Lecture 6 Overview of Basic Separation Techniques Chapter 21 pp. 447 457 Principles of Chromatography Chapter 22 pp. 479 489 HPLC (Problems 22:8,9,11,12) (Note order of chapters) Anal Chem 251 Lect 6 Chromatography 1 Tools of the Laboratory Basic Separation Techniques Filtration : Separates components of a mixture based upon differences in particle size. Normally separating a precipitate from a solution, or particles from an air stream. (You filtered your seawater) Crystallization : Separation is based upon differences in solubility of components in a mixture. (Organic Lab) Distillation : separation is based upon differences in volatility. Extraction : Separation is based upon differences in solubility in different solvents (major material). Chromatography : Separation is based upon differences in affinity in a solvent (mobile) versus a stationary phase. Anal Chem 251 Lect 6 Chromatography 2 Tools of the Laboratory Basic Separation Techniques Figure B2.3 Filtration Figure B2.4 Crystallization Anal Chem 251 Lect 6 Chromatography 3 Tools of the Laboratory Figure B2.5 Anal Chem 251 Lect 6 Chromatography 4 Tools of the Laboratory Figure B2.6 Anal Chem 251 Lect 6 Chromatography 5 History of Chromatography During the beginning of the 20th century, a Russian botanist named Mikail S. Tsweet developed the chromatography process. Tsweet originally used the process to separate plant pigments. &quot;Chroma&quot; means color and &quot;graphein&quot; means to write. Anal Chem 251 Lect 6 Chromatography 6 p.923 What is Chromatography Chromatography is the general name applied to a series of methods that employ a system with two phases of matter: a stationary phase and a mobile phase. The stationary phase is a solid or a liquid adsorbed on a solid, and the mobile phase is either a liquid or a gas. The separation process occurs because the components of the mixture have different affinities for the two phases and thus move through the system at different rates. A component with a high affinity for the mobile phase moves relatively quickly through the chromatographic system, whereas one with a high affinity for the solid phase moves more slowly. Anal Chem 251 Lect 6 Chromatography 7 Classification of Chromatographic Methods Planar Chromatography: The stationary phase is supported on a flat plate or in the pores of a paper. Here the mobile phase moves through the stationary phase by capillary action or under the influence of gravity. Column Chromatography: The stationary phase is held in a narrow tube, and the mobile phase is forced through the tube under pressure or by gravity. (Lab) Lect 6 Chromatography 8 Anal Chem 251 Paper Chromatography (Planar Chromatography) One simple type of chromatography, paper chromatography, employs a strip of porous paper, such as filter paper, for the stationary phase. A drop of the mixture to be separated is placed on the paper, which is then dipped into a liquid (the mobile phase) that travels up the paper as though it were a wick. This method of separating a mixture is often used by biochemists, who study the chemistry of living systems. Anal Chem 251 Lect 6 Chromatography 9 Column Chromatography Observe the Figure depicting the separation of a mixture of pigments in ink. Anal Chem 251 Figure B2.7 Lect 6 Chromatography 10 Column Chromatography (Continued) One can collect the fractions separately and analyze them separately. Anal Chem 251 Lect 6 Chromatography 11 Fig 30-6b, p.922 Chromatography Terminology Elution is a process in which solutes are washed through a stationary phase by the movement of a mobile phase. The mobile phase that exits the column is termed the eluate. An eluent is a solvent used to carry the components of a mixture through a stationary phase. Chromatogram: If a detector that responds to solute concentration is placed at the end of the column during elution and its signal is plotted as a function of time (or of volume of added mobile phase), a series of peaks is obtained. Such a plot, is called a chromatogram, and is used for both qualitative and quantitative analysis. Anal Chem 251 Lect 6 Chromatography 12 Gas-Liquid Chromatorgraphy (GLC) Many types of chromatography are used to separate a wide variety of substances, from simple gases to biological macromolecules. In gas-<a href="/keyword/liquid-chromatography/" >liquid chromatography</a> (GLC), the stationary phase is made of small particles packed in a long tube. The mobile phase is an inert gas, such as helium, that carries the previously vaporized components into the stationary phase. The components emerge separately and reach a detector to create a chromatogram. A typical chromatogram has numerous peaks of specific position and height, each