Thursday, March 9, 2017

Chromatography - Column Chromatography Basics

Till now, we have discussed about plane chromatography technique. Now, coming to column chromatography. The five major types of column chromatography are as:
1. Adsorption chromatography in which the components of mixture are selectively adsorbed on the surface of packing column material i.e. adsorbent.
2. Partition chromatography in which component is partitioned between the mobile phase and their stationary phase is held stationary on inert solid support.
3. Ion exchange chromatography in which the constituent of sample is selectively retained by exchange resin by replacing ion/s on packing material.
4. Gel chromatography is the method in which column is packed with a permeable gel which brings out separation by sieving (gel permeation )
5. Affinity chromatography utilizes the specificity of a enzyme for its substrate or substrate analogue for the enzyme’s separation.

We will discuss in details all these types of chromatogrpahic techniques. But for now, lets understand that all these chromatographic techniques have a lot of features in common. So, lets discuss what all they share in common. 

The columns are usually made up of glass or polyacrylate plastic. They all are different in dimensions and range from 2 mm in diameter to 70 mm in diameter and lengths vary from 15 cm to 150 cm. The choice of the column is determined by the amount or the volume of the sample.
The commonly used glass column shave a sintered glass disc at the bottom to support the stationary phase. Another affordable alternative is the use of a plug of glass wool with a small amount of quartz sand and glass beads.
Some columns might get affected by the temperature and hence, for such techniques, the columns with thermostat jackets are used. All the columns are provided with an inlet and an outlet.

Packing of the column
The column is held in the upright position and as already mentioned above, the bottom is sealed with glass wool or other such supports and the outlet is closed. Now, the mobile phase is added into the column so that 1/3rd is filled with it. Next, the stationary phase in the form of slurry i.e., degassed gel or adsorbent or resin is gently poured in the column. The slurry is added such that the 3/4th of the column is full. The outlet is now opened and the column is stabilized by adding the mobile phase. Note that a nylon disk is placed on the top of the column so as to prevent the disturbance of the column by the addition of the mobile phase or by the addition of the sample.

Introducing the sample
The addition of the sample is a critical step as it should not disturb the column. Also, the sample should be added in a less volume and the sample should be desalted so as to avoid anomalous adsorption effects.
The sample is mixed with sucrose or ficoll to 1% concentration so as to increase the density of the sample. This sample now sinks below the top layer of the solvent to the surface of the column. Bromophenol blue dye can also be added in place of sucrose which will help in tracking as well.  Alternate method of sample addition is to reach the column surface directly with the help of the syringe or capillary tubing.

The continuous flow of mobile phase (called the "eluant" – mobile phase exiting the column is the eluant) through the packed column separates the components of the sample which is applied to the column. This process is called column development. There are two main techniques of elution:
Isocratic elution: When there is a single solvent acting as an eluant during development, then, this type of process is called isocratic elution.

Gradient elution: However, sometimes, single solvent elution may not be sufficient as the resolution may not be satisfactory. So, in such cases, pH or ionic strength or polarity of the eluant is changed w.r.t. time. This leads to the formation of gradient in the column which leads to a better resolution of the sample components. So, this process where the composition of the mobile phase is changed thereby giving rise to the gradient is called gradient elution. 

This was about the basics of the column chromatography. From next posts on, we will discuss the types of column chromatography in details. 

Wednesday, February 22, 2017

Chromatography - Thin Layer Chromatography

As discussed in earlier post, there are two types of plane chromatography. We have discussed Paper Chromatography in details in the earlier post. In this post, we are going to discuss about Thin Layer Chromatography
Thin Layer Chromatography, abbreviated as TLC, is a type of plane chromatography which is used to separate non-volatile mixtures. Just like all chromatographic procedures have a stationary phase and a mobile phase, TLC also posses the same. 

Preparation of Mobile and Stationary Phase for TLC:
In case of TLC, the stationary phase normally consists of a finely divided adsorbent, generally, silica (SiO2) or alumina (Al2O3) powder. This adsorbent is used in the form of a thin layer (around 0.25mm) on a supporting material (which is usually the glass plate, polyester or aluminium sheet). Also, a binder like gypsum (chemically, calcium sulphate) is mixed into the stationary phase so that it sticks better to the supporting material.  At times, the stationary phase also often contains a substance which fluoresces under short UV (254nm) which is used in later stage for visualizing. 
The mobile phase consists of an organic solvent or a mixture of solvents.

