Revision KNEC Question and Answer on Separation, Extraction and Purification

Vacuum filtration is typically a fast and efficient way of filtering. Gravity filtration relies on downwark sucking through filter paper by gravity,therefore the gravitation pull exerted by the gravity may not be sufficient enough to cause diltration of relatively courseparticles to pass through the filter paper.

In a vacuum filtration, the solution to be filtered is drawn through the filter paper by applying a vacuum to a filter flask with a side arm adaptor (also known as a Buchner flask). This exert relative high sucking presure which causes sucking or filtration of substances through filter paper to be more faster and more efficient

Describe any two reasons for using an oil bath instead of water bath in determining the melting point of a substance

Oil baths are much like water baths, but use silicone or mineral oils is used in order to enable temperatures hotter than the boiling point of water.

Silicone oil baths can be heated to 250^oC, while mineral oil baths can be heated to 300^oC. Mineral oil is composed of mixtures of long-chain alkanes, and so is combustible.

Silicon oil provide a relatively uniform and sustained heat distribution that can be maintained over a longer period that water bath

Explain the importance of using the most minimum amounts of solvent to disolve solid during crystalization process

If too much solvent is added, the solution will not be saturated upon cooling and no crystals will form. Therefore, the solvent is heated to its boiling point and then slowly added to completely dissolve the solute

State two reasons for slow cooling of hot filtrate during recrystalization

The slower the cooling process, the less chance of trapping impurities in the developing crystal lattice.
Slow cooling often leads to purer crystals.
1. Outline the procedure used in vacuum distillation

Vacuum distillation is a special method of separating compounds at pressure lower than the standard atmospheric pressure.

Under this condition, the compounds boil below their normal boiling temperature. Hence, vacuum distillation is best suited for separation of compounds with higher boiling points (more than 392°F), which tend to decompose at their boiling temperature.

Vacuum distillation can be conducted without heating the mixture, as is usually followed in other distillation types.

For the separation of some aromatic compounds, vacuum distillation is used along with steam distillation. When vacuum distillation is combined with fractional distillation method, components of a mixture get separated very easily.

Describe two conditions that a compound must meet to be successfully separated by steam distillation

Steam distillation is used for the purification of mixtures in which the components are heat sensitive; for example, organic compounds.

In this process, steam is introduced to the apparatus and the temperature of the compounds are depressed, by vaporizing them at lower temperature. This way, the heat sensitive compounds are separated before decomposition.

The vapors are collected and condensed in the same way as other distillation types. The resultant liquid consists of two part, water and compound, which is then purified by using simple distillation..

The process requires some initial training and skill to operate the equipment. It also requires periodic maintenance. Steam distillation is widely used for large-scale separation of essential oils, fats, waxes, and perfumes.

Two solvents are equally suitable for recrystalization process. State two preferences in choosing one of them

The solvent should have a high temperature coefficient.

The solvent must not dissolve the compound at low temperatures, but must dissolve the compound at high temperatures. The solute must dissolve in order to rid its lattice of impurities, but must not remain dissolved at room temperature.

b.) using a solvent that dissolves impurities readily or not at all. If the solvent dissolves the impurities readily (even at room temperature) then the impurities will not become trapped in the developing crystal lattice, but will remain dissolved in the solvent. If the impurities do not dissolve (even at elevated temperatures) then they can be easily removed by gravity filtration.

c.) The solvent should not react with the solute. As mentioned earlier, recrystallization does not chemically alter a molecule. No chemical bonds must be broken in the solute molecule. The crystal lattice is dissolved at elevated temperatures, but this only involves overcoming the intermolecular attractive forces.

d.) The solvent should be nonflammable, inexpensive and volatile. Solvents with low boiling points (i.e., volatile) can be easily removed from the resultant crystals by simply allowing the solvent to evaporate.

State three advantages of Soxhlet extraction as a method of separating mixtures

Only minimum amount of solvent is used
Instead of many portions of warm solvent being passed through the sample, just one batch of solvent is recycled.
The non-soluble portion of the extracted solid remains in the thimble, and is easier to discard
After extraction the solvent is removed, typically by means of a rotary evaporator, yielding the extracted compound.

Describe how a Soxhlet apparatus works

The solvent is heated to reflux. The solvent vapour travels up a distillation arm, and floods into the chamber housing the thimble of solid.

The condenser ensures that any solvent vapour cools, and drips back down into the chamber housing the solid material.

The chamber containing the solid material slowly fills with warm solvent. Some of the desired compound dissolves in the warm solvent.

When the Soxhlet chamber is almost full, the chamber is emptied by the siphon.

The solvent is returned to the distillation flask. The thimble ensures that the rapid motion of the solvent does not transport any solid material to the still pot.

This cycle may be allowed to repeat many times, over hours or days. During each cycle, a portion of the non-volatile compound dissolves in the solvent.

After many cycles the desired compound is concentrated in the distillation flask.

After extraction the solvent is removed, typically by means of a rotary evaporator, yielding the extracted compound.

The non-soluble portion of the extracted solid remains in the thimble, and is usually discarded

Explain how fruit juice can be extracted using soxhlet extractor.

The Soxhlet extractor is used for liquid-solid extractions when the compound to be extracted has limited solubility in the chosen solvent and the impurities are insoluble.

During the extraction, solvent vapour will flow up the distillation path, into the main chamber and up into the condenser where it will condense and drip down. The solvent will fill the main chamber, dissolving some of the desired compound from the solid sample. Once the chamber is almost full, it is emptied by the siphon, returning the solvent to the round bottom flask to begin the process again. Each time the extraction is repeated, more of the desired compound is dissolved, leaving the insoluble impurities in the thimble.

Unlike a traditional extraction method, a small amount of solvent is reused to perform an extraction many times. This means that much less solvent is used in a Soxhlet extraction, making it more time and cost effective. Also, the Soxhlet extractor can run continuously without any further operation, making it an excellent choice for extracting compounds over hours or even days.

The Soxhlet extractor is used whenever exhaustive extractions are needed, particularly in the oil and food industries. It is also widely used for extracting bioactive compounds from natural resources which is crucial in environmental analysis of soils and wastes. The Soxhlet extractor will run continuously once set up correctly

How to use a soxhlet extractor

Load the sample material containing the desired compound into the thimble ( the fruit sample in this case)
Place the thimble into the main chamber of the Soxhlet extractor
Add the chosen solvent to a round bottom flask and place onto a heating mantle
Attach the Soxhlet extractor above the round bottom flask
Attach a reflux condenser above the extractor, with cold water entering at the bottom and exiting above

Now the apparatus is set up, heat the solvent to reflux and leave to extract for the required amount of time

State one disadvantage of Soxhlet extraction

Because of its small size, it only allow small scale laboratory extraction of samples but cannot be used in large scale industrial processing

Draw a labelled diagram of Soxhlet extraction apparatus

List the criteria for determining purity of a substance

(a) Physical Comparison with Pure Standard

Physical comparison has been used in understanding the purity of a particular compound. It is the simplest method of checking purity as it only involves comparing a particular chemical with a certified pure sample. There is an argument against the use of physical comparison, but it is obvious that this method can show significant differences between products. Physical comparison is very useful in revealing large impurities such as colored impurities and dirt. Smell tests will also reveal the differences between the chemical undergoing tests and the actual and pure state of a particular compound. However, it’s important to note that only non-toxic chemicals can be tested through touch, taste, and smell.