of which represents the amount of a given component. Anal Chem 251 Lect 6 Chromatography 13 Tools of the Laboratory Figure B2.8 Separation by Gas - <a href="/keyword/liquid-chromatography/" >liquid chromatography</a> Anal Chem 251 Lect 6 Chromatography 14 High Performance (High Pressure <a href="/keyword/liquid-chromatography/" >liquid chromatography</a> ) (HPLC) The principles of HPLC are very similar to gas-<a href="/keyword/liquid-chromatography/" >liquid chromatography</a> . However, in this technique the mixture is not vaporized, so a more diverse group of components, which may include nonvolatile components, can be separated. Anal Chem 251 Lect 6 Chromatography 15 Classifications of Column Chromatographic Methods Anal Chem 251 Lect 6 Chromatography 16 Table 30-4, p.921 Migration Rates of Solutes All chromatographic separations are based on differences in the extent to which solutes are distributed between the mobile and stationary phases. For the solute species A, the equilibrium involved is described by the equation: A (mobile) A (stationary) Kc = CS CM The distribution constant for a solute in chromatography is equal to the ratio of its molar concentration in the stationary phase to its molar concentration in the mobile phase Anal Chem 251 Lect 6 Chromatography 17 Retention Times The dead time (Void time) tM, is the time it takes for an un-retained species to pass through a chromatographic column. All components spend this amount of time in the mobile phase. Separations are based on the different times ts that components spend in the stationary phase 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 tR = ts + tM Anal Chem 251 Lect 6 Chromatography 18 Retention Times Continued A Typical Chromatogram for a two-component mixture. The solute on the left represents a solute that is not retained on the column and so reaches the detector almost immediately after elution has begun. Thus, its retention time TM is approximately equal to the time required for a molecule of the mobile phase to pass through the column. 19 Fig 30-9, p.924 Chromatography Theory -Theoretical Plates In 1952 Nobel Prize was awarded to two Englishmen, A.J.P. Martin and R. L.M. Synge for their work in the development of modern chromatography. In their theoretical studies, they adapted a model that was first developed in the early 1920's to describe separations on fractional distillation columns. Fractionating columns which were first used in the petroleum industry to separate closely related hydrocarbons, consisted of numerous interconneced bubble-cap plates at which vapor-liquid equilibria were established when the column was operated under reflux conditions. Martin and Synge treated a chromatographic column as if it were made of contiguous bubble-cap-like plates within which equilibrium conditions always prevail. Plate and plate height are designators of column efficiency and are retained for historic reasons. Lect 6 Chromatography 20 Anal Chem 251 Figure 13.29 Gasoline vapors Condenser Gas Gasoline 380C Kerosene 1500C Chromatography Theory -Theoretical Plates The process of fractional distillation. Heating oil 2600C Lubricating oil 3150C-3700C In industry, this process is used to separate petroleum into many products. Anal Chem 251 Crude oil vapors from heater Steam Lect 6 Chromatography Residue (asphalt, tar) 21 Chromatography Theory -Theoretical Plates Plates in a Fractionating Column Same concept used for HPLC Anal Chem 251 Lect 6 Chromatography 22 Fig 30-F2, p.930 Theoretical Plates An ideal chromatographic peak has a Gaussian shape, like that in the Figure. If the height of the peak is h, the width at half-height, w , is measured at half height, h. For a Gaussian peak, w , is equal to 2.35, where is the standard deviation of the peak. The w = width of the peak at the baseline is 4. For any peak in Figure, the theoretical plates is computed by measuring the retention time and the width at half-height: Number of plates on column: N= 5.55 tr2 w21/2 Retention and width can be measured in units of time or volume (such as mL of eluate) Lect 6 Chromatography 23 Anal Chem 251 Figure 21-3 Number of Theoretical Plates N = NUMBER OF THEORETICAL PLATES = 5.55 tr2 w21/2 Schematic Gas Chromatogram, showing measurement of retention time and width at half height. The width of the base is found by drawing tangents to the steepest parts of the Gaussian curve and extrapolating down to the baseline. The standard deviation of the curve is . Anal Chem 251 Lect 6 Chromatography 24 Plate Height If a column is divided into N theoretical plates (only in our minds), then the plate height, H is the length of one plate. H is calculated by dividing the length of the column L