Chromatography Principles and Application PDF

PRINCIPLES AND APPLICATION OF CHROMATOGRAP HY History The Russian botanist Mikhail Tswett coined the term chromatography in 1906 to describe his experiments in separating different colored constituents of leaves by passing an extract of the leaves through a column Problem: What would be a good method for determining the following: identity of accelerant at a suspected arson scene amount of caffeine in Coca Cola identifying active ingredient in an illicit drug preparation (LSD is heat sensitive) puriﬁcation and characterization of novel thermophilic plant enzyme from South America identifying explosive materials used in Oklahoma bombing Chromatography A wide variety of separation techniques based on the partitioning or distribution of a sample (solute) between a moving or mobile phase and a ﬁxed or stationary phase. Series of equilibrations between the mobile and stationary phase. Nielsen SS, Food Analysis, Food Science Texts Series Chromatography is a separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium. Chromatography Chromatogram - Detector signal vs. retention time or volume Detector Signal 1 2 time or volume General Principles of Chromatography E.g 1: Paper chromatography Mobile phase? Stationary phase? Supporting medium? General Principles of Chromatography Components of Chromatography?? Mobile phase: a solvent that f lo ws through the supporting medium S tati onary p hase : a l aye r or c oati ng on the supporting medium that interacts with the analytes Supporting medium: a solid surface on which the stationary phase is bound or coated A. Type of Chromatography H o w c a n w e c l a ssi f ie d t he c hro m a to g ra p hi c techniques? 1. The primary division of chromatographic techniques is based on the type of mobile phase used in the system: Type of Chromatography Ty p e o f M o b i l e Phase Gas chromatography (GC) gas Liquid chromatograph (LC) liquid Mobile Phase gas (GC) water (LC) organic solvent (LC) supercritical ﬂuid (SCFC) A. Type of Chromatography 2. Further divisions can be made based on the type of stationary phase used in the system: i. Gas Chromatography Name of GC Method Type of Stationary Phase Gas-solid chromatography solid, underivatized support Gas-liquid chromatography liquid-coated support Bonded-phase gas chromatography chemically-derivatized support Purpose of Chromatography Analytical - determine chemical composition of a sample Preparative - purify and collect one or more components of a sample Chromatography Columns Packed Column: Typical HPLC columns but some gas chromatography columns also (especially older columns). The columns are packed with tiny particles. Capillary Column: Typical gas chromatography column which consists of a small diameter tube coated on the inside with stationary phase. Characteristics of Different Chromatographic Methods A. Column Chromatography, B. Planar Chromatography Classiﬁcation based on Mobile Phase Gas Chromatography Pyrolysis GC - heat solid materials to 500 - 10000C so they decompose Gas - solid Gas - liquid into gaseous products Stationary Phase Sample MUST be volatile at temperatures BELOW 3500C Classiﬁcation based on Mobile Phase Liquid chromatography (LC) Column High performance Thin layer (gravity ﬂow) (pressure ﬂow) (adsorption) Classiﬁcation based on Attractive Forces Adsorption - for polar non-ionic compounds Ion Exchange - for ionic compounds Anion - analyte is anion; bonded phase has positive charge Cation – analyte is cation; bonded phase has negative charge Partition - based on the relative solubility of analyte in mobile and stationary phases Normal – analyte is nonpolar organic; stationary phase MORE polar than the mobile phase Reverse – analyte is polar organic; stationary phase LESS polar than the mobile phase Size Exclusion - stationary phase is a porous matrix; sieving Detectors UV-vis Refractive Index (RI) Mass spectrometry (MS) Electrochemical (EC) amperometric NMR - novel Thin-Layer Chromatography Sorbents and Mode of Separation Column Liquid Chromatography A system for low-pressure column liquid chromatography. In this diagram, the column efﬂuent is being split between two detectors in order to monitor both enzyme activity (at Right) and UV absorption (at Left). The two tracings can be recorded simultaneously by using a dual-pen recorder. The