(b) Boiling and Melting Point Determination

It is obvious that the physical properties of a chemical can be used to indicate whether it is pure or not. Most of the chemical products have defined boiling and melting points. Pure chemicals are known to boil and melt at specific temperatures, and that is documented. However, if the melting point of a particular chemical is lowered, there is a great sense that the chemical is not pure. The boiling point will be higher than normal. This is a simple method of determining the purity of a product and is highly applied in various chemical manufacturing plants.

(c) Colorimetric Methods

The colorimetric method is another widely used strategy of determining the purity of a chemical compound. Biochemically, some chemical compounds change into a particular color when they are exposed to a certain chemical. This is widely known, documented, and used as a standard way of determining the purity of various products. One of the major applications of the colorimetric method is in determining the presence of specific illegal drugs such as cocaine and heroin. Colorimetric purity testing is highly preferred because it does not only indicate the purity of a chemical but also the percentage purity.

(d) Analytical Testing

Although other methods of testing purity are highly appropriate and advanced, analytical testing is the most accurate and highly preferred method of testing chemical purity. Analytical testing involves a standard procedure that is used in testing various chemicals. These procedures are applied in drug and chemical industries, and they indicate the presence and the number of impurities. Some of the analytical purity testing methods include titration, infrared spectroscopy, paper chromatography, and optical rotation, among others.

These are some of the strategies used in testing the presence of impurities in chemical compounds. However, the method that one applies depends on the resources available, the magnitude of the entire process, and the chemical under consideration. However, any method is likely to indicate the presence of impurities in a particular chemical.

Define chromatography It is technique for rapid and efficient separation of components of a mixture and purification of compounds. It is based on differential migration of the various components of a mixture through a stationary phase under the influence of a moving phase.

Explain the basis (principle) of chromatographic process ?
It is based on the differential migration of the individual components of a mixture through a – stationary phase under the influence of a moving phase.

Explain the following terms as used in chromatography

Eluent: The mobile phase is used to perform a separation.
Elution” The process of passing mobile phase through the column to transport solutes down a column called
Solvent: A the term sometimes referring to the liquid stationary phase in partition chromatography.
Absorption: The process of retention in which the solute partitions into a liquid-like coating.
Adsorbent: Packing used in adsorption chromatography. Silica gel and alumina are the most frequently used adsorbents in high-performance liquid chromatography (HPLC)
Analyst: The compound of interest to be analyzed by injection into and elution from an HPLC Column
Asymmetry: Factor describing the shape of a chromatographic peak. The chromatographic theory assumes a Gaussian shape and that peaks are symmetrical. A quantitative measure is the peak asymmetry factor, which is the ratio of the distance from the peak apex to the backside of the chromatography curve over the distance from the peak apex to the front side of the chromatography curve at 10 % of the peak height.
Buffer: A solution that maintains constant PH by resisting changes in PH from dilution o addition or small amounts of acids and bases.
Chromatography: Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (Stationary Phase) while the other (The mobile phase ) moves in a definite direction.
Chromatogram: A plot of detector signal output or sample concentration versus time or elution volume during the chromatographic process.
Adsorbate
Resolution
Location
Development

define solvent

A solvent’s molecules pull apart the solute’s molecules, and eventually the solute’s molecules become evenly distributed throughout the solvent. This homogeneous mixture cannot be separated physically.

That means heat or another chemical process must be applied to the solution in order to separate the solvent from the solute.

Popular solvents include water and organic compounds like benzene, tetrachloroethylene and turpentine.

Explain solvent extraction

Solvent extraction is a process in which compounds are separated based on their relative solubilities. Solvent extraction is the act of removing something or separating it.

This must be done through force and this process occurs over the course of two different immiscible phases.

This treatment method involves using a solvent – a fluid that has the ability to dissolve another substance. Solvent extraction does not destroy a compound. It instead separates it, a process that can provide several valuable benefits depending on the industry in which the process is used.

Immiscible liquids (liquids that do not dissolve in one another) form layers when put together. This is because each liquid differs in polarity, or orientations. The order of the phases, whether a particular liquid is on top or on the bottom, is determined by its density.

For example, if you use ether and water during the extraction process, water has a higher density than ether. Therefore, water will be the bottom phase.

Your method for extraction may vary depending on your equipment, but in its most simplistic form, extractions often use some kind of separatory funnel. The liquid mixture that is to be separated is first added to the funnel. When the two extraction solvents are then added to the funnel, they should separate into two phases.

After forcibly shaking the funnel and allowing it to stand for several minutes, the two layers can be collected. In the end, the target molecule for extraction will be located in one of the solvents.

State four requirements of an extracting solvent

Immiscible pair of solvents: water and low polarity organic solvents

The extracting solvent must be immiscible with the solution to be extracted.

Good solubility of the target compound

The compound(s) to be extracted, which are present in a solution, should also be soluble in the extracting solvent.

Poor solubility of impurities

Major impurities (e.g., from a reaction) should not be soluble in the extracting solvent.

Volatility of the extraction solvent

The extracting solvent should be sufficiently volatile so that it can be removed easily from the extracted material by distillation.

Toxicity and safety properties of the extraction solvent

It is usually desirable if the solvent is non-toxic and not flammable.

Unfortunately, few solvents are known to meet both criteria…

11.Define the term partition coefficient

A partition coefficient is the ratio of the concentration of a substance in one medium or phase (C1) to the concentration in a second phase (C2) when the two concentrations are at equilibrium; that is, partition coefficient = (C1/C2)equil. The units of C1 and C2 may be different.

11..Briefly describe the technique of partition extraction

This method is often called “extraction.” Extraction means drawing a compound out of a mixture using a solvent. Solvent partitioning is more specific. It means compounds have a “choice” of two solvents that they can dissolve in. Some compounds dissolve in one solvent. Some compounds dissolve in the other solvent. That way the compounds in the mixture become separated into two groups.

Solvent partitioning depends on solubility. It depends on the solubility in two different solvents, though. It depends on an equilibrium: does the compound dissolve more in solvent A, or solvent B?

Solvent partitioning requires two solvents that are not miscible in each other. Usually one of the solvents is water. The other solvent is a liquid that does not dissolve very well in water, such as diethyl ether (this is the most common type of ether, and it is often called simply “ether”). If you look closely at a mixture of ether and water, you will see two layers because the two compounds do not dissolve very well in each other.

It’s important that the two solvents are immiscible, because then it is easy to separate them from each other. The top liquid can be drawn off with a pipet, or the bottom layer can be drained out via a stopcock. The compounds that dissolved in the ether have thus been separated from the water-soluble compounds. Because ether evaporates very easily, the compounds that dissolved in the ether can also be separated from the ether (see “distillation”). As a result, purer compounds can be obtained.

The formula for the partition coefficient is,

Kd=Cs

Where,

In the stationary phase, the concentration of the solute is C_s,

In the mobile phase, the concentration of the solute is C_m.

12..The concentration of solute in the mobile phase is 5.00M and 7.00M in the stationary phase. Calculate the partition coefficient K_d between water and hexane.

Given

C_s = 7.00M

C_m = 5.00M

Apply the numerics in the corresponding formula given below

Kd=Cs

Kd=7.00

5.00

Kd = 1.4

Using an illustrate , explain the multistage of solvent extraction.

Multistage Liquid Liquid Extraction (LLE) is a process where extraction steps are repeated in order to increase the recovery of a product. This process is required when, due to a small partition coefficient, the recovery in a single extraction step is insufficient.