The sample containing the mixture of components/compounds is applied onto the stationary phase i.e.; on the adsorbent as a small spot. This TLC plate is then placed vertically in a closed container with its edge having the sample spot placing down touching the solvent as shown in the diagram. The solvent which is in the bottom of the container travels up the layer of the adsorbent by capillary action. It passes over the sample spot and it continues to move up and moves the compounds of the mixture up the plate at different rates, thereby resulting in the separation of the compounds. If the compound is soluble in the mobile phase (solvent), it will travel up the TLC plate. If the compound is not soluble in the mobile phase, it gets adsorbed on the stationary phase meaning, it will stick to it and will not move much far on the TLC plate.

Steps of TLC
There are three major steps in TLC which is used for the separation of components as:
1. Spotting the sample
2. Development
3. Visualization

1. Spotting the sample: The sample, containing a mixture of compounds, which is to be analysed is dissolved in a suitable volatile solvent to produce a very dilute solution. A pencil line can be drawn near the bottom (around 1 cm from bottom) of the plate where the sample will be placed. (Remember, not to use the pen while marking, as the dye from the ink will interfere with the separation of the components and give erroneous results). Using a micro-pipette, a small amount of the sample is then transferred at the point marked on the TLC plate. The spotting solvent evaporates quickly leaving behind a small spot.

2. Development: Once the spot is dried, the TLC plate is placed vertically into an air-tight jar containing the solvent, to a depth less than 1 cm (so as, not to soak the sample spot point). The solvent now travels up the plate and tries to take the components of the sample along with it. As the solvent travels up, over the spot, equilibrium is set up as the solvent and TLC plate (silica coating) competes for the components of the sample. The silica gel binds to the solute and the solvent tries to dissolve it away from the sample spot as the solvent travels up the plate. The result as how much the components will travel will depend on the polarity of the three components – polarity of the plate, the polarity of the development solvent and the polarity of the components in the spotting sample.
If the solvent is polar enough, the components of the sample will move some distance with the solvent from its original location. Different components will have different polarities and hence will move at different distances from original spotting location and will appear as different spots.  When the solvent has traveled almost to the top of the plate, the plate is removed and solvent front is marked with the pencil and solvent is allowed to evaporate.
Lets try to understand how the development is taking place at the molecular level. 
Development at molecular level:
To understand the development, here, we will assume that the plate is coated with silica gel. The three-dimensional structure of silica gel is somewhat like this:

It has a network of thousands of alternating silicon and oxygen bonds with hydroxide (O-H) groups on the outside surface. So, this structure is highly polar and is capable of hydrogen bonding.
As the solvent travels up over the spot, there is a balance of inter-molecular forces which helps in determining the position of equilibrium and thus the ability of the solvent to move the components of the sample up the plate. For example, if the sample has two components, then the more polar will tend to stick more tightly to the plate as the O-H bonds of the silica gel, being polar will bind with the polar components of the sample more tightly while the less polar will move along with the solvent.

3. Visualization: The visualization of colored compounds is easy as the colored spots can be directly seen on the silica get plate. However, most of the compounds are colorless, and so a visualization method is required to see the separated components. Iodine is the most common stain which leads to staining of the components as brown colored spots. Other stains used for visualizing separated components are as follows:

Analysis of TLC plate:
The components visible as separated spots can be analyzed by comparing the distance traveled by the component to that of the reference values. As mentioned, once the solvent reaches the top, the plate is removed and the distance traveled by the solvent and the distance traveled by the component is measured. Then, Rf value is calculated as shown for one component in the above diagram "steps of chromatography"
Advantages of TLC:
TLC is simple to use and is inexpensive.
The solvents for the TLC plate can easily be changed and it is possible to use different solvents.
Purity of the compound can be ensured by using TLC. The purity can be checked by using UV-light.

Disadvantages of TLC:
They do not have long stationary phases. Thus, the length of separation is limited compared to other chromatographic technique. It operates in a somewhat open system, so factors like moisture and temperature can interfere with the separation process thereby affecting the chromatogram.