INTRODUCTION TO CHROMATOGRAPY

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

Chromatography Web Dictionary: Analytic technique to discover chemical components: a method of finding out which components a gaseous or liquid mixture contains that involves passing it through or over something that absorbs the different components at different rates

Chromatography Column Oven Carrier Gas Injector Port Detector Flow Control Injector Port Column Column Oven Detector Recorder

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.

Chromatography Theory

Partition Coefficient Remember from the solvent lecture………. 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 molar concentration in mobile phase K =

Partition Coefficient etc. concentration in stationary phase concentration in mobile phase K = k = mass in the stationary phase 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 Vol. Stationary Phase: 2 mL K = 4 b = k = grams in mobile phase = Compound A: mass = 1 mg Vol. Mobile Phase: 1 mL Vol. Stationary Phase: 1 mL Mobile Phase K = 4 b = k = grams in mobile phase = 1 4 0.2 0.5 8 0.11 Stationary Phase 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 0.16 mg 0.83 mg 0.69 mg 0.14 mg 0.69 mg 0.12 mg 0.58 mg 0.06 mg 0.28 mg 0.10 mg 0.08 mg 0.07 mg 1.0 mg

Partitioning in a Mobile Phase 0.23 mg 0.05 mg 0.03 mg 0.13 mg 0.13 mg 0.29 mg 0.06 mg 0.23 mg 0.12 mg 0.32 mg 0.06 mg 0.29 mg 0.10 mg 0.28 mg 0.16 mg 0.14 mg 0.12 mg 0.10 mg 0.08 mg 0.07 mg 0.06 mg

Partitioning in a Mobile Phase 0.83 mg 0.00 mg 0.83 mg 0.00 mg 0.69 mg 0.69 mg 0.00 mg 0.58 mg 0.00 mg 0.00 mg 0.01 mg 0.05 mg 0.17 mg 0.28 mg 0.34 mg 0.28 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 mg 0.00 mg 0.83 mg 0.00 mg 0.69 mg 0.69 mg 0.00 mg 0.58 mg 0.00 mg 0.00 mg 0.01 mg 0.05 mg 0.17 mg 0.28 mg 0.34 mg 0.28 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 can be measured from the peaks on a chromatogram.. N in Practical Terms... N can be measured from the peaks on a chromatogram.. N = 5.54 tr w1/2 ( ) 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)

Rs = ¼ (a-1/a) (k/k+1) N½ The effect on Rs of: Resolution Efficiency Selectivity Retention The effect on Rs of: increasing a…? increasing k…? increasing N…?

Rs = 2 (tR-B – tR-A)/(wb-A + wb-B) 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.

The value of N is greatly dependent on the value of H. 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 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. Flow Direction 2 1 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. Flow Molecules diffuse from areas of high concentration to areas of low concentration. Over time….

Equilibrium between the mobile and stationary phases is never realized 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.

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.

van Deemter Plot Plate Height, H A + B/u + Cu Linear Velocity, u Multipath Term, A Mass Transfer (both), Cu Longitudinal diffusion, B/u A + B/u + Cu