Draw a labeled diagram of gravity filtration techniques

Put folded filter paper cone into glass filter funnel inserted into neck of conical (Erlenmeyer) flask.
Add liquid suspension from the glass beaker with a spout into the funnel with paper filter.
Collect filtrate in the conical flask and solid on the paper filter in the funnel.

Explain the type of solvents are generally employed in chromatography ?
Generally solvents having low viscosities are employed in chromatography. This is due to the fact that the rate of flow of a solvent varies inversely as its viscosity.
Give four chromatographic techniques.

Paper chromatography, column chromatography, thin layer chromatography, gas chromatography.

16.Explain the mobile and stationary phases in paper chromatography
In paper chromatography, the mobile phase and the stationary phase are two important components that play a crucial role in separating the components of a mixture.

The mobile phase refers to the solvent or mixture of solvents that moves through the paper during the chromatographic process. It carries the mixture being analyzed and allows it to travel along the paper. The choice of mobile phase depends on the specific application and the nature of the compounds being separated. Common mobile phases include water, alcohol, or a combination of both. The mobile phase should have the ability to dissolve the components of the mixture to facilitate their movement.

The stationary phase, on the other hand, refers to the paper used in chromatography, which acts as a support medium. It is typically a special type of paper called chromatography paper, which is composed of cellulose fibers. The stationary phase is called “stationary” because it remains fixed or stationary while the mobile phase moves through it. The stationary phase provides a surface for the components of the mixture to interact with as the mobile phase passes through. The cellulose fibers in the paper have a high affinity for certain compounds, causing them to adhere or “stick” to the paper to varying degrees. This differential adhesion leads to the separation of the components of the mixture as they migrate at different rates along the paper.

As the mobile phase moves through the stationary phase, it carries the components of the mixture along with it. However, the extent to which each component interacts with the stationary phase determines its rate of movement. Components that have a stronger affinity for the stationary phase will move more slowly, while those with a weaker affinity will move faster. This differential movement based on the affinity for the stationary phase leads to the separation of the mixture into distinct bands or spots on the paper, which can be visually observed or further analyzed.

In summary, the mobile phase is the solvent or mixture of solvents that carries the mixture being analyzed, while the stationary phase is the chromatography paper that provides a surface for the components of the mixture to interact with, leading to their separation.

Explain the term developing in chromatography

In chromatography, the term “developing” refers to the process of allowing the mobile phase to move through the stationary phase, resulting in the separation and visualization of the components of a mixture.

Once the stationary phase (such as chromatography paper) is prepared with the sample mixture applied to it, the development process begins. The mobile phase, which is the solvent or mixture of solvents, is carefully added to the bottom of the chromatography paper or applied to the starting point. The mobile phase then moves through the paper due to capillary action or by applying external pressure.
As the mobile phase progresses through the stationary phase, it carries the mixture’s components along with it. The individual components interact with the stationary phase to different extents based on their affinity for the stationary phase. Components that have a higher affinity for the stationary phase will be retained or slowed down, causing them to move more slowly along the paper. Conversely, components with a lower affinity for the stationary phase will be less retained and will move more quickly with the mobile phase.
As the mobile phase continues to move, the different components of the mixture separate into distinct bands or spots along the chromatography paper. This separation occurs due to the differential partitioning between the mobile phase and the stationary phase. The extent of separation depends on the composition of the mobile phase, the characteristics of the stationary phase, and the chemical properties of the components being analyzed.
Once the developing process is complete, the chromatography paper is removed from the mobile phase and allowed to dry. The separated components on the paper can then be visualized by various techniques such as ultraviolet (UV) light, staining, or spraying with a suitable reagent. The resulting chromatogram, which shows the separated bands or spots, can be analyzed and used for identification or quantification of the components in the mixture.

In summary, developing in chromatography refers to the process of allowing the mobile phase to move through the stationary phase, leading to the separation and visualization of the components of a mixture on the chromatography paper.

Explain how does the liquid rise through the filter paper
The rise of liquid through filter paper occurs due to a phenomenon called capillary action, also known as capillarity. Capillary action is the ability of a liquid to flow upward in narrow spaces against the force of gravity.

Filter paper is made up of cellulose fibers, which have a high affinity for water molecules. When a drop of liquid, such as water or a solvent, is placed on the filter paper, the paper’s fibers act as small capillary tubes. The following steps explain the process of liquid rising through filter paper:
Adhesion: The liquid droplet adheres to the cellulose fibers of the filter paper. Adhesion refers to the attractive forces between the liquid molecules and the solid surface of the filter paper. The water molecules in the droplet form hydrogen bonds with the cellulose fibers, causing them to stick to the paper.
Cohesion: Cohesion refers to the intermolecular forces between the liquid molecules themselves. In this case, the water molecules in the droplet are attracted to each other, creating a cohesive force. Cohesion allows the liquid to form a continuous column or meniscus.
Capillary action: Capillary action occurs due to the combined effect of adhesion and cohesion. The cohesive forces within the liquid pull adjacent liquid molecules upward. At the same time, the adhesive forces between the liquid and the cellulose fibers pull the liquid upward along the paper. This creates a capillary rise of the liquid through the small channels or capillaries formed by the cellulose fibers.

The rise of the liquid through the filter paper continues until the forces of adhesion, cohesion, and gravity reach an equilibrium. The liquid will rise higher in filter paper with smaller pores or finer fibers, as they provide more capillary channels for the liquid to travel.
Capillary action is also responsible for the spreading of ink in paper, the absorption of water by plants’ roots, and various other phenomena involving liquids in narrow spaces.

In summary, the rise of liquid through filter paper is facilitated by capillary action, which is the result of the combined forces of adhesion and cohesion. The liquid adheres to the filter paper’s cellulose fibers and is pulled upward through small capillary channels, defying the force of gravity.

Explain the term R_fvalue

The term “Rf value” stands for “retention factor value” and is a measurement used in chromatography to assess the relative migration or movement of a particular component within a mixture. It is a dimensionless quantity that helps in the identification and comparison of different compounds.

The Rf value is calculated by dividing the distance traveled by the component by the distance traveled by the solvent front (the leading edge of the mobile phase) on the chromatography medium, such as a chromatography paper or a thin-layer chromatography (TLC) plate. Mathematically, it is expressed as:
Rf = (Distance traveled by the component) / (Distance traveled by the solvent front)
The Rf value can range from 0 to 1. A value of 0 indicates that the component did not move at all and remained at the starting point, while a value of 1 indicates that the component moved the same distance as the solvent front.
The Rf value is influenced by several factors, including the nature of the mobile phase, the characteristics of the stationary phase, and the specific interactions between the components of the mixture and the stationary phase. Each component within the mixture may have a unique Rf value under specific chromatographic conditions, allowing for their differentiation and identification.
The Rf value serves as a characteristic property of a compound under a given set of experimental conditions. It can be used to compare the behavior of different compounds, aid in compound identification, and verify the purity of a substance. However, it’s important to note that the Rf value is not a definitive or absolute measurement and can vary depending on the specific experimental setup and conditions used.

In summary, the Rf value in chromatography is a ratio that represents the distance traveled by a component relative to the distance traveled by the solvent front. It is a useful parameter for comparing and identifying compounds within a mixture.

