the efficiency of the column. docsity.com Separation Efficiency and Peak Width  The peak width is an indication of peak sharpness and, in general, an indication of the column efficiency. However, the peak width is dependent on a number of parameters: – column length – flow rate – particle size  In absence of the specific interactions or sample overloading, the chromatographic peak can be represented by a Gaussian curve with the standard deviation . The ratio of standard deviation to the peak retention time  /tR is called the relative standard deviation, which is independent of the flow rate. docsity.com Theoretical Plates  A “plate”: an equilibration step (or zone) between the analytes, mobile phase, and stationary phase (comes from distillation theory)  Number of theoretical plates (N): the number of plates achieved in a separation (increases with longer columns)  Plate “height” (H): a measure of the separation efficiency of e.g. the column – Smaller H is better – Also known as HETP (height equivalent to a theoretical plate) – Measures how efficiently the column is packed  Plate equation: N LH  docsity.com Band Broadening Theory  Column band broadening originates from three main sources: – multiple paths of an analyte through the column packing (A) – molecular diffusion (B) – effect of mass transfer between phases (C)  In 1956, J.J. Van Deemter introduced the first equation which combined all three sources and represented them as the dependence of the theoretical plate height (H) and the mobile phase linear velocity (u) docsity.com Relationship Between Plate Height and Separation Variables Remember: Mt Lu  tM = retention time of mobile phase (“dead time”)  The Van Deemter equation is made up of several terms: Cu u BAH  docsity.com Van Deemter “A” Term  The “A” Term: Eddy diffusion – molecules may travel unequal distances in a packed column bed – particles (if present) cause eddies and turbulence – “A” depends on size of stationary particles (small is best) and their packing “quality” (uniform is best) docsity.com Van Deemter “B” Term u D u BH m2  In this equation, Dm is the analyte diffusion coefficient in the mobile phase,  is a factor that is related to the diffusion restriction by the column packing (hindrance factor), and u is the flow velocity. – The higher the eluent velocity, the lower the diffusion effect on the band broadening – Molecular diffusion in the liquid phase is about five orders of magnitude lower than that in the gas phase, thus this effect is limited for LC, but important for GC  The longitudinal diffusion (along the column long axis) leads to band broadening of the chromatographic zone. This process may be described by the equation: docsity.com mobile phase Stationary phase (SP) analyte attracted onto SP movement onto SP movement off SP Van Deemter “C” Term  Resistance to Mass Transfer: – The analyte takes a certain amount of time to equilibrate between the stationary phase and the mobile phase – If the velocity of the mobile phase is high, and an analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase – The band of analyte is broadened – The higher the velocity of the mobile phase, the worse the broadening becomes docsity.com where dp is the particle diameter, df is the thickness of the film, DM and DS are the diffusion coefficients of the analyte in the mobile/stationary phases, and u is the flow velocity Van Deemter “C” Term u D dkf u D dkf uCuCH M p S f MS 22 )(')(   The C term is given by two parts (for MP and SP):  The slower the velocity, the more uniformly analyte molecules may penetrate inside the particle, and the less the effect of different penetration on the efficiency.  On the other hand, at the faster flow rates the elution distance between molecules with different penetration depths will be high. docsity.com Resolution   BA ARBR s WW ttR    )()(2               k kNRs 11 4   (Eq. 26-24 in Skoog et al. 6th edition)  The selectivity factor, , describes the separation of band centers but does not take into account peak widths. Another measure of how well species have been separated is provided by measurement of the resolution.  The resolution of two species, A and B, is defined as  Baseline resolution is achieved when Rs = 1.5  The resolution is related to the number of column plates (N), the selectivity factor () and the average retention factor (k) of A and B: docsity.com Improving Resolution  For good resolution in separations, the three terms can be optimized Poor Rs ~ 0.8 Increase k Rs > 1.5 Increase N Rs > 1.5 Change  Rs > 1.5               k kNRs 11 4    Increasing k (retention factor) – Change temperature (GC) – Change MP composition (LC)  Increasing N (number of plates) – Lengthen column (GC) – Decrease SP particle size (LC)  Increasing  (selectivity factor) – Changing mobile phase – Changing column temperature – Changing stationary phase docsity.com