Throughout the development of Science, chromatography is a common method used for analysis and research. Especially in pharmaceutical companies, chromatography is used for separation, identification, and purification of components of mixtures, both qualitatively and quantitatively. It is prominently used in Drug Purification and to trace contaminants.
Chromatography is a biophysical technique to separate different components in a mixture. The molecules travel through the medium, each component may be separated on basis of molecular weight, polarity, size, shape, and binding capacity. Technically there are different types of chromatography namely, Column chromatography, Ion-exchange chromatography, Gel-permeation (molecular sieve) chromatography, Affinity chromatography, Paper chromatography, Thin Layer Chromatography, Gas chromatography, Dye-ligand chromatography, Hydrophobic interaction chromatography, Pseudo affinity chromatography, and High-pressure liquid chromatography (HPLC). On the whole, all types of Chromatographic separation work on the same principle. Among these Column chromatography and Thin Layer Chromatography are performed widely on a commercial scale.
What is Column Chromatography?
Column Chromatography is a method used to isolate single compounds to form a mixture. It is primarily based on differential adsorption to the adsorbent material (e.g. Silica gel and Aluminum oxide). The mixture is stacked into the column filled with adsorbent and a blend of solvents is allowed to pass. This process is also known as a solid-liquid technique.
Before Separation in column chromatography, it is preferred to be optimized first in Thin Layer Chromatography. This can distinctly show the results of how it will perform in column chromatography.
Phases of the Thin Layer Chromatography
There are primarily two phases of Thin Layer Chromatographic separation.
- Stationary phase
The stationary phase is the solid phase. The most common solids used in this phase are silica gel and Aluminium Oxide. Silica gel is more advantageous because of multiple reasons.
- Mobile phase
The mobile phase is liquid. Varied blends of solvents that moves through the column or Thin Layer Chromatography plate. It acts as a developing agent and introduces the mixture into the column.
Now, the Success of the chromatography is based on the selection of the stationary phase. Few things to keep in mind before the preparation of the stationary phase are the Number of components to be separated, affinity difference between the components, and quantity of the adsorbent used.
Silica Gel preparation for TLC
Thin Layer chromatography plate preparation is done by selecting the optimal size of plates for the application of silica gel. The slurry is prepared by mixing silica gel and a little amount of gypsum and water. The slurry is applied on the plate by pouring, dipping, spreading, or spraying method. It is dried and activated by heating in an oven for 30 minutes at 110°C.
Generally, Silica gel is preferred commonly for the stationary phase in Column and Thin Layer Chromatography. Silica gel is an amorphous form of silica (SiO2). It is very porous. It is formed by aggregation of colloidal silica or by polymerization of silicic acid. The aggregate acts as an excellent adsorbent with pore size in the range of 6- 25 nm and great surface area which makes ideal for Thin Layer Chromatography plate preparation.
Here listed are few advantages of silica gel for chromatographic separation.
Advantage of Silica gel
- Silica powder mesh sizes are available a whole range of sizes (30 - 600).
- It offers a good stream rate and best adsorption capacity than any other counterparts.
- It gives a quality result at a cost-effective rate.
- It isolates the components in the blend in a distinct way.
- The Batch and cluster reports are reproducible; hence it is reliable for research and commercial scale.
- Low cost of instrumentation.
Silica Gel has a novel place in the pharmaceutical and food industries. May it is a simple separation of biomolecules or drug purification, Silica gel is an integral component in chromatographic separation.
Sorbead India is one of the leading manufacturers of high-quality silica gel desiccants. The quality of our desiccants, the wide range of availability, and the ease of product access has helped us remain the trusted partner in all kinds of desiccant requirements for our clients across the globe. For more information, feel free to reach us.
Wherever mixture of compounds are involved, Chromatography emerge as the most widely used methods of separation which involves the use of chromatographic adsorbents as stationary phase through which mobile phase is run to cause the separation of the compounds present in the mixture. In all types of chromatographic techniques, what remains of prominent importance to carry out the process successfully and to obtain desired results is the selection of adsorbent “the stationary phase”. The most common adsorbents used in chromatography are Alumina (Al2O3) and Silica gel (SiO2), they also happen to be the widely used adsorbents as they work well across a wide range of compounds.
Stationary phase is generally referred to as chromatographic media in modern chromatographic separations. It is either packed in a chromatography column or added to the separation apparatus. The media is always chosen depending upon the type of molecule that is to be separated.
Silica gel as an adsorbent showcases an excellent affinity for water, hence it can be used for carrying out the isolation and purification of a variety of molecules. Silica gel also comes with high adsorbing capacity making it a good choice by plenty of industries. Swambe Chemicals manufactures the premium quality of silica gel that offers the lowest moisture content, tightest particle size distribution, and minimal presence of impurities.
Another major feature of silica gel which should be considered before choosing it for chromatographic purifications is its mesh size. Swambe chemicals supplies silica gel ranging from mesh sizes 35 to 800. The choice of mesh size differs on various parameters such as the complexity of the mixture of compounds, the amount of water present in it and chemical nature of the compound in question. Generally, adsorbent with higher mesh size is used for those chromatographic methods in which solvent uses positive air pressure to run through the column and smaller mesh size is used for those, where solvent runs down the column on its own due to gravity. These are the features that signify the quality of silica gel and assure one of the desired results including reproducibility.