State factors that the R_fvalue of a compound depend

The Rf (retention factor) value of a compound in chromatography depends on various factors, including:

Nature of the compound: The chemical and physical properties of the compound, such as its polarity, molecular size, and solubility, can influence its interaction with the stationary phase. Compounds with higher polarity tend to have lower Rf values, as they interact more strongly with the stationary phase and move at a slower rate. Conversely, non-polar compounds typically have higher Rf values, as they interact less with the stationary phase and move more rapidly.
Mobile phase composition: The choice of solvent or mixture of solvents used as the mobile phase affects the Rf value. The polarity of the mobile phase can impact the degree of interaction between the compound and the stationary phase. If the mobile phase is more polar than the compound, it may compete with the compound for interactions with the stationary phase, resulting in a lower Rf value. Conversely, if the mobile phase is less polar than the compound, it may have minimal interactions with the stationary phase, leading to a higher Rf value.
Stationary phase characteristics: The nature and composition of the stationary phase can significantly impact the Rf value. Different stationary phases have varying affinities for different compounds based on their chemical properties. For example, a polar stationary phase will interact more strongly with polar compounds, resulting in lower Rf values, while a non-polar stationary phase will have less interaction, leading to higher Rf values for non-polar compounds.
Temperature: The temperature at which the chromatographic separation is performed can influence the Rf value. Changes in temperature can affect the solubility and mobility of compounds, thereby impacting their interactions with the stationary phase and the mobile phase.

Chromatography technique: Different chromatographic techniques, such as paper chromatography, thin-layer chromatography (TLC), or high-performance liquid chromatography (HPLC), can yield different Rf values. The specific experimental conditions and parameters used in each technique, including the type of stationary phase, mobile phase, and separation time, can affect the Rf value.

It’s important to note that the Rf value is not an absolute value and can vary under different experimental conditions. Therefore, it is crucial to establish consistent and controlled experimental conditions when comparing Rf values for different compounds.

Give the biochemical uses of chromatography.

Chromatography plays a crucial role in various biochemical applications and is widely used in many areas of biochemistry. Some of the important biochemical uses of chromatography include:

Separation and purification of biomolecules: Chromatography is extensively used to separate and purify biomolecules such as proteins, nucleic acids, carbohydrates, lipids, and metabolites.
Protein and enzyme analysis: Chromatography is widely used in protein and enzyme analysis. It enables the separation, purification, and quantification of proteins and enzymes from complex mixtures.
Drug discovery and pharmaceutical analysis: Chromatography plays a crucial role in drug discovery and pharmaceutical analysis. It is used to separate and analyze drug compounds, metabolites, and impurities. Chromatographic techniques are employed to determine the purity, potency, and stability of pharmaceutical products and to characterize drug compounds during the drug development process.
Environmental analysis: Chromatography is utilized in environmental analysis to detect and quantify pollutants, contaminants, and toxic compounds in air, water, soil, and biological samples.
Forensic analysis: Chromatography is employed in forensic analysis to analyze biological samples, such as blood, urine, and DNA samples, for the detection and identification of drugs, toxins, and other substances. Chromatographic techniques aid in the separation, identification, and quantification of compounds of forensic interest.

Define the term loading (or spotting)

In chromatography, the term “loading” or “spotting” refers to the process of applying a sample or a mixture onto the stationary phase of the chromatography medium. It involves depositing a small amount of the sample onto a specific location on the chromatography plate or paper.
Loading is typically performed using a micropipette or a capillary tube. The sample is carefully placed as a small spot or line at a specific starting point on the chromatography medium. The size and concentration of the spot can vary depending on the desired separation and detection sensitivity.
The loading process is a critical step in chromatography as it ensures that the sample is evenly and accurately distributed onto the stationary phase. A properly loaded sample allows for efficient separation and analysis of the components within the mixture during the chromatographic process.
After loading, the chromatography medium is allowed to dry to ensure that the sample spot is immobilized and does not spread during the subsequent development phase. The mobile phase is then introduced, and as it moves through the chromatography medium, the components of the sample begin to separate based on their interactions with the stationary phase.
Loading or spotting is an essential technique in various chromatographic methods, including paper chromatography, thin-layer chromatography (TLC), and column chromatography. It facilitates the separation, identification, and quantification of components within a mixture and allows for subsequent analysis and characterization of the sample.

State essential characteristics of the substance used as a developer

The substance used as a developer in chromatography should possess certain essential characteristics to ensure effective separation and visualization of the components. The key characteristics of a developer substance are as follows:

Solubility: The developer substance should be readily soluble in the chosen mobile phase or solvent system. This ensures that it can easily dissolve and interact with the components being separated, facilitating their movement along the stationary phase.
Selectivity: The developer substance should have selectivity towards the components of interest. It should interact differently with each component, leading to distinct separation and visualization. Different developer substances may be required for specific analytes or classes of compounds to achieve optimal separation and detection.
Stability: The developer substance should be stable under the chromatographic conditions, including the mobile phase composition and temperature. It should not chemically react or degrade during the separation process, as this could lead to unwanted changes or interference in the chromatogram.
Visibility: The developer substance should provide a suitable visual contrast to the components being separated, enabling their visualization and analysis. It should generate clear and distinct color reactions, fluorescence, or other detectable signals to facilitate identification and quantification.
Compatibility: The developer substance should be compatible with the chromatography medium being used, such as paper or TLC plates. It should not interfere with the separation process or affect the interaction between the components and the stationary phase.
Sensitivity: The developer substance should be sensitive enough to detect and visualize the separated components, even at low concentrations. It should provide a clear indication of the presence and relative abundance of the components for accurate analysis.
Non-toxicity: The developer substance should be non-toxic and safe to handle. It should not pose any health risks to the user or interfere with the biological or chemical properties of the analytes being separated.

The specific requirements for a developer substance may vary depending on the chromatographic technique used and the nature of the components being analyzed. Therefore, careful consideration of these essential characteristics is important in selecting an appropriate developer substance for a given chromatographic application.

What are the main differences between high performance liquid chromatography and gas chromatography?
- In HPLC the mobile phase is a liquid whereas in Gas Chromatography the mobile phase or carrier is a gas.
- HPLC is useful for analysis of samples which are liable to decompose at higher temperatures. GC involves high temperatures so compounds are stable at such temperatures.
- Gas Chromatography is applied for analysis of volatile compounds whereas non volatile compounds can be easily analyzed on HPLC
- Gas Chromatography cannot be used for analysis of high molecular weight molecules whereas HPLC has applications for separation and identification of very high molecular weight compounds
- HPLC requires higher operating pressures than GC because liquids require higher pressures than gases for transport through the system
- HPLC columns are short and wide in comparison to GC columns

What Is The Basic Principle Of Paper Chromatography?

The basic principle of paper chromatography is based on the differential migration of components in a mixture through a porous medium, such as chromatography paper, due to their differential affinity for the mobile phase and the stationary phase.The principle of paper chromatography is based on the fact that different components of a mixture have different affinities for the stationary phase and the mobile phase, leading to their differential migration and separation on the chromatography paper. This technique is widely used for qualitative analysis, identification of unknown compounds, and the separation of complex mixtures in various fields, including chemistry, biochemistry, forensics, and environmental sciences.