process of passing the mobile phase through the column is called elution, and the portion that emerges from the outlet end of the column is sometimes called the eluate (or efﬂuent). Elution may be isocratic (constant mobile-phase composition) or a gradient (changing the mobile phase, e.g., increasing solvent strength or pH) during elution in order to enhance resolution and decrease analysis time. As elution proceeds, components of the sample are selectively retarded by the stationary phase based on the strength of interaction with the stationary phase, and thus they are eluted at different times. A. Type of Chromatography ii. Liquid Chromatography 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 Aﬃnity chromatography support with immobilized ligand A. Type of Chromatography 3.) Chromatographic techniques may also be classif ied based on the type of support material used in the system: Column chromatography (Packed bed) Capillary chromatography (Open tubular) Planar chromatography (Open bed) B. Theory of Chromatography 1. Typical response obtained by chromatography (i.e., a chromatogram): chromatogram - concentration versus elution time Wh Wb Inject 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 B. Theory of Chromatography What factors can affects the separation of solutes? (From the chromatogram) i ii Note: The separation of solutes in chromatography depends on two factors: (a) a difference in the retention of solutes (i.e., a difference in their time or volume of elution (b) a suﬃciently narrow width of the solute peaks (i.e, good eﬃciency for the separation system) A similar plot can be made in terms of elution volume instead of elution time. If volumes are used, the volume of the mobile phase that it takes to elute a peak off of the column is referred to as the retention volume (VR) and the amount of mobile phase that it takes to elute a non- retained component is referred to as the void volume (VM). B. Theory of Chromatography 2. Solute Retention: A solute’s retention time or retention volume in chromatography is directly related to the strength of the solute’s interaction with the mobile and stationary phases. Retention on a given column pertain to the particulars of that system: - size of the column - ﬂow rate of the mobile phase Capacity factor (k’): more universal measure of retention, determined from tR or VR. 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. B. Theory of Chromatography The value of the capacity factor is useful in understanding the retention mechanisms for a solute, since the fundamental deﬁnition of k’ is: moles Astationary phase k’ = moles Amobile phase k’ is directly related to the strength of the interaction between a solute with the stationary and mobile phases. Moles A st at ionar y phase and moles A m obile phase represents the amount of solute present in each phase at equilibrium. Equilibrium is achieved or approached at the center of a chromatographic peak. When k' is = 1.0, separation is poor When k' is > 30, separation is slow When k' is = 2-10, separation is optimum B. Theory of Chromatography 3. Eﬃciency: peak width Eﬃciency is related experimentally to a solute’s peak width. - an eﬃcient system will produce narrow peaks - narrow peaks  smaller difference in interactions in order to separate two solutes Estimate  from peak widths, assuming Gaussian shaped peak: Wh Wb = 4 Wh = 2.354 This is Wb/2 Wb = peak width at baseline not a Gaussian Wh = peak width at half height shaped   standard deviation peak Dependent on the amount of time that a solute spends in the column (k’ or tR) B. Theory of Chromatography Eﬃciency: Theoretical plates (Plate Model) Number of theoretical plates (N): compare eﬃciencies of a system for solutes that have different retention times N = (tR/)2 Wb = 4 or Wh = 2.354 N = 16 (tR/Wb)2 or N = 5.54 (tR/Wh)2 The larger the value of N is for a column, the better the column will be able to separate two compounds. - the better the ability to resolve solutes that have small differences in retention - N is independent of solute retention - N is dependent on the length of the column B. Theory of Chromatography Plate height or height equivalent of a theoretical plate (H or HETP): compare eﬃciencies of columns with different lengths: H = L/N where: L = column length N = number of theoretical plates for the column Note: H simply