Aluminium oxide as an adsorbent provides reproducible results hence they are widely used for carrying out the separation and purification of dyes, organic solvents, alkaloids, steroids, lipids, amino acids, hormones and vitamins.
Swambe Chemicals manufactures a range of tailor-made chromatographic products to address the needs of different industries. It manufactures Silica Gel for column chromatography, Silica Gel Powder for column chromatography, and Aluminium Oxide for column chromatography. Since chromatographic purifications have gained much importance owing to its accurate results, column chromatography has become the first choice for industries like Life Sciences, API Production plants, Herbal Extraction plants, Dyes Manufacturing plants, DNA fingerprinting, Nutraceuticals, and Plant-based Nutraceuticals. Swambe Chemicals is the chromatographic adsorbent supplier and manufacturer of variants of chromatographic adsorbents such as Silica gel and Alumina to address the needs of various industries.
Column Chromatography is one of the widely used techniques for carrying out the purification of drug molecules. This method allows the separation of targeted molecules from the mixture of compounds. As this method provides high accuracy, it is also used for the analysis of complex organic mixtures. Chromatography is a physical method of separation which helps in separating the components based on their distribution between two phases: stationary phase and mobile phase. Stationary phase can be in the form of porous bed, bulk liquid or thin layer of the adsorbent and mobile phase is the fluid that runs through the stationary phase.
Two most commonly used stationary phase are Silica gel (SiO2) and Alumina (Al2O3) which is packed in a column and mobile phase passes through this column. Silica gel is the most widely used adsorbent for carrying out phytochemical investigations. It is observed that most of the phytochemical separations use silica gel as the adsorbent and the choice of solvents for mobile phase is dependent on the polarity of molecules that are to be separated from the mixture of compounds.
Swambe Chemicals manufactures a range of tailor made chromatographic products to address the needs of different industries. It manufactures Silica Gel for column chromatography, Silica Gel Powder for column chromatography, and Aluminium Oxide for column chromatography. Since chromatographic purifications have gained much importance owing to its accurate results, column chromatography has become the first choice for industries like Life Sciences, API Production plants, Herbal Extraction plants, Dyes Manufacturing plants, DNA fingerprinting, Nutraceuticals and Plant-based Nutraceuticals.
Most commonly used Adsorbents for chromatographic purifications are silica gel and aluminium oxide. Aluminium oxide as an adsorbent provides reproducible results hence they are widely used for carrying out isolation and purification of antibiotics, for carrying separations through preparative column chromatography and, separation and purification of dyes, organic solvents, alkaloids, steroids, lipids, amino acids, hormones and vitamins.
Silica gel as an adsorbent showcases an excellent affinity for water, hence it can be used for carrying out the isolation and purification of a variety of molecules. Silica gel also comes with high adsorbing capacity making it a good choice by plenty of industries. Swambe Chemicals manufactures the premium quality of silica gel that offers lowest moisture content, tightest particle size distribution, and minimal impurities. Another major feature of silica gel which should be considered before it being used for chromatographic purifications is its mesh size. Swambe Chemicals manufactures silica gel in varying mesh sizes that ranges between 35 and 800. These are the defining features when it comes to quantifying the quality of silica gel for analyzing the desired reproducibility.
Chromatography is essentially a physical method of separation in which the components of a mixture are separated by their distribution between two phases; one of these phases in the form of a porous bed, bulk liquid, layer or film is called stationary phase while the other is a fluid that flows through the stationary phase is called as mobile phase.
Chromatography works because of the differences in the properties of molecules in materials, their mobility, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase.
- Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on the surface solid support”.
- Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
- Separated molecules
Column Chromatography is one of the most important and widely used techniques for the separation and analysis of complex organic mixtures. It consists of two phases: the mobile phase and the stationary phase. The stationary phase is solid and the mobile phase is liquid. The compound mixture moves along with the mobile phase as it flows through the stationary phase and separation of molecules occurs depending on their affinity with the stationary phase.
High-quality silica gel offers the lowest moisture content, tightest particle size distribution, minimal impurities. This results in greater reproducibility of the separation process. Another parameter that affects the successful completion of the process is mesh size of silica gel used as the stationary phase.
Mesh size of silica gel is given by the value which refers to the number of holes in the mesh that is used to sieve the absorbent. Thus higher mesh values such as "silica gel 230–400" have more holes per unit area and correspondingly smaller particles than "silica gel 60". Typically, 70–230 mesh size of silica gel is used for gravity column chromatography and 230–400 mesh for flash column chromatography.
Scale-up Chromatography: The scale-up of a chromatographic separation is in principle a simple procedure since the process parameters are scalable in a linear fashion and the process is scaled by increasing the column diameter while keeping the bed height, the velocity, and the volumes of the different phases (measured in column volumes) constant.