Here are the key steps and principles involved in paper chromatography:

Preparation of the stationary phase: A strip or sheet of chromatography paper is used as the stationary phase. The paper is typically made of cellulose fibers, which create capillary channels through which the mobile phase can flow. The paper is cut into a suitable size and prepared by marking a baseline near one end.
Application of the sample: A small spot or line of the sample mixture is applied to the baseline using a capillary tube or micropipette. The sample should be applied accurately and in a concentrated form.
Development: The paper containing the applied sample is placed in a suitable container with a shallow layer of the mobile phase. The mobile phase is a solvent or a mixture of solvents that can migrate through the paper by capillary action. As the mobile phase moves through the paper, it carries the components of the sample mixture along with it.
Separation: As the mobile phase moves through the paper, the different components of the sample mixture interact with the stationary phase (the cellulose fibers) to varying degrees. Components that have a higher affinity for the stationary phase will be retained or slowed down, causing them to move more slowly along the paper. Conversely, components with a lower affinity for the stationary phase will be less retained and will move more quickly with the mobile phase. This differential migration results in the separation of the components into distinct bands or spots along the paper.
Visualization: Once the mobile phase has migrated to a desired distance or has reached the top of the paper, the paper is removed from the container. The separated components on the paper are then visualized by various techniques, such as UV light, staining, or spraying with a suitable reagent. This allows for the identification and analysis of the separated components.

Why The Developing Solvent Mixture Is Prepared Fresh Before Use?

The developing solvent mixture in chromatography is often prepared fresh before use to ensure the accuracy and reliability of the separation process. Here are a few reasons why a fresh solvent mixture is preferred:

Consistency of composition: The composition of the developing solvent mixture is critical for achieving reproducible and reliable results in chromatography. Over time, the solvent mixture may undergo changes due to evaporation, degradation, or contamination, leading to variations in the solvent composition. By preparing the solvent mixture fresh before each use, one can ensure that the solvent composition is consistent and accurately matches the intended formulation, allowing for consistent and reliable separations.
Solvent strength: The effectiveness of the solvent in eluting the components from the stationary phase depends on its strength and composition. Solvents can lose their strength over time due to evaporation or chemical degradation. Freshly prepared solvent mixtures ensure that the solvent is at its optimum strength, maximizing the separation efficiency and preventing potential loss or smearing of the components.
Contamination prevention: During storage, the solvent mixture may be susceptible to contamination from impurities present in the air, dust particles, or even residues from previous experiments. Contaminants can interfere with the separation process, leading to distorted or inaccurate results. By preparing the solvent mixture fresh, one can minimize the risk of contamination and maintain the purity of the solvent, ensuring reliable and uncontaminated separations.
Stability: Some solvent mixtures may exhibit instability over time, leading to changes in their properties, such as pH, polarity, or selectivity. These changes can significantly impact the separation process and the behavior of the components being analyzed. By preparing the solvent mixture fresh, one can ensure the stability of the solvent and maintain its desired properties, allowing for consistent and predictable separations.

Overall, preparing the developing solvent mixture fresh before each use helps to maintain the consistency, reliability, and accuracy of the chromatographic separation. It minimizes variations in solvent composition, prevents contamination, maximizes solvent strength, and ensures stability, all of which contribute to obtaining reliable and reproducible chromatographic results.

27.Why Is It Necessary To Cover The Developing Chamber During The Paper Development?

This is essential as the environment inside the chamber should remain saturated with the solvent vapour. Development times can vary from about an hour to several hours and a saturated environment prevents losses due to evaporation.

What Are The Common Techniques Used For visualizing spots or Detecting Colourless Spots?

There are several common techniques used for visualizing spots or detecting colorless spots in chromatography. These techniques are employed to aid in the identification and analysis of separated components. Some of the commonly used techniques include:

UV (Ultraviolet) Visualization: Ultraviolet light can be used to visualize spots on chromatography plates or paper. Many compounds absorb UV light and become visible under UV lamps or UV cabinets. The chromatogram is observed under UV light, and the spots appear as dark spots against a fluorescent background. UV visualization is particularly useful for compounds that exhibit natural fluorescence or can be treated with fluorescent reagents.
Iodine Staining: Iodine staining is a common technique used to visualize spots on chromatography plates or paper. The chromatogram is exposed to iodine vapor, which reacts with various compounds, forming visible brown or purple spots. This technique is especially effective for visualizing non-polar or weakly polar compounds.
Ninhydrin Spray: Ninhydrin is a reagent that reacts with amino acids, amines, and proteins, forming colored products. The chromatogram is sprayed with a ninhydrin solution, and when heated, the spots containing these compounds turn purple or blue. This technique is useful for visualizing amino acids in chromatography, such as in amino acid analysis or peptide mapping.
Silver Nitrate Staining: Silver nitrate staining is used to visualize spots containing organic compounds, including lipids and fatty acids. The chromatogram is treated with a silver nitrate solution, and upon exposure to light, the spots containing organic compounds turn brown or black. This technique is commonly used in lipid analysis and lipid profiling.
Dye Sprays or Reagents: Various dye sprays or reagents can be used to visualize spots on chromatography plates or paper. These dyes react with specific classes of compounds, forming colored products. For example, anisaldehyde spray can be used to visualize spots containing carbohydrates, while Fast Blue B salt can be used to visualize spots containing lipids.
Chemiluminescence: Chemiluminescent compounds can be used to detect colorless spots in chromatography. The chromatogram is treated with a chemiluminescent reagent, and the spots containing specific compounds react with the reagent, emitting light. The emitted light can be captured using specialized equipment for detection.

It’s important to note that the choice of visualization technique depends on the nature of the compounds being separated and detected, as well as the specific requirements of the analysis. Each technique has its advantages and limitations, and the appropriate technique should be selected based on the specific application and the desired detection sensitivity.

Why should the samples have reasonable solubility which is neither too high or too low in the developing solvent mixture?

The samples should have a medium solubility in the developing solvent mixture.Too high a solubility will lead to transfer of the component alongwith the solvent front and on the other hand if the solubility is too low the component will not be carried by the solvent mixture and will remain close to the initial applied spot. In either case the resolution of the mixture components will be low. Thus reasonably good resolution can be obtained for medium solubility of compounds in the solvent mixture.

What Information You Get From The Retardation Factor Value?

Retardation factor Rf is a measure of the separation of a particular component. It is expressed as

Rf = distance moved by the component spot/ distance moved by solvent front

Rf is a unit less quantity and lies between 0and 1.A value of 0 indicates no separation has taken place and 1 represents that the component has moved entire length alongwith the solvent front. In case two spots have same value of Rf it indicates that they are not resolved. At least a difference of 0.05 is necessary to discern the separation between two spots.

Explain Various Paper Chromatography Techniques?

Paper chromatography separations are classified in accordance with the direction of flow of mobile phase along the filter paper.

Ascending paper chromatography – the carrier liquid moves from bottom upwards.
Descending paper chromatography – the carrier liquid trough is on top and mobile phase moves downward on the filter paper.
Ascending – descending paper chromatography – The paper is rolled downward over the rod at the top. On reaching the top in ascending mode it starts downward movement in the next phase.

State the Essential Criteria For Selection Of Suitable Solvents For Paper Chromatography?

Solvents are selected on the basis of solubility of the sample components. In general it is advisable to keep in mind:
Solvents are not toxic or carcinogenic.
Solvent constituents of mixture should not react with any of the sample constituents.
Solvents selected should not interfere in detection of separated spots.
Solvents should not be highly volatile as loss of components can result in change of mixture composition.
1. Describe the principle of liquid- liquid extraction

Liquid-liquid extraction, also known as solvent extraction or partitioning, is a separation technique based on the differential solubility of a solute in two immiscible liquid phases. The principle of liquid-liquid extraction relies on the fact that a solute will distribute itself between two immiscible solvents in proportion to its solubility in each solvent.

By carefully selecting the solvents and manipulating their distribution, it is possible to separate and extract specific solutes from a mixture.