gives the length of the column that corresponds to one theoretical plate H can be also used to relate various chromatographic parameters (e.g., ﬂow rate, particle size, etc.) to the kinetic processes that give rise to peak broadening: B. Theory of Chromatography The plate model – assumes that EFFICIENCY equilibrium is inﬁnitely fast The rate theory of chromatography – takes account of the time taken for the solute to equilibrate between the stationary and mobile phase. According to the rate theory of chromatography, bands spread due to several factors. What are the factors? a. Eddy diffusion b. Mobile phase mass transfer c. Stagnant mobile phase mass transfer d. Stationary phase mass transfer e. Longitudinal diffusion B. Theory of Chromatography a.) Eddy diffusion – a process that leads to peak (band) broadening due to the presence of multiple ﬂow paths through a packed column. 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. Longer path arrives at end of column after (1). C. The Rate Theory of Chromatography b.) Mobile phase mass transfer – 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 cha nnel f ir st lea ding to ba nd- broadening The degree of band-broadening due to eddy diffusion and mobile phase mass transfer depends mainly on: 1) the size of the packing material 2) the diffusion rate of the solute B. Theory of Chromatography c.) Stagnant mobile phase mass transfer – band-broadening due to differences in the rate of diffusion of the solute molecules between the mobile phase outside the pores of the support (ﬂowing mobile phase) to the mobile phase within the pores of the support (stagnant mobile phase). Since a solute does not travel down the column when it is in the stagnant mobile phase, it spends a longer time in the column than solute that remains in the ﬂowing mobile phase. The degree of band-broadening due to stagnant mobile phase mass transfer depends on: 1) the size, shape and pore structure of the packing material 2) the diffusion and retention of the solute 3) the ﬂow-rate of the solute through the column B. Theory of Chromatography d.) Stationary phase mass transfer – band-broadening due to the movement of solute between the stagnant 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 B. Theory of Chromatography e.) Longitudinal diffusion – band-broadening due to the diffusion of the solute along the length of the column in the ﬂowing mobile phase. 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 B. Theory of Chromatography Van Deemter equation: relates ﬂow-rate or linear velocity to H: H = A + B/ + C where:  = linear velocity (ﬂow-rate x Vm/L) H = total plate height of the column A = constant representing eddy diffusion & mobile phase mass transfer B = constant representing longitudinal diffusion C = constant representing stagnant mobile phase & stationary phase mass transfer Number of theoretical plates(N) (N) = 5.54 (tR/Wh)2 peak width (Wh) H = L/N B. Theory of Chromatography Plot of van Deemter equation shows how H changes with the linear velocity (ﬂow-rate) of the mobile phase  optimum Optimum linear velocity (opt) - where H has a minimum value and the point of maximum column eﬃciency: B. Theory of Chromatography 4. Measures of Solute Separation: i. Separation factor () – parameter used to describe how well two solutes are separated by a chromatographic system:  = k’2/k’1 k’ = (tR – tM)/tM where: The selectivity (or separation) factor (α) is k’1 = the capacity factor of the ﬁrst solute the ability of the k’2 = the capacity factor of the second solute, chromatographic system with k’2 = k’1 to ‘chemically’ distinguish between A value of  >1 is usually indicative of a good separation sample components. High α values indicate good separating power and a good separation between the APEX of each peak. Does not consider the effect of column eﬃciency or peak widths, only retention. Physicochemical principles of chromatography. PHYSICOCHEMICAL PRINCIPLES OF CHROMATOGRAPHIC SEPARATION Adsorption (Liquid–Solid) Chromatography Partition (Liquid–Liquid) Chromatography Ion-Exchange Chromatography Size-Exclusion Afﬁnity Chromatography Chromatography Van der Waals forces Electrostatic forces Hydrogen bonds Hydrophobic interactions separates aromatic or aliphatic nonpolar compounds, based primarily on the type and number of functional groups present. based upon