Chromatography: Methods, Phases, and Separation Efficiency
Chromatography:
Physical method of separation. Compounds are distributed between stationary phase and mobile phase – the chromatographic process occurs as a result of repeated equilibration events during the movement of the sample components along the stationary phase bed. The separation occurs due to differences in their affinity for the stationary and mobile phases. As the components exit the system, a detector identifies them based on specific properties of the desired analytes.
Stationary Phase:
Can be solid/liquid coated onto a solid support (flat surface – PLANAR eg TLC/packed into a column COLUMN eg HPLC, GC/coated onto the inner walls of a very narrow column OPEN TUBULAR eg capillary GC)
Mobile Phase:
Liquid/gas (fluid). Often called the eluent. When emerges from end of column – eluate. Elution- the process of passing liquid or gas through the column
2 Types of Stationary Phase Format
Columnar: HPLC – columns contain monolith (channels running through SP) or particle structure, made of stainless steel to withstand high pressure. GC – smaller diameter, longer than HPLC. Strong durable coating (such as a polyamide coating fused with silica) and needs to be flexible due to the high temperatures and length. To minimize the impact of longitudinal diffusion, increase the length of the column. Planar – used for small scale reactions.
Chromatogram:
Output received from the detector as a signal response vs time. For electrically driven separations these are termed electropherograms.
Modes of Chromatography:
- Adsorption – normal phase, reversed-phase
- Partition – PDMS in open tubular GC, RP
- Ion exchange – IEX, IIC
- Molecular exclusion – gel permeation
- Affinity – IMAC, immunoaffinity
Retention and Equilibrium:
Retention time is the time taken for a solute to pass over through the fill length of stationary phase bed from the point of injection to detection at the other end. The migration rate is dependent on the partition coefficient ratio K given by K=Cs/Cm. The higher K is the more retention occurs. Cs and Cm represent the amount of analyte in the SP and MP respectively.
Adjusted retention time: Tr: time from t0 to tr
Retention factor k: a measure of retention across formats
Resolution Rs: the separation of two gaussian bands
The dip in a chromatogram means that the sample has reached the column after injection
Good peak shape: Gaussian – evenly distributed around a mean central point this is the tr marker
Non gaussian: poor peak shape: tailed, fronted or split
Retention factor, the longer a component is retained on the column the greater the retention factor. If k<1, compounds are eluted too quickly and you should alter the method. K is used to interpret retention across chromatographic formats, lengths, diameters. It’s a reference point to which researchers can relate different methods.
Selectivity factor a- important measure of band or peak proximity
- where for 2 adjacent bands having the k values k1 and k2: a =k2/k1. In general, a must be at least 1.005-1.05 for acceptable separations for GC and 1.05-1.1 for good separations in LC.
- Substitution of the retention times into the previous equation gives an expression that permits the determination of a from an experimental chromatogram
- is relatively independent of flow rate and can be used to identify peaks when the flow rate changes
- a=selectivity/separation factor/relative retention
k=tr0-t0/t0=tr/t0
perform calculation to determine separation efficiency
determine separation efficiency using triangulation method
perform calculation to determine resolution between two adjacent peaks
evaluate whether the peak resolution is acceptable
Separation Efficiency:
Difference in retention times between peaks
Broadness of peaks or width wider peaks poorer separation
Theoretical Plates:
Is an area within the column where an equilibrium between the analyte adsorption and desorption with the SP has been establishes, the more theoretical plates, the more opportunities for this to occur and the better the separation. A column with 256 plates will not produce well resolved peaks because it’s a low number of plates. Ideal number of plates is in the thousands.
Key equations used to describe the quality of a column:
Increasing the column length also increases its cost. For column length L the plate height H is given by H=L/N where N is the number of theoretical plates. N can be calculated from a chromatogram