by the number of theoretical plates on the column: Plate height H = L/N The smaller the plate height, the narrower the peaks The ability of a column to separate components of a mixture is improved by decreasing plate height. Different solutes behave as if the column has somewhat different plate heights, because different compounds equilibrate between the mobile and stationary phases at different rates. In HPLC, plate heights are about 10 m. Lect 6 Chromatography 25 Anal Chem 251 Example Calculation of N (Number of Theoretical Plates) and H (Plate Height) A solute with a retention time of 407 s has a width at half-height of 8.1 s on a column 12.2 m long. Find th number of plates and the plate height. Solution: number of plates N = 5.55 x 4072 = 1.40 x 104 8.12 5.55 tr2 w21/2 = Plate height H = L/N = 12.2 m/1.40 x 104 plates Convert to mm , H = 0.87 mm Anal Chem 251 Lect 6 Chromatography 26 The resolution between neighboring peaks is equal to the peak separation (tr) divided by the average peak width (wav) measured at the base as in Figure Resolution = tr wav The better the resolution, the more complete is the separation between neighboring peaks. w , is equal to 2.35 Therefore = w / 2.35 w = 4 (width of the peak at the baseline) Resolution Substitute for in the above equation for w Resolution = 0.589 tr / w1/2av Resolution is proportional to column length Anal Chem 251 Lect 6 Chromatography 27 Figure 21-4 Resolution of Gaussian peaks of equal area and amplitude. Dashed lines show individual peaks; solid lines are the sum of two peaks. Overlapping area is shaded. Resolution Overlap is 16% Overlap is 2.3% Anal Chem 251 Lect 6 Chromatography 28 Why Do Bands Spread? If a solute is applied to a column in an ideal manner as an infinitesimally thin band, the band will broaden as it moves through a chromatography column Figure 21-6 Broadening of an initially sharp band of solute as it moves through a chromatography column Anal Chem 251 Lect 6 Chromatography 29 Why Do Bands Spread?Continued Broadening occurs because of: 1)Diffusion (The faster the flow rate, the sharper the peaks) 2)Slow equilibration of solute between the mobile and stationary phases (The faster the flow rate, the broader the peaks) 3)Irregular flow paths on the column (Independent of flow rate) Anal Chem 251 Lect 6 Chromatography 30 Van Deemter Equation Figure - GC of a gas at a particular temperature using three different gases Optimum resolution (that is minimum plate height) occurs at an intermediate flow rate Optimum resolution, that means minimum plate height occurs at an intermediate flow rate. Anal Chem 251 Lect 6 Chromatography 31 Van Deemter equation describes the broadening of a band of solute as it passes through a chromatography column. If the band has some finite width when it is applied to the column, the eluted band emerging from the other end will be broader than we predict. where: H = plate height u = flow rate Bands can broaden outside the column if there is too much A = constant due to multiple flow paths tubing to flow through. Best B= constant due to longitudinal diffusion of solute results are obtained in chromatography if the lengths C = constant due to equilibration time of solute and diameters of all tubing between phases outside the column are kept to a Anal Chem Lect 6 Chromatography 32 minimum. 251 High Performance <a href="/keyword/liquid-chromatography/" >liquid chromatography</a> HPLC has become an indispensable analytical tool. The crime labs in forensic and police dramas such as CSI, Law and Order often use HPLC in the processing of evidence. The following photo shows actress Marg Helgenberger (Catherine Willows) of CSI in the lab preparing samples for HPLC. Anal Chem 251 Lect 6 Chromatography 33 HPLC The following photo shows actress Marg Helgenberger (Catherine Willows) of CSI in the lab preparing samples for HPLC Anal Chem 251 Lect 6 Chromatography 34 Fig 32-CO, p.973 HPLC The eluent is pumped through the column Pumping pressures of several hundred atmospheres are required to achieve reasonable flow rates with packings in the 3- to 10- m size range, which are common in modern <a href="/keyword/liquid-chromatography/" >liquid chromatography</a> A modern HPLC is equipped with one or more glass reservoirs, each of which contains 500 mL or more of a solvent Provisions are often included to remove dissolved gases and dust from the liquids. Anal Chem 251 Lect 6 Chromatography 35 HPLC Continued Sparging is a process in which dissolved gases are swept out of a solvent by bubbles of an inert, insoluble gas. An isocratic elution in HPLC is one in which the solvent composition remains constant. A gradient elution in HPLC is one in which the composition