Read this article to learn about the basics, principles and theories of chromatography.
Chromatography involves a sample (or sample extract) being dissolved in a mobile phase (which may be a gas, a liquid or a supercritical fluid).
The mobile phase is then forced through an immobile, immiscible stationary phase. The phases are chosen such that components of the sample have differing solubilities in each phase.
A component which is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase.
As a result of these differences in mobilities, sample components will become separated from each other as they travel through the stationary phase.
Chromatography may be preparative or analytical. Preparative chromatography seeks to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography normally operates with smaller amounts of material and seeks to measure the relative proportions of analytes in a mixture. The two are not mutually exclusive.
Before moving into the details of the topic it would be advisable that we should first get acclimatized with various technical terminologies of use in the topic:
i. The analyte is the substance which is to be separated during chromatography.
ii. Analytical chromatography is used to determine the existence and possibly also the concentration of analyte(s) in a sample.
iii. A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.
iv. A chromatogram is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.
v. A chromatograph is an equipment that enables a sophisticated separation, e.g., gas chromatographic or liquid chromatographic separation.
vi. The effluent is the mobile phase leaving the column.
vii. An immobilized phase is a stationary phase which is immobilized on the support particles, or on the inner wall of the column tubing.
viii. The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC and CEC), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC).
ix. Preparative chromatography is used to nondestructively purify sufficient quantities of a substance for further use, rather than analysis.
x. The retention time is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions.
xi. The sample is the matter analysed in chromatography. It may consist of a single component or it may be a mixture of components. When the sample is treated in the course of an analysis, the phase or the phases containing the analytes of interest is/are referred to as the sample whereas everything out of interest separated from the sample before or in the course of the analysis is referred to as waste.
xii. The solute refers to the sample components in partition chromatography.
xiii. The solvent refers to any substance capable of solubilizing other substance, especially the liquid mobile phase in LC.
xiv. The stationary phase is the substance which is fixed in place for the chromatography procedure, e.g., the silica layer in thin layer chromatography.
Basic Principles:
Distribution of analytes between phases:
The distribution of analytes between phases can often be described quite simply. An analyte is in equilibrium between the two phases;
A mobile ↔ A stationary
The equilibrium constant, K, is termed the partition coefficient, defined as the molar concentration of analyte in the stationary phase divided by the molar concentration of the analyte in the mobile phase. The time between sample injection and an analyte peak reaching a detector at the end of the column is termed the retention time (tR). Each analyte in a sample will have a different retention time. The time taken for the mobile phase to pass through the column is called tM.
A term called the retention factor, k’, is often used to describe the migration rate of an analyte on a column. You may also find it called the capacity factor. The retention factor for analyte A is defined as
kA‘= (tR-tM)/tM.
tR and tM are easily obtained from a chromatogram. When an analytes retention factor is less than one, elution is so fast that accurate determination of the retention time is very difficult. High retention factors (greater than 20) mean that elution takes a very long time. Ideally, the retention factor for an analyte is between one and five.
We define a quantity called the selectivity factor, α, which describes the separation of two species (A and B) on the column
When calculating the selectivity factor, species A elutes faster than species B. The selectivity factor is always greater than one.
Band broadening and column efficiency:
To obtain optimal separations, sharp, symmetrical chromatographic peaks must be obtained. This means that band broadening must be limited. It is also beneficial to measure the efficiency of the column.
The Theoretical Plate Model of Chromatography:
The plate model supposes that the chromatographic column contains a large number of separate layers, called theoretical plates. Separate equilibrations of the sample between the stationary and mobile phase occur in these “plates”. The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next.
It is important to remember that the plates do not really exist; they are a figment of the imagination that helps us understand the processes at work in the column. They also serve as a way of measuring column efficiency, either by stating the number of theoretical plates in a column, N (the more plates the better), or by stating the plate height; the Height is equivalent to a theoretical plate (the smaller the better).
If the length of the column is L, then
HETP = L/N.
The number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution.
N = 5.55 t2R /w21/2
where W1/2 is the peak width at half-height.
As can be seen from this equation, columns behave as if they have different numbers of plates for different solutes in a mixture.
The Rate Theory of Chromatography:
A more realistic description of the processes at work inside a column takes account of the time taken for the solute to equilibrate between the stationary and mobile phases (unlike the plate model, which assumes that equilibration is infinitely fast). The resulting band shape of a chromatographic peak is, therefore, affected by the rate of elution.
It is also affected by the different paths available to solute molecules as they travel between particles of stationary phase. If we consider the various mechanisms which contribute to band broadening, we arrive at the Van Deemter equation for plate height.
HETP = A + B/u + Cu,
where u is the average velocity of the mobile phase. A, B, and C are factors which contribute to band broadening.
(A) Eddy diffusion:
The mobile phase moves through the column which is packed with stationary phase. Solute molecules will take different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths.
(B) Longitudinal diffusion:
The concentration of analyte is less at the edges of the band than at the centre. Analyte diffuses out from the centre to the edges. This causes band broadening. If the velocity of the mobile phase is high, then the analyte spends less time on the column, which decreases the effects of longitudinal diffusion.
(C) Resistance to mass transfer:
The analyte takes a certain amount of time to equilibrate between the stationary and mobile phases. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase, the worse the broadening becomes.
Van Deemter plots:
A plot of plate height vs. average linear velocity of mobile phase. Such plots are of considerable use in determining the optimum mobile phase flow rate.
Resolution:
Although the selectivity factor, α, describes the separation of band centers, it does not take into account peak widths. Another measure of how well species have been separated is provided by measurement of the resolution. The resolution of two species, A and B, is defined as
R = 2[(tR)B-(tR)A] / WA+WB
Baseline resolution is achieved when R = 1.5.
It is useful to relate the resolution to the number of plates in the column, the selectivity factor and the retention factors of the two solutes,
R = √N/4(α-1/α)(1+kB’/ kB’)
To obtain high resolution, the three terms must be maximised. An increase in N, the number of theoretical plates, by lengthening the column leads to an increase in retention time and increased band broadening—which may not be desirable. Instead, to increase the number of plates, the height equivalent to a theoretical plate can be reduced by reducing the size of the stationary phase particles.
It is often found that by controlling the capacity factor, k’, separations can be greatly improved. This can be achieved by changing the temperature (in Gas Chromatography) or the composition of the mobile phase (in Liquid Chromatography).
The selectivity factor, α, can also be manipulated to improve separations. When α is close to unity, optimising k’ and increasing N is not sufficient to give good separation in a reasonable time. In these cases, k’ is optimized first, and then α is increased by one of the following procedures:
1. Changing mobile phase composition
2. Changing column temperature
3. Changing composition of stationary phase
4. Using special chemical effects (such as incorporating a species which complexes with one of the solutes into the stationary phase).