The principle of liquid-liquid extraction can be explained as follows:

Selection of immiscible solvents: Two immiscible solvents are chosen, typically an organic solvent and an aqueous solvent, that do not mix together. The choice of solvents depends on the solute to be extracted and their relative solubilities in the two phases.
Contacting the solvents: The solute-containing mixture is first dissolved or suspended in one of the immiscible solvents, known as the initial solvent. The initial solvent is usually the one in which the solute has a higher solubility. The solvents are then mixed or shaken together to allow for contact and distribution of the solute between the two phases.
Partitioning of the solute: When the two solvents are mixed, the solute molecules distribute themselves between the two phases based on their solubilities. The solute molecules will partition or transfer from the initial solvent into the second solvent phase to an extent determined by the solute’s partition coefficient, which is the ratio of its solubility in the two solvents.
Separation of the phases: After sufficient mixing, the two solvents are allowed to separate into distinct layers based on their immiscibility and differences in density. The layers form due to the natural phase separation caused by gravity or centrifugation. The solute of interest will be present in one or both of the solvent phases, depending on its solubility characteristics.
Extraction and recovery: The solvent phase containing the desired solute is separated from the other solvent phase. This can be done by carefully decanting or using a separating funnel. The solute is then typically recovered from the solvent phase by evaporation, distillation, or other suitable methods.

Liquid-liquid extraction is widely used in various applications, such as pharmaceutical, chemical, and environmental industries, for the separation, purification, and recovery of compounds from complex mixtures. It is a versatile technique that can be optimized by adjusting factors such as solvent selection, pH, temperature, and agitation to achieve efficient and selective extractions.

State Nernst distibution law

It states that ” At constant temperature, when two solutions of a solute in two immiscible solvents are in contact with each other, then solute distributes itself to keep a fixed ratio of concentration in the two immiscible solvents at equilibrium.

“ if a solute X distributes itself between two immiscible solvents A and B at constant temperature and X is in the same molecular condition in both solvents.”

Give the advantages of paper chromatography over the other Advanced Instrumental Techniques?

Chromatographic technique of analysis has seen an impressive growth over time. Such advances have increased laboratory throughputs lowered limits of detection and has made forays into new areas of applications. Paper chromatography has retained its ground till date and is popular in laboratories across the world. Some of the reasons for this are:

Low cost of analysis and freedom from maintenance.
Separated spots are visible for coloured compounds and colourless compounds can be viewed by using alternate techniques.
Minimum operation and training requirements.Solvent consumption is much less as compared to more sophisticated techniques.
Paper chromatography serves as a good demonstration of basic concepts of separation for school and undergraduate students.

Give the Limitations Of Paper Chromatography Technique?

Paper chromatography has some limitations such as:

Semi-quantitative in nature.
Overlapping of spots of components having close Rf values.
Higher concentration of components often leads to streaking instead of well-defined spots.
Errors in Rf calculations can result from uneven flow of solvent front. This can be caused by running out of solvent at the bottom of the chamber, uneven cutting of the filter paper or unevenness of the bottom of the development chamber.
Improper sample spotting, spotting below the marked line resulting in dipping into the solvent or accidental dipping of spot into solvent while inserting the paper into the solvent chamber.
1. Explain the separation principle in size exclusion chromatography?

In size exclusion chromatography the separation does not involve chemical interactions between eluting molecules and stationary phase. The separation takes place on the basis of molecular size with larger molecules eluting first and small molecules in the end. Small molecules are retained longer in the pores of the stationary phase therefore they get eluted last.

Explain why it is necessary to degass the mobile phase?

Mobile phases entrap air from the atmosphere and this trapped air gets released as small bubbles under high pressures encountered during the HPLC analysis. Such bubbles can lead to noise in detector response or hinder flow of mobile phase through columns. In order to overcome such problems degassing of mobile phase becomes essential.

What is the basis (principle) of chromatographic process?

It is based on the differential migration of the individual components of a mixture through a — stationary phase under the influence of a moving phase.

State the type of solvents are generally employed in chromatography?

Generally solvents having low viscosities are employed in chromatography. This is due to the fact that the rate of flow of a solvent varies inversely as its viscosity.

What Are The Moving And Stationary Phases In Paper Chromatography?

Water absorbed on cellulose constituting the paper serves as the stationary phase and organic solvent as moving phase.

What Is Meant By The Term R_fValue?

The term “Rf value” stands for “retention factor” or “relative front.” It is a quantitative measure used in chromatography to describe the relative migration of a component within a chromatographic system. The Rf value is calculated as the ratio of the distance traveled by the component to the distance traveled by the solvent front.

The formula for calculating the Rf value is:

Rf = (Distance traveled by the component) / (Distance traveled by the solvent front)

In paper chromatography, the Rf value is determined by measuring the distance the component moves from the baseline to the center of the spot or peak, divided by the distance the solvent front travels from the baseline to the center of the spot or peak. The Rf value is a dimensionless quantity and is always between 0 and 1.
The Rf value is specific to a particular chromatographic system and conditions, including the type of chromatography, the composition of the mobile phase, and the nature of the stationary phase. It is primarily used as a means of identification and comparison of components within a mixture. By comparing the Rf values of known compounds to those of unknown compounds under identical conditions, one can gain insights into the identity and composition of the unknown compounds.

It is important to note that the Rf value is influenced by various factors, including the polarity of the components, the composition of the mobile phase, the type of stationary phase, and the temperature. Hence, Rf values should be used as a qualitative tool for comparison rather than an absolute measure.

41.What are the advantages of chromatography over other techniques?

It can be used for a mixture containing any number of components.
Very small quantities of the substances can be effectively detected and separated from a mixture.

42.Describe how a thin layer chromatography is prepared for laboratory work

The experiment is conducted on a sheet of aluminium foil, plastic, or glass which is coated with a thin layer of adsorbent material.

The stationary phase that is applied to the plate, which is usually used is aluminium oxide, cellulose, or silica gel. is made to dry and stabilize on a clean glass slide .

To apply sample spots, thin marks are made at the bottom of the plate with the help of a pencil.

Apply sample solutions to the marked spots.

Pour the mobile phase into the TLC chamber and to maintain equal humidity, place a moistened filter paper in the mobile phase.

Place the plate in the TLC chamber and close it with a lid. It is kept in such a way that the sample faces the mobile phase.

Immerse the plate for development. Remember to keep the sample spots well above the level of the mobile phase. Do not immerse it in the solvent.

Wait till the development of spots. Once the spots are developed, take out the plates and dry them. The sample spots can be observed under a UV light chamber.

44.Explain four qualities of solvents suitable for solvent extraction

The solvent should be able to dissolve at least one component to a large extent than the rest of the components in the mixture.
The solvent should not be miscible with the liquid to be extracted but not with the other components of the mixture or react with the solute.
The reaction taking place should be stable and irreversible. Reversible reactions can bring back the dissolved components in their previous form and the extraction will not be completed successfully.
The boiling point of the solvent should be low enough ( well below the melting point of the solute) such that it can be

Evaporated easily after collection.

The compound formed after the reaction should be easily separated from the extracted compound so that it can be reused.
The density of the compound should be different from the required component to help the separation readily.
. It should have a favorable temperature coefficient
.It should be inexpensive and cost-effective.
The solvent should not be toxic or corrosive as it can harm the extraction instruments.
Outline Thin Layer Chromatography

Here a sheet of alumina is taken ( 0.2 mm thick) over which a small spot of the mixture is placed and it is kept in a suitable solvent. The solvent rises due to capillary action and the constituents also rise with the solvent depending on their differential adsorption, and thereby, they are separated.

