Introduction to Analytical Separations 1.) Sample Purity Many chemical analysis are not specific for one compound Actually respond to many potential interferences in the sample Often it is necessary to first purify the compound of interest Remove interfering substances before a selective analysis is possible This requires a separation step. 2.) Techniques available for Chemical Separations: Extraction Distillation Precipitation Chromatography Many others (centrifugation, filtration, etc) Extractions and Chromatography are especially useful in analytical methods

Introduction to Analytical Separations 3.) Illustration Biological Samples are Composed of Complex Mixtures Analysis of composition and changes help in understanding disease and the development of treatments NMR Spectra of Mouse Urine after treatment with a Drug Analysis of Various Pesticides in Ground water using LC-MS 2D Gel Electrophoresis of total protein extract from E. coli cells Journal of Chromatography A, 1109 (2006) 222–227 Toxicological Sciences (2000) 57:326-337 Electrophoresis (1997) 18:1259-1313

Introduction to Analytical Separations Extractions 1.) Definition The transfer of a compound from one chemical phase to another The two phases used can be liquid-liquid, liquid-solid, gas-solid, etc Liquid-liquid is the most common type of extraction The partitioning of solute s between two chemical phases (1 and 2) is described by the equilibrium constant K Immiscible liquids K is called the partition coefficient

Introduction to Analytical Separations Extractions 2.) Extraction Efficiency The fraction of moles of S remaining in phase 1 after one extraction can be determined The value of K and the volumes of phases 1 and 2 need to be known The fraction of S remaining in phase 1 after n extractions is where: q = fraction of moles of S remaining in phase 1 V1 = volume of phase 1 V2 = volume of phase 2 K = partition coefficient Assumes V2 is constant

Is distributed in both layers Introduction to Analytical Separations Extractions 2.) Extraction Efficiency Illustration Ether layer Water layer 1M UO2(NO3)2 (yellow) After mixing, UO2(NO3)2 Is distributed in both layers After 8 extractions, UO2(NO3)2 has been removed from water

Introduction to Analytical Separations Extractions 3.) What happens as n approaches infinity? Eventually the amount of S remaining in phase 1 becomes zero Solution is infinitely diluted This Situation Created a Strange Saga in Science – Water Memory a founding principal of homeopathic medicine the claim is that water remembers the activity of the drug after it has been removed Nature (1988) 333:816-818 Authors’ claim to still observe antibody activity even after a 1x10120 fold dilution. Less than 1 molecule is present with a 1x1014 dilution A number of subsequent studies have disputed the claim but the controversy is still popular in the press and as alternative medicine, even though the results are consistent with the placebo effect.

Introduction to Analytical Separations Extractions 4.) Example #1: Solute A has a K = 3 for an extraction between water (phase 1) and benzene (phase 2). If 100 mL of a 0.01M solution of A in water is extracted one time with 500 mL benzene, what fraction will be extracted? Solution: First determine fraction not extracted (fraction still in phase 1, q): The fraction of S extracted (p) is simply:

Introduction to Analytical Separations Extractions 4.) Example #2: For the same example, what fraction will be extracted if 5 extractions with 100 mL benzene each are used (instead of one 500 mL extraction)? Solution: Determine fraction not extracted (fraction still in phase 1, q): The fraction of S extracted (p) is: Note: For the same total volume of benzene (500 mL), more A is extracted if several small portions of benzene are used rather than one large portion

Introduction to Analytical Separations Extractions 5.) pH Effects in Extractions For weak acids (HA) and Bases (B) Protonated and non-protonated forms usually have different partition coefficients (K) Charged form (A- or BH+) will not be extracted Neutral form (HA or B) will be extracted Partitioning is Described in Terms of the Total Amount of a Substance Individual concentrations of B & BH+ or HA & A- are more difficult to determine Partitioning is regardless of the form in both phases Described by the distribution coefficient (D)

Introduction to Analytical Separations Extractions 5.) pH Effects in Extractions The distribution of a weak base or weak acid is pH dependent For a weak base (B) where BH+ only exists in phase 1:

Introduction to Analytical Separations Extractions 5.) pH Effects in Extractions The distribution of a weak base or weak acid is pH dependent Substitute definition of KB and Ka into D: (partition coefficient) (equilibrium constant) D is directly related to [H+]

