Typically, column chromatography is set up with peristaltic pumps, flowing buffers and the solution sample through the top of the column. The solutions and buffers pass through the column where a fraction collector at the end of the column setup collects the eluted samples. Prior to the fraction collection, the samples that are eluted from the column pass through a detector such as a spectrophotometer or mass spectrometer so that the concentration of the separated samples in the sample solution mixture can be determined.
For example, if you were to separate two different proteins with different binding capacities to the column from a solution sample, a good type of detector would be a spectrophotometer using a wavelength of 280 nm. The higher the concentration of protein that passes through the eluted solution through the column, the higher the absorbance of that wavelength.
Because the column chromatography has a constant flow of eluted solution passing through the detector at varying concentrations, the detector must plot the concentration of the eluted sample over a course of time. This plot of sample concentration versus time is called a chromatogram.
The ultimate goal of chromatography is to separate different components from a solution mixture. The resolution expresses the extent of separation between the components from the mixture. The higher the resolution of the chromatogram, the better the extent of separation of the samples the column gives. This data is a good way of determining the column's separation properties of that particular sample. The resolution can be calculated from the chromatogram.
The separate curves in the diagram represent different sample elution concentration profiles over time based on their affinity to the column resin. To calculate resolution, the retention time and curve width are required.
Retention time is the time from the start of signal detection by the detector to the peak height of the elution concentration profile of each different sample.
Curve width is the width of the concentration profile curve of the different samples in the chromatogram in units of time.
A simplified method of calculating chromatogram resolution is to use the plate model. The plate model assumes that the column can be divided into a certain number of sections, or plates and the mass balance can be calculated for each individual plate. This approach approximates a typical chromatogram curve as a Gaussian distribution curve. By doing this, the curve width is estimated as 4 times the standard deviation of the curve, 4σ. The retention time is the time from the start of signal detection to the time of the peak height of the Gaussian curve.
From the variables in the figure above, the resolution, plate number, and plate height of the column plate model can be calculated using the equations:
Resolution (R):
:R = 2(t – t)/(w + w),
where:
:t = retention time of solute B
:t = retention time of solute A
:w = Gaussian curve width of solute B
:w = Gaussian curve width of solute A
Plate Number (N):
:N = (t)/(w/4)
Plate Height (H):
:H = L/N
where L is the length of the column.