Silica gel adsorbents are widely accepted as one of the best adsorbents that are used in column chromatography. One of the main benefits is that it has a huge affinity for absorption; besides, it is very easily available commercially in many different forms and sizes and there is a lot of research and information that is provided by the manufacturers on its numerous uses, especially in chromatography.
The Many Advantages of Using Silica Gel in Chromatography
One of the important criteria for opting for silica gel in chromatography is that it remains neutral and does not interact with any of the substances that are passed through it. It maintains its own stable structure throughout a process. Importantly, it can be regenerated or reused many times, cutting the costs of purification considerably. All it needs is to be heated to a specific temperature (about 150 * C) when it releases all the substances absorbed by it.
Silica gel is a polar adsorbent and being slightly acidic in nature, it has a powerful capacity to absorb basic contents that may be present in the material that needs separation or purification. It is also well known for its role in reversed-phase partition chromatography. It has a wide range of applications that include the purification of lipids, steroids, amino acids, several dyes and alkaloids, and many other pharmaceutical processes.
Another important aspect of using silica gel in column chromatography is that it is possible to get the exact required size of the particle size for a specific procedure.
Chromatography has spread to various other dimensions and modifications for the separation and analysis of complex mixtures, and in most cases, silica gel is the most used. Over the years, chromatography has evolved tremendously in the process of separation and identification of a substance contained in a mixture. Silica gel is acknowledged as one of the most versatile and effective agents that can be used in chromatography.
One of the other advantages is that the separation of compounds, that are produced either synthetically or that occur in their normal state in nature, can be effectively achieved by the use of silica gel. The basic process involves that the mixture that needs purification is passed over a silica gel chromatography that separates the different components.
Analytical Separation and isolation of chemical compounds from a mixture is called Chromatography and purification of such compounds in larger quantities is achieved prominently using Column Chromatography. Column Chromatography is a separation technique that is applied in various fields among which Pharmacy is the one, where preparation and dispensing of drugs are performed based on this technique, to ensure the effectiveness of the drug. In Column Chromatography, there are different types of columns such as Gravity Columns, Flash Columns, Low and Medium pressure Columns, Vacuum Columns and High-Pressure Columns. But the similar point in all these columns is that all require an Absorbent, which acts as a Stationary Phase through which samples containing different compounds flow down at differing rates. On the other hand, the Adsorbent is applied to the Column in 2 ways namely-Slurry packing method and Dry packing Method. The slurry packing method is often used for macro scale separations and Dry packing method for microscale separations.
Silica Gel for Column Chromatography
Silica gel is often used for macro scale separations using the Slurry pack method where the adsorbent is mixed with a small amount of Non-polar solvent until a consistent paste is formed and then poured in the column. In the dry pack method, a steady stream of the absorbent is poured through a funnel into the column by tapping the sides of the column so that packing remains even in the column. Silica gel for Column Chromatography is considered as the best absorbent in column packing for both dry pack and Slurry pack method, as it gets eventually distributed and forms a packed Stationary phase. The sample is then mixed with a polar solvent and added to the column where the component molecules which are to be separated are either adsorbed on the particle surface or adsorbed into the particle pores. Thus, the different components of a sample get split into separate bands in the column and get eluted at different rates.
The elution is at different rates because of the particle size and pores of Silica gel absorbent which are available in different sizes called Silica Gel mesh with small size and more pores which act as a Sieve. These Silica gel mesh sizes chromatography is typically used in Column as it retains the compounds for a longer time thereby enhancing the purification of the product. Silica gel for Column chromatography mesh size refers to the number of holes present in the mesh, per unit area of the absorbent to be used in the Column. Silica Gel 100, 200 mesh Merck are employed in Gravity Columns, whereas higher mesh Merck is used in Flash Columns.
Gravity Columns are mainly preferred in Gel Permeation Chromatography and are followed by Adsorption chromatography technique containing Silica gel 100-200 mesh adsorbent gives the higher efficiency of separations, improved reproducibility and require low solvent consumption. Whereas, Flash Columns containing Silica gel (250-400 mesh) with small particle size, restricts the flow of solvent, therefore pressurized gas is applied to drive the solvent resulting in high resolution. The amount of Silica gel to be used for these Columns mainly depends on the amount of the sample. It means nearly 30 to 100 grams of Silica gel is required for easy separations but for difficult separations, more than 30:1 ratio of silica gel is required. With the increase in the quantity of Silica gel, the time consumption for the separation also increases.
Column Chromatography Applications
Purification of the Reaction Mixture in Chemical Synthesis such as –
Through Flash column chromatography using Silica gel 60(220-240 mesh), β-Ketoester and Desired alcohol were purified, Purification of Polychlorinated biphenyls (PCB) from traces of sulfur using Silica gel 60(70-230 mesh) and Silver nitrate impregnated silica gel. Silica gel (100-200 mesh) for Organ chlorine Pesticides, Phenols, and Polynuclear aromatic compounds
Purification of Biomolecules such as Proteins for Pharmaceutical Research :
Synthesis of Pramlintide which is an analog of Amylin, a peptide hormone, for treating type 1 and type 2 Diabetics is also purified using Silica gel. Purification of bioactive glycolipids, showing antiviral activity towards HSV-1(Herpes Virus)is also performed by Column chromatography using Silica gel as absorbent.