differences in afﬁnity of the exchangers for the ions (or charged species) to be separated. Relationship between Kav and log (molecular weight) for globular proteins chromatographed on a column of Sephadex G-150 Superﬁne. Principles of bioselective afﬁnity chromatography. (a) The support presents the immobilized ligand to the analyte to be isolated. (b) The analyte makes contact with the ligand and attaches itself. (c) The analyte is recovered by the introduction of an eluent, which dissociates the complex holding the analyte to the ligand. (d) The support is regenerated, ready for the next isolation. Chromatograph y Theory Partition Coeﬃcient Extraction refers to the transfer of a solute from one liquid phase to another K = Co/Cw Co is concentration in the organic phase (solvent) Cw is the concentration in the aqueous phase (water) Partition Coeﬃcient K = Co/Cw Co is concentration in the organic phase (solvent) Cw is the concentration in the aqueous phase (water) molar concentration in stationary phase K= molar concentration in mobile phase Partition Coeﬃcient etc. concentration in stationary phase K= concentration in mobile phase mass in the stationary phase k = mass in the mobile phase b= volume of mobile phase volume of stationary phase Partition Coeﬃcient etc. If mass = volume x concentration then: k = K/b Example: Compound A: mass = 1 mg Vol. Mobile Phase: 1 mL Compound A: mass = 1 mg Vol. Stationary Phase: 2 mL Vol. Mobile Phase: 1 mL Vol. Stationary Phase: 1 mL Mobile K=4 Phase b= 1 K=4 k= 4 b = 0.5 grams in k = 8 Stationary mobile phase = 0.2 grams in Phase mobile phase = 0.11 If the mobile phase is moving, in which situation will compound A move faster through the column? Partitioning in a Mobile Phase Theoretical Plates 0.83 mg 0.69 0.83 mg 0.69 mg 0.58 0.28 mg 0.16 mg 0.14 mg 0.12 mg 0.10 mg 0.08 mg 0.07 mg 0.06 mg 1.0 mg Partitioning in a Mobile Phase 0.13 mg 0.13 mg 0.23 0.23 mg 0.29 0.29 mg 0.32 0.28 mg 0.16 0.03 mg 0.05 mg 0.14 0.12 mg 0.06 0.10 mg 0.06 0.08 mg 0.07 mg 0.06 mg mg Partitioning in a Mobile Phase 0.83 0.00 mg mg 0.69 0.00 mg 0.83 mg 0.58 0.69 0.01 mg mg 0.05 mg 0.17 mg 0.28 mg 0.34 0.28 mg 0.00 mg 0.00 mg 0.00 mg 0.03 mg 0.04 mg 0.07 mg 0.06 mg 1.0 mg Note: These equilibrium steps to do not actually take place in the column, it is a continuous process. Analyte Peaks in the Mobile Phase 0.83 0.00 mg mg 0.69 0.00 mg 0.83 mg 0.58 0.69 0.01 mg mg 0.05 mg 0.17 mg 0.28 mg 0.34 0.28 mg 0.00 mg 0.00 mg 0.00 mg 0.03 mg 0.04 mg 0.07 mg 0.06 mg 1.0 mg How would you make this broad peak more narrow? Analyte Peaks in the Mobile Phase Separation of Peaks Retention k = (tr – to)/ to Where tr = the retention time of the compound, and to = the dead time Higher values of k mean the analyte will stay in the column longer. The longer it stays, the more time there is for the peak will widen. Selectivity a = kB/kA the selectivity factor α and is an indication of how well the compounds will separate. Higher α means larger difference in retention time and more separation Eﬃciency Eﬃciency is a factor that is typically used to describe peak width. High Eﬃciency - narrow peaks Eﬃciency The term that is generally used to describe column eﬃciency is “number of theoretical plates” or N N = L/H Where: L =column length H = plate height (both in the same units) N in Practical Terms... N can be measured from the peaks on a chromatogram.. ( ) tr 2 N = 5.54 w1/2 Units for tr and to….? Units for W1/2 …..? Resolution The purpose of chromatography is to separate or resolve compounds. The separation or distance between two peaks is known as their resolution and is a function of the 3 factors discussed previously: retention (the time it takes for the analytes to elute, related to k), selectivity (how different the analytes are from each other and related to α), and eﬃciency (how good the column is, related to N) Resolution Eﬃciency Rs = ¼ (-1/) (k/k+1) N½ Selectivity Retention The effect on Rs of: increasing a…? increasing k…? increasing N…? Resolution Rs can also be calculated from actual measurements of peak retention times and measured peak widths Rs = 2 (tR-B – tR-A)/(wb-A + wb-B) Where: A and B are the two peaks tR = retention time and wb = the peak width at the base of each peak Resolution With a resolution value of 1.0, two peaks that overlap by about 4%. Values less than 1.0 indicate peaks