An Introduction to Chromatographic Separations Lecture 33

It was Mikhail Tswett, a Russian botanist, in 1903 who first invented and named liquid chromatography. Tswett used a glass column filled with finely divided chalk (calcium carbonate) to separate plant pigments. He observed the separation of colored zones or bands along the column, hence name chromatography, where Greak chroma means color and graphein means write.

The development of chromatography was slow for reasons to be discussed later and scientists waited to early fifties for the first chromatographic instrument to appear in the market (a gas chromatograph). However, liquid chromatographic equipment with acceptable performance was only introduced about two decades after gas chromatography.

According to the nature of the mobile phase, chromatographic techniques can be classified into three classes: Liquid chromatography (LC) Gas chromatography (GC) Supercritical fluid chromatography (SFC) Other classifications are also available where the term column chromatography where chromatographic separations take place inside a column, and planar chromatography, where the stationary phase is supported on a planar flat plate, are also used.

General Description of Chromatography In a chromatographic separation of any type, different components of a sample are transported in a mobile phase (a gas, a liquid, or a supercritical fluid). The mobile phase (also called eluent) penetrates or passes through a solid or immiscible stationary phase. Solutes (eluates) in the sample usually have differential partitioning or interactions with the mobile and stationary phases. Since the stationary phase is the fixed one then those solutes which have stronger interactions with the stationary phase will tend to move slower (have higher retention times) than others which have lower or no interactions with the stationary phase will tend to move faster.

Therefore, chromatographic separations are a consequence of differential migration of solutes. It should be remembered that maximum interactions between a solute and a stationary phase take place when both have similar characteristics, for example in terms of polarity. However, when their properties are so different, a solute will not tend to stay and interact with the stationary phase and will thus prefer to stay in the mobile phase and move faster; a polar solvent and a non polar stationary phase is a good example.

Elution Chromatography The term elution refers to the actual process of separation. A small volume of the sample is first introduced at the top of the chromatographic column. Elution involves passing a mobile phase inside the column whereby solutes are carried down the stream but on a differential scale due to interactions with the stationary phase. As the mobile phase continues to flow, solutes continue to move downward the column. Distances between solute bands become greater with time and as solutes start to leave the column they are sequentially detected.

The time a solute spends in a column (retention time) depends on the fraction of time that solute spends in the mobile phase. As solutes move inside the column, their concentration zone continues to spread and the extent of spreading (band broadening) depends on the time a solute spends in the columns. Factors affecting band broadening are very important and will be discussed later. The dark colors at the center of the solute zones in the above figure represent higher concentrations than are concentrations at the sides. This can be represented schematically as:

Chromatograms The plot of detector signal (absorbance, fluorescence, refractive index, etc..) versus retention time of solutes in a chromatographic column is referred to as a chromatogram. The areas under the peaks in a chromatogram are usually related to solute concentration and are thus very helpful for quantitative analysis. The retention time of a solute is a characteristic property of the solute which reflects its degree of interaction with both stationary and mobile phases. Retention times serve qualitative analysis parameters to identify solutes by comparison with standards.

Migration Rates of Solutes   The concepts which will be developed in this section will be based on separation of solutes using a liquid mobile phase and an immiscible liquid stationary phase. This case is particularly important as it is a description of the most popular processes.

Distribution Constants Solutes traveling inside a column will interact with both the stationary and mobile phases. If, as is our case, the two phases are immiscible, partitioning of solutes takes place and a distribution constant, K, can be written: K = CS/CM (1) Where; CS and CM are the concentrations of solute in the stationary and mobile phases, respectively. If a chromatographic separation obeys equation 1, the separation is called linear chromatography. In such separations peaks are Gaussian and independent of the amount of injected sample

The time required for an analyte to travel through the column after injection till the analyte peak reaches the detector is termed the retention time. If the sample contains an unretained species, such species travels with the mobile phase where the time spent by that species to exit the column is called the dead or void time, tM. Solutes will move towards the detector in different speeds, according to each solute’s nature.

Retention Times tM = retention time of mobile phase (dead time) tR = retention time of analyte (solute) tR’ = time spent in stationary phase (adjusted retention time) L = length of the column 18

Velocity = distance/time  length of column/ retention times Velocity of solute: Velocity of mobile phase: 19

average linear velocity of a solute, v, can be written as:   v = L/tR (2) Where, L is the column length and tR is the retention time of the solute. The mobile phase linear velocity, u, can be written as: u = L/tM (3) The linear velocity of solutes is a fraction of the linear velocity of the mobile phase. This can be written as: v = u * moles of solute in mp/total moles of solute

21

This can be further expanded by substitution for the moles of solute in mp, CMVM, and the total number of moles of solute, CMVM + CSVS. v = u * CMVM / (CMVM + CSVS) Dividing both nominator and denominator by CMVM we get v = u * 1/ (1 + CSVS/ CMVM) (4)   Now, let us define a new distribution constant, called the capacity or retention factor, k’, as: k’ = CSVS/ CMVM = K VS/VM (5)

Substitution of 1,2 and 5 in equation 4 we get: L/tR = L/tM * {1/(1 + k’)}   Rearrangement gives: tR = tM (1+k’) (6) This equation can also be written as: k’ = (tR – tM)/tM