45.Outline Column Chromatography

Column chromatography is technique in which the substances to be isolated are presented onto a column loaded with an adsorbent (stationary phase), go through the column at various rates that rely upon the affinity of every substance for the adsorbent and the solvent or solvent mixture, and are typically gathered in solution as they pass from the column at various time. The two most common examples of stationary phases for column chromatography are silica gel and alumina while organic solvents are regarded as the most common mobile phases.

46.Column Chromatography principle

The main principle involved in column chromatography is the adsorption of the solutes of the solution with the help of a stationary phase and afterward separates the mixture into independent components. At the point when the mobile phase together with the mixture that requires to be isolated is brought in from the top of the column, the movement of the individual components of the mixture is at various rates. The components with lower adsorption and affinity to the stationary phase head out quicker when contrasted with the greater adsorption and affinity with the stationary phase. The components that move rapidly are taken out first through the components that move slowly are eluted out last. The adsorption of solute molecules to the column happens reversibly. The pace of the movement of the components is communicated as:

R_f = the distance traveled by solute/ the distance traveled by the solvent

Where R_fis called retardation factor

explain the Column Chromatography components

Components of a typical chromatographic system using a gas or liquid mobile phase include:

Stationary phase –Generally it is a solid material having a good adsorption property and should be suitable for the analytes to be separate. It should not cause any hindrance in the flow of the mobile phase.
Mobile phase and delivery system –This phase is made up of solvents that complement the stationary phase.

The mobile phase acts as a solvent, a developing agent (promotes separation of components in the sample to form bands), and an eluting agent (to remove the components from the column that are separated during the experiment).

Column –

For liquid chromatography: 2-50cm long and 4mm internal diameter, fabricated with stainless steel

For gas chromatography: 1-3m long and 2-4mm internal diameter, fabricated either with glass or stainless steel

A column’s material and its dimension are very crucial to support the stationary phase and promote effective separations.

Injector system –Responsible for delivering test samples to the column’s top in a reproducible pattern.
Detector and Chart Recorder – This gives a continuous record of the presence of the analytes in the eluate as they come out from the column.
Detection relies on the measurement of a physical parameter (like visible or UV adsorption).
On the chart recorder, each separated analyte is represented by a peak.

A collector at the bottom is placed at the bottom end of the column set up to collect the separated analytes.

Outline the procedure for preparing Column chromatography

The steps included in the column chromatography are:

Preparation of the column

Mostly the column is comprised of a glass tube with an appropriate stationary phase
The bottom end of the column is packed with a glass wool/cotton wool or an asbestos pad after which the stationary phase is packed.
After packing the column, a paper disc is placed on the top to avoid the disturbance of the stationary phase during the introduction of the sample or mobile phase.
The disturbance in the stationary phase (adsorbent layer) leads to the irregular bands of separation.

Two types of preparing the column, known as packing techniques namely:

Dry packing technique –The amount of absorbent needed is added as a fine dry powder in the column and the solvent flows freely through the column until equilibrium is achieved.
Wet packing technique – The slurry of adsorbent is prepared along with the mobile phase and is poured into the column.
It is regarded as the ideal technique for packaging.

The column should be properly washed and completely dried before in-use.

List four factors that determine column efficiency in colunn chromatography
Column Packing:

Column packing in resolution is yet another significant factor, appropriate column packing is extremely necessary to achieve better results.

The particle size of the adsorbent:

This is also significant factor in the improved separation, if the size of the particles decreases, the separation is improved.

The dimension of the column:

There are different dimensions column are available for the separation, depending on the process, there is a need to select the appropriate column dimensions. Width ratio, height, and length are significant factors, increasing the column length resulting in better separation.

Solvents:

The solvents to be used should be compatible with the columns and the matrix. Usually, a solvent in column chromatography is used with less viscosity, as the viscosity can influence the flow rate of the solvent system and which affects the separation of the component.

Flow Rate:

Changing the flow rate can also affect the column efficiency and it needs to separate the analytes with an appropriate flow rate.

The temperature of the column:

Temperature is also a vital factor in chromatographic separation because some chromatographic columns are damaged at higher temperatures and proteins can also, be degraded at high temperatures. So maintain the suitable temperature to improve the efficiency of column chromatography.

Equilibration and column washing:

Equilibration and washing of the column can also affect efficiency.

These are the key factors that can affect resolution in column chromatography.

Explain three criteria by chromatography techniques can be classified.

Chromatography is a laboratory technique which is frequently employed for the separation of mixtures. In this technique, the mixture that must be separated is first dissolved into a fluid (commonly referred to as the mobile phase). This fluid carries the mixture through a different structure (commonly referred to as the stationary phase). The fluid carries different components of the mixture at different speeds. Therefore, the components of the mixture are separated in the stationary phase of the chromatography setup. The following is the criteria by which chromatographic techniques can be classified.

Classification of Chromatography based on the Chromatographic Bed Shape

Column Chromatography: In this type of chromatography, the stationary phase of the setup is placed inside a tube. Then, the particles of the stationary phase (which is in the solid state) are made to fill the inside with the tube. An unrestricted, open path is then prepared for the mobile phase (somewhere along the middle of the tube).
Planar Chromatography: In this type of chromatography, the stationary phase of the apparatus usually has a planar shape. Different subcategories of planar chromatography include paper chromatography (where the stationary phase is a special type of paper) and thin layer chromatography(usually abbreviated as TLC).
- Classification of Chromatography based on the Physical State of the Mobile Phase
Gas Chromatography: In this type of chromatography, the mobile phase is a substance that exists in the gaseous state. It can be noted that gas chromatography is also known as gas-liquid chromatography, and is often abbreviated to GLC. This type of chromatography almost always involves the use of a packed column.
Liquid Chromatography: This type of chromatography involves the use of a mobile phase that exists in the liquid state. Liquid chromatography, often abbreviated to LC, can be carried out either on a plane or in a column. It can be noted that there exist many subcategories under liquid chromatography such as high-performance liquid chromatography and reversed phase liquid chromatography.
- Classification of Chromatography based on the Mechanism of the Separation
Ion Exchange Chromatography: This type of chromatography is also known as ion chromatography. Ion exchange chromatography involves the separation of the components of the mixture via an ion exchange mechanism. Differently charged components of the mixture are separated with the help of different ions in this separation technique.
Size Exclusion Chromatography: This type of chromatography involves the separation of different components of the mixture based on their sizes. In size exclusion chromatography, components of the mixture are filtered based on their hydrodynamic volume or hydrodynamic diameters. It can be noted that size exclusion chromatography is also known as gel permeation chromatography or gel filtration chromatography.
Expanded Bed Adsorption Chromatography: This type of chromatography is commonly used for biochemical separation processes. It is a specialized technique which is used for the capturing of proteins from the mixture sample.

Explain Types of Column chromatography

Adsorption column chromatography –Technique of separation in which compounds to be separated (solute) is retained or adsorbed on the surface of the adsorbent (stationary phase).
Partition column chromatography – It is based on the variance in partition coefficient of the individual components of the mixture, where the stationary phase and the mobile phase both are in the liquid state.
Gel column chromatography – Here, the separation is carried out through a column packed with gel and possesses a porous stationary phase. It is also referred to as size exclusion chromatography
Ion exchange column chromatography –The basis relies on the charge of the molecules. The separation is done when molecules get attracted to the oppositely charged stationary phase.