that overlap, while at a resolution of 1.5, the peaks are considered fully separated. Going back to N…. N = L/H The value of N is greatly dependent on the value of H. The value of H depends primarily on four factors: 1) the velocity of the mobile phase, 2) eddy diffusion or multipath diffusion, 3) the diffusion of the compound in the mobile phase 4) the transfer of the compound between the stationary phase and the mobile phase. H - Theoretical Plate Height H = A + B/u + (Cs + Cm) u u = the average linear mobile phase velocity A is a term expressing multipath diffusion B/u is the term for longitudinal diffusion Cs is the mass transfer term in the stationary phase Cm is the mass transfer term in the mobile phase A Multipath The amount of spreading is affected by the nature of the column material and how well the column is packed. This factor is generally proportional to the particle size of the packing material. This factor must Flow be taken into account for packed columns, but for capillary columns, this term is not needed since there Direction are no particles. 1 2 Pathways of two molecules during elution. Distance traveled by molecule 1 is longer than that traveled by molecule 2, thus molecule 1 will take longer to elute. B Longitudinal Diffusion At low velocities longitudinal diffusion has a negative effect on resolution, but this effect is negligible at higher velocities. This term is very important in gas chromatography as diffusion coeﬃcients in gasses are orders of magnitude higher than in liquids. In liquid chromatography, this term is typically close to zero relative to the other terms. Molecules diffuse from areas of high concentration to areas of low concentration. Flow Over time…. Flow Mass Transfer Terms Cs & Cm Equilibrium between the mobile and stationary phases is never realized It takes time for analytes to move from the mobile phase into the stationary phase. Because no equilibrium is reached, some of the analytes are swept ahead of the of the main band. It also takes time for molecules to move back out of the stationary phase , and some of the analyte molecules will be left behind by the rapidly moving mobile phase. van Deemter Plot Plate Height, H A + B/u + Cu Mass Transfer (both), Cu Multipath Term, A Longitudinal diffusion, B/u Linear Velocity, u Mass Transfer Terms Cs & Cm The faster the mobile phase moves, the less time there is for equilibrium between the phases and the mass transfer effect on peak broadening is directly related to mobile phase velocity. Column Chromatography Dilution & Peak broadening! Chromatogram Chromatography: Peak separations Chromatography: Peak Resolution Poor resolution More separation Less band spread Chromatography: Distribution Constant (recommended by IUPAC) (old term: partition coeﬃcient) A mobile ↔ A stationary cS K c  stationary cM mobile CS = nS/VS, CM = nM/VM K ~ constant  linear chromatography >>>K >>> Retention in the stationary phase  Retention times How to manipulate K? Chromatography Retention Times tM = retention time of mobile phase (dead time) tR = retention time of analyte (solute) tS = time spent in stationary phase (adjusted retention time) L = length of the column Chromatography: Velocities Linear rate of solute migration! Velocity = distance/time  length of column/ retention times L v  Velocity of solute: tR L Velocity of mobile phase:   tM Chromatography Velocity/Retention time and Kc v    fraction of time in mobile phase moles of solute in mobile phase v    total moles of solute cM V M v    cM V M  c SV S Chromatography Velocity Relationships cM V M v    cM V M  c SV S 1 v    1  c SV S / c M V M cS K  Distributi on Constant cM 1 v    1  K V S /V M Chromatography Retention Factor : are we there yet? 1 v    1  K V S /V M kA  K AV S / V M (Retention Factor) 1 v    1kA L L 1   tR tM 1kA tR  tM Adjusted retention time kA  tM Relative retention time: RRT = tR/tRs tRs = retention time of internal standard Chromatography Selectivity Factor: can you separate from your neighbor B retained more than A   >1 K   B Distribution K A Constant kB   Retention kA factor ( tR ) A  tM ( tR ) B  tM kA  and kB  tM tM ( tR ) B  tM   Retention time ( tR ) A  tM Chromatography Column Eﬃciency - Theoretical Plates Plate and Rate Theories H  plate height N  number of plates L N  H  2 H  L L = length of column packing   standard deviation 2/L variance per unit length. Chromatography Relation between column distance and retention times L  column length (distance)   standard deviation in distance tR  retention time   standard deviation in time    L tR    L / tR Chromatography Relation between column distance and retention times    L tR L   tR W  4 W L ~96%   4 tR Tangent at Inﬂection point  2  2 W 2 L H   2 L 16 t R Chromatography Determining the Number of Theoretical Plates N  number of pates 2 t  N  16  R  W  W1/2 2  tR  N  5. 