Purification of Nucleic acids in vivo and vitro cultures is also done using Silica gel as it absorbs the Nucleic acid.
Analysis of Environmental Samples :
Purification of target chemical from co-extracted, non-chemical samples from the environment is done using Gas chromatography, in which the environmental samples are passed through an inert gas where the target chemical gets desorbed from aqueous phase to gas phase and gets separated from a stream of gas by absorption on the Silica gel mesh. On heating it the trapped chemical gets released and can be analyzed
Extraction of pesticides from solid food samples of animal origin containing lipids, waxes, and pigments as contaminants are performed using Silica gel adsorption chromatography in atmospheric conditions.
The discovery of the Chromatography technique has made a revolution in the field of analytical laboratories for the separation of the chemicals after synthesis. Since the separation process for each compound is different based on their physical interactions with the solute and the absorbent, different methods were discovered for efficient and reliable analysis. For any chromatographic technique, the absorbent remains the same i.e. Silica Gel adsorbent, because it is low acidic and available indefinite particle size ranging from 60-800 mesh size. There are many Chromatography techniques theoretically, but only a few can be operated practically in the laboratories.Among these techniques Column, chromatographic techniques are mostly preferred as it is low in cost as well as requires minimum instrumentation.
In turn-based on the flow of the solvent down the column through the absorbent, Column chromatography is classified into Gravity Column Chromatography and Flash Chromatography. The flow of the solvent in the column is based on the definite pore size of the Silica Gel, which allows smaller molecules into its matrix and thereby excludes the larger molecules which flow down and get eluted faster.
Silica Gel Mesh for Chromatography
If the solvent flows down the column passing the Silica gel absorbent of definite particle size by gravity or fissure, it is referred to as Gravity Column Chromatography. But if the solvent flows down the column passing the Silica gel absorbent of definite particle size by positive air pressure, it is referred to as Flash Chromatography. Therefore the absorbent used in both these techniques is the same, the Silica Gel Mesh. There are many products of Silica Gel mesh available depending on the mesh size such as Silica Gel 60-120 mesh, Silica Gel 60-200 mesh, Silica Gel 70-230 mesh, Silica Gel 200-400 mesh, and Silica Gel 400-800 mesh. Among this Silica Gel 70-230 mesh is used for Gravity Column Chromatography and Silica Gel 200-400 mesh is used in Flash Chromatography. These all are available in a thin white free-flowing powder form with a density of 0.75 gm per ml. Best separation is achieved in Ordinary compounds by Silica Gel Absorbent. Apart from Gravity Column Chromatography and Flash Chromatography, there is another technique in Chromatography named Reversed-phase Chromatography, which is employed for the solvent which is more polar than the Absorbent. Silica gel was the first polymer used formerly in Reversed-phase Chromatography for the purification of small organic molecules but later for the purification of synthesized Peptides. The separation is achieved by the partitioning mechanism between the Stationary phase and the mobile phase in Reversed-phase Chromatography, in which the stationary phase is Silica Gel.
Overall, in each and every Chromatographic techniques the main absorbent is the Silica gel and is considered as the best absorbent in the analytical field, as it is porous, insoluble and its hydrophobic interaction with the solvent makes the elution process progress at a faster rate as the polar components get eluted first. These Silica gels exist in different particle sizes, because of which they are used in many Column Chromatography Techniques and Sorbead being the best Silica gel 200-400 supplier and Silica gel 70-230 mesh supplier.
This entry was posted in Chemicals, Chromatography and tagged silica gel 200-400 mesh supplier, Silica Gel 400-800 Mesh, silica gel 60-120 mesh, Silica Gel 60-200 Mesh, Silica Gel 70-230 Mesh supplier, silica gel adsorbents.
Flash Chromatography – An Introduction
Flash chromatography is also known as “medium pressure chromatography”. This form of chromatography works by air pressure driving the mixture, which is to be separated down the vertical glass column. This type of chromatography is ideal for separations that need to take place in a fast-paced manner.
How Flash Chromatography Works
In the traditional column chromatography, the mixture that has to be separated is placed on the top of the column using a stationary phase like Silica Gel. Then it moves through a mixture of solvents with some help from gravity. The different components will be separated at various levels. While the separation and purification are done in a proper manner, the biggest disadvantage of using column chromatography is its slow rate. This is where the advantages of using flash chromatography come into the forefront. Under flash chromatography, the solvent is forced down the vertical column with the help of positive air pressure.
When air pressure is applied, the rate at which the solvent is flowing increases, thus bringing down to a large extent, the amount of time required to separate any given mixture. If one is to use flash chromatography then the separation process can be completed right under 10-15 minutes. The time saved while purifying the sample is the main reason why companies opt for the flash chromatography. You get the same result as column chromatography with flash chromatography but in lesser time. So one can save time and most importantly, money.
