2 HistoryThe 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
3 Chromatography Web Dictionary: Analytic technique to discover chemical components:a method of finding out which components a gaseousor liquid mixture contains that involves passing itthrough or over something that absorbs the differentcomponents at different rates
4 Chromatography Column Oven Carrier Gas Injector Port Detector Flow ControlInjectorPortColumnColumn OvenDetectorRecorder
5 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.
7 Partition Coefficient Remember from the solvent lecture……….K = Co/CwCo is concentration in the organic phase (solvent)Cw is the concentration in the aqueous phase (water)
8 Partition Coefficient K = Co/CwCo is concentration in the organic phase (solvent)Cw is the concentration in the aqueous phase (water)molar concentration in stationary phasemolar concentration in mobile phaseK =
9 Partition Coefficient etc. concentration in stationary phaseconcentration in mobile phaseK =k =mass in the stationary phasemass in the mobile phaseb =volume of mobile phasevolume of stationary phase
10 Partition Coefficient etc. If mass = volume x concentration then:k = K/b
11 Example: Compound A: mass = 1 mg Vol. Mobile Phase: 1 mL Vol. Stationary Phase: 2 mLK = 4b =k =grams inmobile phase =Compound A: mass = 1 mgVol. Mobile Phase: mLVol. Stationary Phase: 1 mLMobilePhaseK = 4b =k =grams inmobile phase =140.20.580.11StationaryPhaseIf the mobile phase is moving, in which situation will compound A move faster through the column?
12 Partitioning in a Mobile Phase Theoretical Plates0.83 mg0.16 mg0.83 mg0.69 mg0.14 mg0.69 mg0.12 mg0.58 mg0.06 mg0.28 mg0.10 mg0.08 mg0.07 mg1.0 mg
13 Partitioning in a Mobile Phase 0.23 mg0.05 mg0.03 mg0.13 mg0.13 mg0.29 mg0.06 mg0.23 mg0.12 mg0.32 mg0.06 mg0.29 mg0.10 mg0.28 mg0.16 mg0.14 mg0.12 mg0.10 mg0.08 mg0.07 mg0.06 mg
14 Partitioning in a Mobile Phase 0.83 mg0.00 mg0.83 mg0.00 mg0.69 mg0.69 mg0.00 mg0.58 mg0.00 mg0.00 mg0.01 mg0.05 mg0.17 mg0.28 mg0.34 mg0.28 mg0.03 mg0.04 mg0.07 mg0.06 mg1.0 mgNote: These equilibrium steps to do not actually takeplace in the column, it is a continuous process.
15 Analyte Peaks in the Mobile Phase 0.83 mg0.00 mg0.83 mg0.00 mg0.69 mg0.69 mg0.00 mg0.58 mg0.00 mg0.00 mg0.01 mg0.05 mg0.17 mg0.28 mg0.34 mg0.28 mg0.03 mg0.04 mg0.07 mg0.06 mg1.0 mgHow would you make this broad peak more narrow?
18 Retention k = (tr – to)/ to Where tr = the retention time of the compound, and to = the dead timeHigher 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.
19 Selectivitya = kB/kAthe 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
20 Efficiency Efficiency is a factor that is typically used to describe peak width.High Efficiency - narrow peaks
21 Efficiency The term that is generally used to describe column efficiency is “number of theoretical plates” or NN = L/HWhere: L =column lengthH = plate height (both in the same units)
22 N can be measured from the peaks on a chromatogram.. N in Practical Terms...N can be measured from the peaks on a chromatogram..N = 5.54trw1/2()2Units for tr and to….?Units for W1/2 …..?
23 ResolutionThe 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 theanalytes 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)
24 Rs = ¼ (a-1/a) (k/k+1) N½ The effect on Rs of: Resolution Efficiency SelectivityRetentionThe effect on Rs of:increasing a…?increasing k…?increasing N…?
25 Rs = 2 (tR-B – tR-A)/(wb-A + wb-B) ResolutionRs can also be calculated from actual measurements of peak retention times and measured peak widthsRs = 2 (tR-B – tR-A)/(wb-A + wb-B)Where: A and B are the two peakstR = retention time andwb = the peak width at the base of each peak
26 ResolutionWith 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.
27 The value of N is greatly dependent on the value of H. Going back to N….N = L/HThe 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 phase4) the transfer of the compound between the stationary phaseand the mobile phase.
28 H - Theoretical Plate Height H = A + B/u + (Cs + Cm) uu = the average linear mobile phase velocityA is a term expressing multipath diffusionB/u is the term for longitudinal diffusionCs is the mass transfer term in the stationary phaseCm is the mass transfer term in the mobile phase
29 A Multipath Flow Direction 2 1 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 be taken into account for packed columns, but for capillary columns, this term is not needed since there are no particles.FlowDirection21Pathways of two molecules during elution. Distance traveled by molecule 1 is longer than that traveled by molecule 2, thus molecule 1 will take longer toelute.
30 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.FlowMolecules diffuse from areas of highconcentration to areas of low concentration.Over time….
31 Equilibrium between the mobile and stationary phases is never realized Mass Transfer Terms Cs & CmEquilibrium between the mobile and stationary phases is never realizedIt 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.
32 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.
33 van Deemter Plot Plate Height, H A + B/u + Cu Linear Velocity, u Multipath Term, AMass Transfer (both), CuLongitudinal diffusion, B/uA + B/u + Cu