54    W1/2  Summary of Plate Theory Successfully accounts for the peak shapes and rate of movement Does not account for the “mechanism” causing peak broadening No indication of other parameters’ effects No indication for adjusting experimental parameters Rate Theory Zone broadening is related to Mass Transfer processes Column Eﬃciency Kinetic variables Zone Broadening Flow Rate of Mobile Phase Liquid chromatography Gas chromatography Note the differences in ﬂowrate and plates height scales Why GC normalluy has high H, but also high overall eﬃciency? Zone Broadening Kinetic Processes Van - Deemter Equation λ and γ are constants that depend on quality of the packing. B is coeﬃcient of longitudinal diffusion. Cs and Cm are coeﬃcients of mass transfer in stationary and mobile phase, respectively. H  A  B /  (C S  C M ) Zone Broadening Kinetic Processes Van - Deemter Equation H  A  B /  (C S  C M ) Zone Broadening Multiple Pathways Eddy Diffusion: band broadening process results from different path lengths passed by solutes. 1. Directly proportional to the diameters of packing 2. Offset by ordinary diffusion 3. Lower mobile-phase velocity, smaller eddy diffusion Stagnant pools of mobile phase retained in stationary phase. Zone Broadening Longitudinal Diffusion The higher the , the smaller the H Column Much smaller in LC than in GC Diffusion Chromatography Resolution Z Rs  WA /2  WB /2 2 Z Rs  WA  WB 2[( t R ) B  (tR ) A ] Rs  WA  WB Chromatographic Deﬁnitions Chromatographic Relationships Quantitative Analysis Peak areas Peak height Calibration and standards Internal Standard method Gas Chromatography Partitioning must occur between the stationary phase and the mobile phase interactions with the molecules being separated. This is the same equilibrium that is seen between the stationary phase and mobile phase in column chromatography or thin-layer chromatography. Injector Port Flow Control Detector Recorder Column Column Oven Carrier Gas Disadvantage: Destructive Technique – once analyzed by GC, the sample is “lost” ADVANTAGES: Only needs 1 µL to analyze a sample mixture Can analyze any compound that can be vaporized Cheapest, fastest and easiest method for separation, identiﬁcation and analysis of volatile compounds In Gas Chromatography 1. Stationary Phase – the adsorbent inside the Column Solid Support – Steel, Copper or Glass tubing Adsorbent – high-boiling hydrocarbons, waxes, silicone oils, polymeric esters, etc. Based on polarity and molecular weight, the compounds being separated adhere to the column’s adsorbent to varying degrees. The adsorbent in the column determines the maximum limit to how hot the column can be heated. Too hot and you can boil away the adsorbent, right out of the column 2. Mobile Phase – the Gas Gases commonly used for GC would include inert gases like Helium, Argon or Nitrogen. separates compounds based on their boiling points  affected by polarity and weight of the compounds. Lower boiling compounds : lightest in weight and least polar and travel the fastest, spending more time in _____, less time “stuck” to the stationary phase, taking less time to pass through the detector. Example: Compound A has a boiling point of 50ºC and Compound B has a boiling point o 105ºC. Which one will vaporize fastest? Compound Which one will spend more time in the mobile phase? Compound The main reason why different compounds can be separated this way is the interaction of the compound with the stationary phase“(like-dissolves-like” -rule). The stronger the interaction is the longer the compound remains attached to the stationary phase, and the more time it takes to go through the column (=longer retention time). Polarity of the stationary phase Polar compounds interact strongly with a polar stationary phase, hence have a longer retention time than non-polar columns. First, a needle is used to inject the liquid (or dissolved solid) into the Injection port, where the liquid solution is vaporized. The gas (mobile phase) then picks up the two compounds and moves them into the column. The higher boiling compound (BLUE) begins to adhere more to the sides of the column while the lower boiling compound (RED) stays vaporized and in the mobile phase, moving through the column. By the end of the process, the lower boiling compound is moving out of the column separated from the higher boiling compound. 