Across the world, flash chromatography is fast replacing the slow-moving gravity-dependent chromatography. If one is to look closely at how flash chromatography functions, you can see that this form of chromatography makes use of slightly smaller Silica Gel molecules. This would mostly be in the range of 250-400 mesh. Another main point about flash chromatography is that because of the restricted movement of the solvent caused by the small gel particles, pressurized gas is generally used to drive the solvent through the stationary or solid-phase column.
Silica Gel in Flash Chromatography
As mentioned earlier, Silica Gel is one of the desiccants used as the stationary phase in the flash chromatography process. It is widely used because of the two most important characteristics – its numerous interconnected pores and large surface area. These two features help the Silica Gel stationary phase absorb the different components of the mixture in a fast and timely manner.
The amount of silica gel adsorbents being used in flash chromatography depends on the Rf difference of the compound, which are up for separation and on the amount of sample used. For easier separations, ratios closer to 30:1 are highly effective. While for difficult separations, more silica gel adsorbents are used by companies. However, by using more silica gel, one also extends the length of time required for the chromatography process to be completed. Silica Gel for Flash chromatography is available with Silica Gel 70-230 Mesh suppliers like Sorbead India.
Understanding Thin layer Chromatography
Thin-layer chromatography is a widely used form of chromatography. This type of chromatography mainly finds use in the separation process of non-volatile mixtures. Thin-layer chromatography consists of both a stationary phase as well as a mobile phase. The stationary phase is usually an adsorbent like Silica Gel, Aluminium Oxide or Cellulose. The thin-layer chromatography process works by the molecular adsorbent like Silica Gel absorbing the different components of the mixture, which has to be separated, at different levels. The adsorbents for thin-layer chromatography are always decided in such a manner that they will quicken as well as improve the separation as well as the subsequent purification process.
Thin-layer chromatography works by applying the sample on the plate. After that, the solvent or mobile phase is passed through the plate using capillary action. Different constituents move apart at different levels of the thin-layer chromatography plate and thus the separation process is complete.
Role of Silica Gel in Thin-layer Chromatography
As mentioned earlier, Silica Gel has a very important role to play in the thin-layer chromatography process. The reason why out of all the adsorbents for thin-layer chromatography, Silica Gel is the most preferred is very clear. Silica Gel has calcium sulfate as a binder and this makes it the best adsorbent available in the market. Silica Gel is well-known for its top-notch separation qualities and works as an excellent separation tool in the thin-layer chromatography process. When the Silica Gel adsorbent is placed on top of the thin-layer chromatography plate then it will speed up the separation and subsequent purification process.
With Silica Gel being the stationary phase, it will require a mobile phase, which has completely different properties from the stationary phase. As Silica Gel is a highly polar substance, then for the mobile phase it is recommended one use non-polar substances like heptane. The mobile phase is usually made as a mixture so changes can be made accordingly.
As an adsorbent for thin-layer chromatography, Silica Gel has produced good results in the separation and purification of a wide range of substances like alkaloids, amino acids, oils and fats, vitamins, plant pigments, drugs, and sugars.
This entry was posted in Chromatography, Science and tagged adsorbents for thin-layer chromatography, chromatography adsorbents material supplier, silica gel supplier for thin-layer chromatography, thin-layer chromatography.
Chromatography – A Brief Introduction
Chromatography is a method widely used in the separation of different compounds, solid and liquid. This method is very helpful in segregating and purifying various components of mixtures, which when separated can be analyzed individually. The chromatography process is a very simple method of separating compounds using a solid as well as the liquid phase. Silica gel, alumina oxide is mainly used as the solid phase, which adsorbs all the unwanted particles as well as all kinds of impurities. When the mixture passes through the mobile phase, then only the required compounds move forward while the unwanted components of the main mixture are left behind.
Silica Gel for Column Chromatography:
The main characteristics of Silica Gel are that it is a good adsorbent and has a very large surface area. These two characteristics help it in adsorbing small impurities and allow only the desired molecules to pass through. This property works towards making Silica Gel a highly popular desiccant and adsorbent. Silica Gel finds use in almost all kinds of chromatography. These include analytical, preparative, process and gravity chromatography. In all these forms of chromatography, the solid or stationary phase (Silica Gel) effectively adsorbs the different components at various levels to achieve higher levels of purification. Described below are some of the chromatography methods, which make effective use of Silica Gel as an adsorbent.
Analytical chromatography, as the name suggests, is conducted to have a detailed analysis of the various components of mixtures. Analytical chromatography is mostly done when only small quantities of substances have to be separated for further analysis. This form of chromatography also finds use in measuring the different ratios of analytes present in the concerned mixtures.
Preparative chromatography is another important form of chromatography wherein the different components of any given mixture are mainly separated for more advanced purposes. After being separated using the Silica gel solid phase and the liquid phase, the various components are subjected to advanced analysis. Preparative chromatography also finds use in many purification processes as well.
Process chromatography is mainly used in industrial and business establishments for the separation and purification of mixtures on a large scale and advanced basis.