Introduction to Analytical Separations Extractions 5.) pH Effects in Extractions A similar expression can be written for a weak acid (HA) The ability to change the distribution ratio of a weak acid or weak base with pH is useful in selecting conditions that will extract some compounds but not others. Use low pH to extract HA but not BH+ (weak acid extractions) Use high pH to extract B but not A- (weak base extractions) where:

Introduction to Analytical Separations Extractions 6.) Example Butanoic acid has a partition coefficient of 3.0 (favoring benzene) when distributed between water and benzene. Find the formal concentration of butanoic acid in each phase when 100 mL of 0.10 M aqueous butanoic acid is extracted with 25 mL of benzene at pH 4.00 and pH 10.00

Introduction to Analytical Separations Chromatography 1.) Definition A separation technique based on the different rates of travel of solutes through a system composed of two phases A stationary phase A mobile phase Detect compounds emerging in column by changes in absorbance, voltage, current, etc Chromatogram (not spectrum)

Introduction to Analytical Separations Chromatography 2.) System Components and Process Stationary Phase: the chemical phase which remains in the column (chromatographic system) Mobile Phase (eluent): the chemical phase which travels through the column Support: a solid onto which the stationary phase is chemically attached or coated Solute are separated in chromatography by their different interactions with the stationary phase and mobile phase

Introduction to Analytical Separations Chromatography 2.) System Components and Process Solutes which interact more strongly with the stationary phase take longer to pass through the column Strongly Retained Weakly Retained Solutes which only weakly interact with the stationary phase or have no interactions with it elute very quickly

Introduction to Analytical Separations Chromatography 3.) Chromatogram Chromatogram: graph showing the detector response as a function of elution time. Retention time (tr): the time it takes a compound to pass through a column Retention volume (Vr): volume of mobile phase needed to push solute through the column Retention time Non-retained solute (void volume) The strength or degree with which a molecule is retained on the column can be measured using retention time or retention volume.

Introduction to Analytical Separations Chromatography 4.) Fundamental Measures of Solute Retention Adjusted retention time (tr’): the additional time required for a solute to travel through a column beyond the time required for non-retained solute Relative Retention (a): ratio of adjusted retention time between two solutes Greater the relative retention the greater the separation between two components where: tm = minimum possible time for a non-retained solute to pass through the column where: tr2’ > tr1’ , so a > 1

Introduction to Analytical Separations Chromatography 4.) Fundamental Measures of Solute Retention Capacity factor (k’): The longer a component is retained by the column, the greater the capacity factor Capacity factor of a standard can be used to monitor performance of a column Capacity factor is equivalent to: where: Vs = volume of the stationary phase Vm = volume of the mobile phase K = partition coefficient Capacity factor is directly proportional to partition coefficient

Introduction to Analytical Separations Chromatography 4.) Fundamental Measures of Solute Retention Example: The retention volume of a solute is 76.2 mL for a column with Vm = 16.6 mL and Vs = 12.7 mL. Calculate the capacity factor and the partition coefficient for this solute.

Introduction to Analytical Separations Chromatography 5.) Efficiency of Separation The width of a solute peak is important in determining how well one solute is separated from another One measure of this is the width of the peak at half-height (w½ ) or at its baseline (wb)

Introduction to Analytical Separations Chromatography 5.) Efficiency of Separation The separation of two solutes in chromatography depends both on the width of the peaks and their degree of retention The separation between the two solutes is given by their Resolution (Rs)

Introduction to Analytical Separations Chromatography 5.) Efficiency of Separation Resolution (Rs) is defined as: Or Want Rs ≥ 1.5 for complete separation Rs ≥ 1.0 usually adequate for analysis where: tr2,tr1 = retention times of solutes 1 and 2 (tr2 > tr1) wb2,wb1 = baseline widths of solutes 1 and 2 where: N = number of theoretical plates g = t2/t1 (g>1)

Introduction to Analytical Separations Chromatography 6.) Measure of Column Efficiency Number of Theoretical Plates (N) Similar to number of extractions performed in an extraction separation As N increase (number of separating steps)  greater the separation between two compounds where: wb = baseline width of peak (in time units) w1/2=half-height peak width