4. Gas Flow Rate – Determines the amount of time a compound may spend in the mobile phase. >> fast: no time to develop the equilibrium required – no partitioning and the compounds rush through the column together, not separating but forming thin, narrow overlapped peaks. > high : destroy the column by boiling off the adsorbent itself from inside the column >> cool : compounds condensing inside the column and never coming out. Too hot: all the compounds will vaporize and be carried rapidly through the column, with no partitioning effect. Thin, narrow peaks, still overlapped in the middle. Too cool: compounds may condense more than they should, spending too much time in the stationary phase. The ﬁrst compound may adhere too much and not be completely out of the column prior to the second one coming out of the column. Broad and overlapped peaks as a result. Two Types of Analysis: 1. Qualitative Analysis – Identiﬁcation of Compounds Retention time: (similar to Rf values for TLC) -Amount of time it takes for the compound to travel through the column, from the point of vaporization to the detector. Example: Retention Times: Compound X = 3.1 minutes Compound Y = 4.8 minutes Compound Z = 5.3 minutes Now compare Unknown Compound Q, run under identical conditions as X, Y and Z. Q has a retention time of 3.1 minutes. Which compound is it? 2. Quantitative Analysis – Determination of Relative Amounts of each Compound (ratio of compounds) Step 1: Calculate the area under the peaks using the equation: Area = (height of peak) x (width at half the height) First Peak: A x B Second Peak: C x D Step 2: Calculate the Percent Composition (Relative Amounts) Take each individual area and divide by sum total of all areas x 100. Should add up to 100. Assume AxB = 20 and CxD = 5 so the total of the two areas is 25. Percent Composition First Peak: 20/25 x 100 = 80% Percent Composition Second Peak: 5/25 x 100 = 20% Internal Standard a compound that is added, in a known and constant concentration, to the sample. ratio of its retention time to the retention times of the target compounds ratio of its peak area to the peak areas of target compounds is determined for various concentrations of the target compounds External Standard known amounts of analytes are run in a separate analysis a standard run, and the resulting peak areas are used to obtain calibrated response factors that are stored in a calibration library. In later runs, these response factors are used to calculate analyte concentrations. a known and constant quantity of a compound that is not one of the analytes is added to the sample ratio of its retention time to the retention times of the analytes has been establ the unknown concentrations of the analytes in samples are then calculated, using the area of the internal standard peak as a reference. Response factors are included in the calculation to compensate for differences in sensitivity of the detector to different analytes. Analysis by an internal standard is the preferred technique whenever practical because it corrects for errors in sample preparation and variations in the amount sample injected. The concentrations reported for the peaks of interest are affected only by the quantities of the various components and the quantity of internal standard added. Internal standards should be of the same family as the target compounds (for example, phenols) but their retention times should generally not overlap those of targets. An exception is isotopically labeled internal standards. external standard method known amounts of analytes are run in separate analyses (the standard runs), and the resulting peak areas are used to obtain calibrated response factors. In subsequent analyses of samples with unknown concentrations, the concentrations of the analytes are calculated by applying the response factors obtained from the