The column chromatography method, which makes use of the gravitational force instead of air pressure to make the mixture move down the vertical chromatography Column, is known as gravity chromatography.
What is Thin Layer Chromatography?
Similar to Silica Gel Chromatography, Thin Layer Chromatography is another procedure used to separate individual components from a mixture. Thin Layer Chromatography also consists of a solid or stationary phase like a silica gel plate and a mobile phase. The Silica Gel acts as an adsorbent for thin-layer chromatography. The mobile portion can be a solvent or a solvent mixture, which moves upwards using capillary action. The components that are to be separated are adsorbed at different levels and finally collected at the lower end of the Thin Layer Chromatography or TLC plate.
How the TLC Procedure Works
In Thin Layer Chromatography, first, a small quantity of the solvent mixture is inserted into a developing vertical glass column. The solvent mixture must be to such an extent that the mobile portion sits separate from the samples placed at the bottom of the plate. In the next step, place the silica gel plate in the developing vertical column and cover it completely. After some time, the capillary action will take place thus starting the separation process. For better results, one can also place filter paper soaked in the solvent inside the vertical column. Both these steps, the closure of the column and the placement of the filter paper, go a long way in securing the solvent’s presence in the column.
One has to next wait for the solvent to rise to 1 centimeter below the top of the TLC plate and when this happens, remove it from the glass column. The plate will be dry in some time and if the chromatography process is a success, spots will appear on it. The separated components are then taken away for different uses.
Applications of TLC
Thin Layer Chromatography has many uses in varied fields like pharmaceuticals, traditional medicine, insecticides, pesticides among others. The uses are explained in detail below:
- In the Pharmaceutical Industry: TLC is widely utilized in the pharmaceutical industry. It is mainly used as a means to find out the low levels of impurities present in medicines. In this process, the medicinal substance is administered on the TLC plate and after chromatography, secondary spots appear on the plate. These spots are compared for parameters like size and intensity with earlier spots of smaller loadings of known impurities. The latter underwent a similar chromatography process earlier.
- In Traditional Medicine: Traditional medicines make extensive use of plant extracts. In order to know what constitutes these plant extracts, the process of TLC proves very useful. When the samples of a plant extract are separated using TLC, a clearer understanding of the components of the plant can be arrived at. This procedure is also used to study plants of the same species but grown in different environmental conditions.
- In Pesticides: In the pesticide industry, TLC has great importance. It is used to separately identify and determine the different components of high-grade pesticides. This method is also used to segregate metabolites or enzyme inhibitors.
After the process of chemical synthesis, the compounds of interest need to be purified in a proper manner. Some of the popular purification techniques are Crystallization, Separation, Filtration, Liquid-Liquid cleanup and Distillation. All these techniques are vastly used in the laboratories. But there is another purification method that is used for separating two almost the same natural substances. This process is known as Flash Chromatography.
It is a much quicker form of column chromatography. This process is based on pre-packed columns through which the solvent is pumped at a high flow rate. This process is much more effective and also a very economical alternative of column chromatography. It was the year 1978 when this process was first mentioned in the Journal of Organic Chemistry as a substitute for the conventional column chromatography process.
In order to purify complex organic compounds, this process is used since it is a much quicker alternative of column chromatography and also very inexpensive as well. W. C. Stills of Columbia University was the man who first introduced this process to the world in 1978. Now it is one of the most widely used purification processes applied in the labs.
This well-known process uses a column, plastic column basically that is filled with a solid support mostly silica gel 230-400 mesh or silica gel 200-400 mesh. The sample that is to be separated is normally placed on the top of the support (silica gel). The rest of the body of the column is covered with an isocratic solvent. This very solvent makes the sample that is placed on the top of the support run through the column and get separated. In the beginning, air pressure was used for the sample to run through the column. Now pumps are used for this process. This technique is used to separate a few mg to hundreds of grams of sample.
So, what are the differences between flash chromatography and column chromatography? It has been mentioned earlier that the former process is much faster and less time consuming than the latter. Unlike column chromatography, flash columns can be used multiple times which definitely makes an impact on the overall cost of the process. Other than that, the labor cost required is also very less compared to the conventional column chromatography process. All these factors make this process a much widely used in industrial areas.
When it comes to the application of this process, there are a number of applications that include drug discovery, cleaning up of samples, purifying natural products and so many more. There is a relation between flash chromatography and TLC and these two processes are often used in a combined manner prior to and even after the separation using the flash process.
In Industries the synthesis of bio-molecules requires a special technique that does not denature the characteristics of the bio-molecules during Separation and Purification. Column Chromatography is one such technique having different modes that are used on a large scale at the Industrial level as it is bio-compatible and requires a relatively short time for separation and purification. The Column Chromatography Principle is mainly based on size, Charge, and Hydrophobicity of the sample which in turn lead to various Chromatographic techniques such as-Size exclusion chromatography or +++Gel permeation chromatography, Adsorption chromatography, Ion exchange chromatography, and Affinity Chromatography. Each technique is based on the biological interaction between the sample bio-molecules and the packing material such as Alumina or Silica gel.