of the solvent is changed continuously or in a series of steps. Anal Chem 251 Lect 6 Chromatography 36 HPLC Continued Anal Chem 251 Lect 6 Chromatography Block Diagram Showing the Components of a typical HPLC 37 Fig 32-3, p.975 HPLC DETECTOR A UV-VIS Detector for HPLC Anal Chem 251 Lect 6 Chromatography 38 Fig 32-8, p.979 HPLC Detectors Continued HPLC detectors will depend on the nature of the sample. The most widely used detectors for <a href="/keyword/liquid-chromatography/" >liquid chromatography</a> are based on the absorption of ultraviolet or visible radiation. The detectors often make use of the 254- and 280-nm lines from a mercury source because many organic functional groups absorb in the region. Anal Chem 251 Lect 6 Chromatography 39 HPLC The most widely used type of HPLC is partition chromatography, in which the stationary phase is a second liquid that is immiscible with the liquid mobile phase. Partition chromatography can be subdivided into liquid-liquid and liquid-bonded phase chromatography. In liquid-liquid partition chromatography, the stationary phase is a solvent that is held in place by adsorption on the surface of packing particles. The liquid coat might itself be soluble in the mobile phase which will cause the column to &quot;bleed&quot;. To prevent loss of stationary phase, in liquid-bonded-phase partition chromatography, the stationary phase is an organic species that is attached to the surface of the packing particles by chemical bonds. Lect 6 Chromatography 40 Anal Chem 251 HPLC Bonded Phase Packings Most bonded phase packings are prepared by reaction of an organochlorosilane with the OH groups formed on the surface of silica particles by hydrolysis in hot, dilute hydrochloric acid. The product is an organosiloxane. The reaction for one such SiOH site on the surface of a particle can be written as: Where R is often: octadecyl (CH2)17CH3 octyl phenyl _ (CH2)7CH3 _ (CH2)3C6H5 C18 C8 amino cyano diol _ (CH2)3NH2 _ (CH2)3C=N _ (CH2)OCH2CHCH2 OH OH Anal Chem 251 Lect 6 Chromatography 41 Normal and Reversed-Phase Packings Two types of partition chromatography are distinguishable based on the relative polarities of the mobile and stationary phases. In normal phase chromatography, the stationary phase is polar (H2O) and the mobile phase non-polar (hexane). Eluent strength is increased by adding a more polar solvent. In reversed-phase, the polarity of these phases is reversed. The stationary phase is non-polar (hydrocarbon) and the mobile phase is polar (H2O, methanol, acetonitrile). Eluent strength is increased by adding a less polar solvent. Anal Chem 251 Lect 6 Chromatography 42 Problem 22-8 Non-Polar aromatic compounds were separated by HPLC on a bonded phase containing octadecyl groups _ (CH2)17CH3 covalently attached to silica particles. The eluent was 65% by volume methanol in water. How would the retention times be affected if 90% methanol were used instead? Solution: Because non-polar compounds are more soluble in methanol than in water, the retention time will be shorter in 90% methanol. Anal Chem 251 Lect 6 Chromatography 43 Problem 22:12 A 15 cm-long HPLC column packed with 5- m particles has an optimum plate height of 10.0 m in Figure 22-16. a) What will be the half-width of a peak eluting at 10.0 min? B)If the particle size were 3 m and the plate height were 5.0 m, what would be the half-width of a peak eluting at 10.0 min.? Solution: a) Step 1: Calculate the number of plates N = plates = 15 cm/ (10.0 m x 10-4 cm/plate) = 1.5 x 104 Step 2: Calculate the w1/2 at tr = 10.0 min from the N equation, N= 5.55 tr2 w21/2 w1/2 = tr 5.55/N w1/2 = (10.0 min) 5.55 / 1.5 x 104 = 0.192 min Anal Chem 251 Lect 6 Chromatography 44 Problem 22:12 Continued b) Step 1: Calculate the number of plates N = plates = 15 cm/ (5.0 m x 10-4 cm/plate) = 3.0 x 104 Step 2: Calculate the w1/2 at tr = 10.0 min from the N equation, N= 5.55 tr2 w21/2 w1/2 = tr 5.55/N w1/2 = (10.0 min) 5.55 / 3.0 x 104 = 0.136 min Anal Chem 251 Lect 6 Chromatography 45 Power of Separation Science! Petroleum Refinery Anal Chem 251 Lect 6 Chromatography 46 Fig 30-CO, p.906 Lecture 6 Perspective Applications of chromatography are broad. It is a powerful and versatile tool for separating closely related chemical species. It can be employed for the qualitative identification and quantitative determination of separated species Purity of organic compound Overdose on a prescription drug Job offer contingent per urine analysis Different sugars in food products Sunscreens in personal care products Quality Control technique ...............endless......................... Various ions in Seawater as per your lab experiments Anal Chem 251 Lect 6 Chromatography 47