standard runs. there is no standard peak whose area changes with variations in injection size, the sample injection size must be reproducible from run to run. external standard methods are best used with an autosampler. They are not recommended for manual injection. Quantitation in Chromatography Detector Response Detector Signal Depends on concentration of analyte or mass of analyte reaching detector Most (but not all) detectors give linear response over portion of detectable range Detector Noise Present in all detectors High and low frequency types Ability to Detect Small Quantities Depends on Signal (Peak Height) to Noise Ratios Quantitation in Chromatography Levels of Detection and Quantiﬁcation Noise can have high and low frequency parts Ways of deﬁning noise peak to peak (roughly 5σ) standard deviation (more accurate way) Signal = peak height high frequency peak to peak noise component low frequency component Quantitation in Chromatography Data Smoothing Data should be digitized with a frequency ~20/peak width High frequency noise (where fnoise >> fsignal) can be removed by ﬁltering see example below note: overﬁltering results in reduction of signal and loss of resolution overﬁltering result also can occur if detector response is too slow (or cell volume is too large Diﬃcult to remove noise with frequency similar to or lower than peaks Quantitation in Chromatography Integration Integration of peak should give: peak height peak area peak width (often just peak area/peak height) Diﬃculty comes from determining if a peak is a peak but not these (or just noise), and when to we want to pick noise spikes “start” the peak and “end” the up this peak peak. Can use “auto integration” or “manual integration” Quantitation in Chromatography Integration Other issues in integration (besides noise peaks) start and ends to peaks how to split overlapping peaks Quantitation in Chromatography Integration Peak Height vs. Peak Area Reasons for using peak area peak area is independent of retention time (assuming linear response), while the peak height will decrease with an increase in retention time peak area is independent of peak width, while the peak height will decrease if the column is overloaded (non-linear response) Reasons for using peak height Integration errors tend to be smaller if samples are close to the detection limits Quantitation in Chromatography Calibration Methods External Standard most common method standards run separately and calibration curve Area prepared samples run, from peak areas, concentrations are determined best results if unknown concentration comes out in calibration standard range Internal Standard Common for GC with manual injection Concentration (imprecisely known sample volume) Useful if slow drift in detector response Standard added to sample; calibration and sample determination based on peak area ratio AX/AS F = constant where A = area and C = conc. (X = analyte, S = internal standard) A X / AS F  C X /C S Conc. X (constant conc. S) Quantitation Additional (Recovery Standards + Questions) Recovery Standards Principle of use is similar to standard addition Standard (same as analyte or related compound) added to sample, then measured (in addition to direct measurement of sample) amount 100  amount - amount  100 % recovered  recovered  total unknown amount expected amount expected Useful for determining losses during extractions, derivatization, and with matrix effects Quantitation Some Questions/Problems 1. Rank the following compounds in terms of their expected retention times on GC (OV-101 column) and their Rf values on TLC 2. Why is the internal standard calibration more common when using manual injection than injection with an autosampler? 3. Study the chromatograph (below) of a mixture of Compounds A and B, run on the GC 1. What is the retention time of compound A? Compound B? 2. Which compound is present in a larger amount? 3. Which compound has the lower boiling point? 4. What would happen to the retention times of compounds A and B if the column temperature were raised? 5. You suspect that compound B is octane. What can you do to provide supporting evidence for this hypothesis? 4. Consider the following compounds If they are run on an DEGS GC column, which column will have the lowest retention time Highest?

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