Types of chromatography and their Applications
- Size exclusion chromatography:
It is known as molecular sieve chromatography separates analyses according to their size as they pass on through the stationary phase namely Silica which is porous and acts as a hydrophilic surface.
- In Adsorption chromatography:
The stationary phase is considered as the Adsorbent which is used in separating Non-volatile mixtures. In this method, the Adsorbent used can be Silica, Alumina or Cellulose.
- Ion exchange chromatography:
It is used in the separation of polar molecules and ions such as proteins, amino acids containing both positive and negative charged chemical groups. In this method, the stationary phase used is the slurry of either Silica or Alumina.
- Affinity chromatography:
Affinity chromatography is used for the separation of recombinant proteins and enzymes and here the stationary phase act as a Support medium. Chromatography techniques are widely used-In Biochemical research applications to separate and identify the compound of biological origin, In Petroleum industries to control the production of saturated carbons, In Chemical Industries to purify the chemicals, In Sugar factories to purify sugar from Molasses.
For any technique, if the packing material employed is Silica gel than the Silica gel column chromatography Procedure mainly depends on packing the column without cracks and air bubbles as it leads to poor separation and purification. Whereas if the packing material used in these techniques is Alumina Column Methanol, it contains Methanol, which is considered as a volatile eluting solvent and therefore used in Alumina Column Chromatography. Apart, from methanol to improve the effectiveness of purification and to adjust the alkalinity it is treated with either dilute acid or base considering the components nature of the Column. After adjusting the alkalinity of Alumina it is characterized as Neutral, Acidic and Basic Alumina. Among this Neutral Alumina is used in chromatographic cleanup procedures and also for the separation of Aliphatic, Aromatic and Polar fractions. Purification of free base Porphyrins and Metalloporphyrins is also performed using Neutral Alumina. When it is used as a stationary phase in the Column, for the preparative separation of the mixture having basic properties, it does not require a mobile phase modifier.
Uses of Chromatography in Medicine
Lack of efficiency of medicine results in lethal accidents and has become a major concern. Therefore Chromatography opts as a detection tool in identifying and separating the desired medicine from the contaminants. The following are some of the medicines that are developed using Chromatography techniques
Synthesis of Potential Anti Pertussis medicine involves the Chromatography technique.
Separation of enantiomers that are useful in drug discovery is progressed through different Chromatography techniques.
Chromatography is also applied in the batch Fermentation process to determine the improvement of the process during antibiotic production.
Separation often different H1-Antihistamines which were structurally related was achieved by Chromatography, impregnating Silica gel with transition metal ions.
Purification of Proteins and Peptide hormones is attained either by Ion-exchange Chromatography or Gel Permeation Chromatography.
By Size exclusion chromatography using Alumina nanoparticles size separation of DNA molecules was achieved.
Application of Chromatography in Chemistry
In chemistry one of the major aspects is to analyze the residue of all major classes which include Food, water, and air. It is mainly essential to emphasize the quality and safety of these classes or else lead to an adverse effect on human health. Therefore, this analysis of residues is accomplished by different Chromatography Techniques. The following are some of the techniques used in chemistry to separate and analyze the components which are essential in day to day life.
Separation of Protein and small Oligonucleotides made easy by using Ion-exchange chromatography.
It is also used for the separation of Aldehydes, Ketones, Quinones, Esters, Lactones, and Glucosides.
The environmental agency made Chromatography a method to test the quality of the Drinking water.
It is also used to monitor air quality.
It is employed in the separation of organic as well as Inorganic compounds.
It is an indispensable method to study the heavy oils containing the hydrocarbon group.
Analysis of pesticide residues on fruits and vegetables is also carried by this analytical technique.
In today’s arena, Herbal medicine or medicine extracted from medicinal plants have found their way in ailing many diseases when compared with Synthetic drugs. In recent years their utilization has increased rapidly and accounted for a significant percentage in the medicine market. Even the WHO (World Health Organization) supports the use of medicinal plants and introduced measures to ensure the quality of Herbal medicines using modern techniques such as Chromatography and for manufacturing practices.
Isolation of Natural Products From Plants
Natural products are the components or substances that are produced from natural sources such as plants. These products which occur naturally have an impact on human life and therefore, used as Medicines, dietary supplements, and cosmetics.
The components of plants that have medicinal value and healing properties are called as Bioactive compounds and they undergo a series of the process called Extraction, Isolation, and Characterization. Column Chromatography procedure for plant extracts is one such technique for isolating and identifying the Bioactive compound using a stationary phase such as Silica gel or Alumina. Before Isolation and extraction, identification of the bioactive compound is done based on a method called Fingerprint which defines the character and gives complete information about a specific plant. Fingerprint analysis gives an accurate identification with similar peaks and is helpful in determining the intrinsic quality of the Bioactive compound.
Isolation of Bioactive Compounds From Plants.