Introduction to Analytical Separations Chromatography 6.) Measure of Column Efficiency Height Equivalent of a Theoretical Plate (H or HETP) The distance along the column that corresponds to one “theoretical” separation step or plate (N) As H decreases, more separation steps per column length are possible Results in a narrower peak width and better separation between two neighboring solutes where: L = length of column N = number of theoretical plates H

Introduction to Analytical Separations Chromatography 6.) Measure of Column Efficiency H is affected by: Flow-rate of mobile phase Size of support: decrease size decrease H Diffusion of solute: increase diffusion  decrease H Strength of retention Others Improved resolution by increasing column length

Introduction to Analytical Separations Chromatography 6.) Measure of Column Efficiency Example: Two compounds with partition coefficients of 15 and 18 are to be separated on a column with Vm/Vs = 3.0 and tm = 1.0 min. Calculate the number of theoretical plates needed to produce a resolution of 1.5

Introduction to Analytical Separations Chromatography 7.) Why Bands Spread? Remember: Efficiency is dependent on peak width A band of solute spreads as it travels through the column described by a standard deviation (s) Factors include: Sample injection Longitudinal diffusion Finite equilibration between phases Multiple flow paths others

Introduction to Analytical Separations Chromatography 7.) Why Bands Spread? Sample injection – sample is injected on the column width a finite width, which contributes to the overall broadening Similar broadening may occur in the detector Longitudinal diffusion – band slowly broadens as molecules diffuse from high concentration in band to regions of lower concentration

Introduction to Analytical Separations Chromatography 7.) Why Bands Spread? Finite Equilibration Time Between Phases – a finite time is required to equilibrate between stationary and mobile phase at each plate Some solute is “stuck” in stationary phase as remainder moves forward in mobile phase Results in band broadening Distribution of solute between mobile and stationary phase Solute in mobile phase moves down column  broader peaks

Introduction to Analytical Separations Chromatography 7.) Why Bands Spread? Multiple Flow Paths – As solute molecules travel through the column, some arrive at the end sooner then others simply due to the different path traveled around the support particles in the column that result in different travel distances. Molecules exit the column at different times due to different path lengths Molecules enter the column at the same time

Introduction to Analytical Separations Chromatography 8.) Description of Band Spread Plate height (H) is proportional to band width The smaller the plate height, the narrower the band Van Deemter equation Multiple paths equilibration time Longitudinal diffusion where: mx = linear flow rate A,B,C = constants for a given column and stationary phase

Introduction to Analytical Separations Chromatography 9.) Types of Liquid Chromatography Adsorption Chromatography Solutes are separated based on their different abilities to adsorb to the support’s surface Uses an underivatized solid support (stationary phase = solid support) Oldest type of chromatography, but not commonly used

Introduction to Analytical Separations Chromatography 9.) Types of Liquid Chromatography Partition Chromatography Solutes are separated based on their different abilities to partition between the stationary phase and mobile phase. Uses a solid support coated or chemically derivatized with a polar or non-polar layer Most common type of liquid chromatography at present. Good for most organic compounds Reversed Phase: stationary phase is non-polar Normal Phase: stationary phase is polar

Introduction to Analytical Separations Chromatography 9.) Types of Liquid Chromatography Ion-Exchange Chromatography Used to separate ions based on their different abilities to interact with the fixed exchange sites. Uses a solid support containing fixed charges (exchange sites) on its surface Cation-Exchange: support with negative groups Anion-Exchange: support with positive groups

Introduction to Analytical Separations Chromatography 9.) Types of Liquid Chromatography Size Exclusion Chromatography Separates large and small solute based on their different abilities to enter the pores of the support Uses a porous support that does not adsorb solutes Commonly used to separate biological molecules or polymers which differ by size (MW)

Introduction to Analytical Separations Chromatography 9.) Types of Liquid Chromatography Affinity Chromatography Separates molecules based on their different abilities to bind to the affinity ligand Uses a support that contains an immobilized biological molecule (affinity ligand) Commonly used to purify and analyze biological molecules Most Selective type of Chromatography