The Development of analytical techniques has to lead to a qualitative as well as quantitative analysis of Isolation of bioactive compounds from Plants which are herbs with a complicated system of mixtures formed through various pathways. Chromatography is one such analytical technique used for the authentication and identification of bioactive compounds in a plant and is readily available. High-Performance Liquid Chromatography (HPLC) is the most extensively used application in Pharmaceutical industries for the isolation and purification of bioactive compounds from medicinal plants. This application requires larger stainless steel Columns packed with normal Phase Silica which not only isolates but also gives information about the sample containing new synthetic products. This is a very helpful technique for the pharmaceutical industry to introduce a new product into the market within less timeframe.
Many more feasible techniques are emerging to control the quality of the bioactive compounds by using Solid phases such as Silica Gel with less particle size and shorter Column size. Ultra High-Performance Chromatography(UHPLC) is such a technique that is gaining momentum because of its enhanced selectivity and decreased analysis time, in isolating bioactive compounds from medicinal plants.
Some of the Bioactive Compounds and Medicinal Plants
Atropine is a Bioactive compound isolated from Atropa Belladonna by HPLC technique for preparing analgesic and sleeping potions.
Isolation of extraction of Quinidine quinine, a bioactive compound from Cortex cinchona by HPLC method for treating Arthritis.
Isolation and extraction of Ephedrine Norephedrine bioactive compound from Ephedra Sinica by Liquid Chromatography Technique for treating Asthma, Narcolepsy, and Obesity.
Isolation and extraction of Quercetin Kaempferol bioactive compound from Ginko Biloba by Column Chromatography on Silica gel or HPLC for cancer treatment.
Isolation and extraction of Rhein Emodin bioactive compound from Rheum Palmatum by HPLC analysis which is used for Inflammatory diseases and hepatitis.
Chromatographic analyses of natural volatiles from Tasmanian blue gum and fennel for resolving breathing problems
Through Ion exchange chromatography, Hydrophilic and Lipophilic extraction of bioactive compound from Conabiola for treating skin problems
Isolation of Secondary Metabolites From Plants
The metabolites which are essential for plant growth and development are considered as Primary metabolites, whereas the metabolites produced by the plant which are highly diverse in the structure are considered as Secondary Metabolites. These metabolites are bio-synthetic in origin and are classified into four different categories.
- Alkaloids: These metabolites contain Nitrogen group and are extracted and isolated by HPLC Column Chromatography.
- Phenylpropanoids: These metabolites contain aromatic compounds and are isolated by various Chromatographic techniques.
- Polyketides: These metabolites are biosynthetic in origin, and their isolation and detection are carried by Liquid Chromatography or HPLC.
- Terpenoids: These metabolites are the largest class of natural products in plants, and is extracted and isolated by Silica gel Column Chromatography.
Pigment Extraction From Plants
The pigment is a molecule that has an ability to absorb the color and reflect the color which is abundantly found in Plant Tissues. Pigments obtained from plants are of utmost importance in day to day life as they are used -for coloring foods, in medicines, in plastics, in fabrics, in cosmetics. Many types of research have also proved that consumption of diet which is rich in plant pigment slows cellular aging. They have features such as resistant to high heat, chemical agents and different weather conditions that made them popular and lead to extensive usage. Therefore Pigment extraction from plants can be accomplished through Analytical techniques among which Chromatography is considered as the best technique and the pigments which are of interest in Pharmaceutical and Nutritional research are said to be –Anthocyanins, Beta-carotene, Curcumin, Lutein, Lycopene, Zeaxanthin.
Column Chromatography is said to be a method that is utilized to isolate segments inside any substance. It is fundamentally a biological term with additionally figures the synthetic synthesis of any substance. This separation process consists of two phases that are a mobile and a stationary phase. The basic working is done on the stationary phase when adsorption is done and then the mobile phase is passed into it.
Know More in Details:
This is the procedure which helps in the separation of the constituent mixtures. It is isolated based on its solubility and absorption rate. Chromatography is of two types - Column Chromatography and adsorption chromatography. Absorption chromatography is also divided into Thin Layer Chromatography and Column Chromatography. Column chromatography is also divided into two types. They are paper and gas chromatography.
Among all the methods, mostly people prefer Column Chromatography as the most appropriate one and commonly used for the separation of mixed organic substances. In the case of silica gel column chromatography, it is available in many types of required mesh sizes – 60-120 mesh, 30-60 mesh, 70-230 mesh, 100-200 mesh, 400-600 mesh and many more. These adsorbents are used in different mesh sizes which are selected based on the water content present in it as well as on the complexity of the mixture and molecules of the desired compound. It is usually used for the purification of organic compounds like steroids, alkaloid and pharmaceutical work as well.
In Silica Gel Column Chromatography, there are mainly four types of methods used, which is analytical chromatography which is used to analyze that how much percentage of mixture is to be purified. This is a very fast and cost-effective method for separation. Another is preparative chromatography, which is used to optimize the opportunities of the process and also improves the process of separation.
Next, process chromatography where the separation is taken up to a biologics level and separation of proteins, viruses, hormones, and anti-bodies is now possible. The last is gravity chromatography which is said to be the manual process. The mixture is allowed to move around by gravitational force.