Describe techniques used in purification of substances after separation

The purification of organic compounds is necessary, though complex, step after its extraction from a natural source or synthesis in the laboratory. The method of purification of the organic compound depends mainly on the nature of the compound and the impurities present.

One easy method to check the purity of an organic compound is to either melt or boil it as organic compounds tend to have sharp melting and boiling points.

Purification means the removal of unwanted impurities present in an organic compound. The general methods of purification are:

Sublimation

Some solids can directly pass to the vapour state without going through the liquid phase. The purification technique which exploits this property is called sublimation. It is helpful in separating sublimable compounds from non-sublimable ones.

Methods of Purification – Sublimation

The substance is heated in a china dish above which an inverted funnel is kept to collect the sublimable compounds. The funnel is kept cool so as to hasten the process. Vapours of the substance solidify on the funnel.

Crystallisation

The principle here is that the compound and the impurities have different solubilities in a solvent. A solvent is chosen where the compound to be purified is sparingly soluble, that is, it is sparingly soluble at lower temperature and soluble at a higher temperature. The solution is heated to get a saturated solution, and on cooling, the crystals of the compounds are removed via filtration.

For example, crystals of benzoic acid can be crystallised with water. Benzoic acid is sparingly soluble in cold water and soluble in hot water.

If the mixture contains impurities that have the same solubility as of the compound to be purified, repeated crystallisation is performed.

Distillation

The underlying principle behind distillation is that the mixture of liquids can be separated by the difference in their boiling points. The boiling point is defined as that temperature at which the vapour pressure of the liquid is equal to the atmospheric pressure. This method separates volatile liquids from non-volatile liquids. The setup is given below.

Purification of Liquids – Distillation

The mixture is taken in the round bottom flask and boiled. The more volatile or the component with lower boiling point evaporates faster and is collected in a separate container. A condenser is used to hasten the process of condensation.

For example, a mixture of chloroform and aniline can be separated by distillation. The boiling point of chloroform is 60°C and that of aniline is 189°C. Therefore distillation can be used to separate a mixture of chloroform and aniline.

Fractional Distillation

This method is employed when the difference between the boiling points of the liquids isn’t much. Since the vapours of such liquids might condense together, a fractionating column is fixed to the mouth of the RB.

Explain the principle of fractional distillation

Fractional distillation is used to separate miscible liquids with different but very close boiling point. This method is more efficient than simple distillation.

Fractionating column is used to separate constituents whose volatility is nearly similar or differs by 77°F (25°C, at 1 atmosphere pressure) and which cannot be separated by simple distillation.

A fractionating column is introduced between the distillation flask and the condenser. A simple fractionating column is a tube packed with glass beads. The beads provide surface for the vapors to cool and condense repeatedly.

The upper portion of the column, which is closer to the condenser, is cooler than the lower portion and hence, only gases with the same temperature as the upper portion are allowed to pass on to the condenser. On the other hand, the gases with higher boiling points will condense and flow back to the bottom into the distillation flask, and is heated into a gas again. At the end, liquid with the lowest boiling point will be the first to boil and hence the first to be distilled out and collected.

When vapors of a mixture are passed through the fractionating column, because of the repeated condensation and evaporation, the vapors of the liquid with the lower boiling point first pass out of the fractionating column, condense and are collected in the receiver flask. The other liquid, with a slightly higher boiling point, can be collected in similar fashion in another receiver flask.

It is a more effective and easier to use process, compared to simple distillation method. It is, however, relatively expensive than any other types of distillation.

Vacuum Distillation

Since the boiling point is dependent on the atmospheric pressure, the liquids will boil at a temperature lesser than their boiling points if they were distilled in an atmosphere having lower pressure. This is achieved by using a vacuum pump. Since the atmospheric pressure is reduced, the liquids also boil faster and hence the whole process of distillation is made fast.

Process of Vacuum Distillation

Steam Distillation

In this variant, steam is passed into the flask containing the liquids to be separated. The principle here is that the liquids will boil faster because of aqueous tension (vapour pressure of water) helps in equalising the atmospheric pressure.

Total pressure = Aqueous tension + vapour pressure of liquid components

Purification of Liquids by Steam Distillation

In the absence of aqueous tension, the process of boiling would have been continued until it equalises the atmospheric pressure. Now with the addition of steam, that process is expedited

52.Explain the basic principles involved in steam distillation

Steam distillation is used for the purification of mixtures in which the components are heat sensitive; for example, organic compounds.

Differential Extraction

This method is used for immiscible liquids, that is, liquids that do not mix together. For example, oil and water are immiscible.

The immiscible liquids are taken in a separating funnel and left undisturbed. After a while, they separate out according to their specific gravities, with the heavier liquid at the bottom. Then they are later collected.

Substances can also be separated according to their preferential solubilities in the liquid. For example, if phenol is to be extracted, it can be preferentially extracted using NaOH solution as one of the liquids used.

53.Outline the procedure used to obtain dry crystals from a hot solution by recrystallyzation techniques :

The crude impure solid is dissolved in hot solvent. If some solid remains undissolved after adding solvent, it is likely to be an impurity and should be removed by filtrering the hot solution.

Typical problems: Adding too much solvent so that the product does not crystallize later. Filtering the hot solution too slowly so that the solvent cools and the solid starts crystallizing in the funnel and/or on the sides of glassware.

The solution is allowed to stand without being disturbed. The temperature is allowed to gradually drop, leading to growth of large crystals. The flask should not be placed on a surface (it will shock-cool the solution), but either placed in an insulated jar, or clamped.

Typical problems: Crystals do not form at all (too much solvent), precipitate forms instead of crystals (temperature has dropped too quickly, or an oil forms).

The solution is allowed to stand until crystallization is complete.

Typical problems: Crystallization can be a slow process, and impatience can lead to low recovery.

The solution is placed in an ice-water bath to lower the temperature even further, and allow more crystals to form. At this point, most crystals should already have formed.
The crystals are filtered and air-dried.

54.Explain how to determine if a recrystallization process success. Simple visual inspection is a good start: The crystals should have shiny surfaces and catch the light. They should appear uniform, and you should have crystals of similar structure and size. A melting point analysis should also show a narrower and elevated melting point range compared to the crude material.

Like any purification technique, recrystallization has some limitations. First of all the compound you crystallize should be a solid at standard conditions. Greases, waxes and oils cannot be crystallized at standard conditions. Secondly, the crude material should be mostly pure. There is not any minimum purity standard for any crude material, because the success of any recrystallization depends on the identities of the other constituents and their respective solubilities, but in general the crude material should contain about 80% of the desired compound.

The crude material is transferred to a suitable crystallization vessel. The crude material is dissolved in a solvent, and gently heated.

The solution is allowed to gently and slowly cool down. Notice the crystals growing in the solution. The solution is cooled to room temperature, leading to the formation of large crystals.

55.Describe how ethanol can be obtained a mixture of ethanol and water

Fractional distillation is a method for separating a liquid from a mixture of two or more liquids. For example, liquid ethanol can be separated from a mixture of ethanol and water by fractional distillation. This method works because the liquids in the mixture have different boiling points. When the mixture is heated, one liquid evaporates before the other.

A water and ethanol mixture is heated in a flask. Vapour forms in the air above the mixture in the flask.

One way to check the purity of the separated liquids is to measure their boiling points. For example, pure ethanol boils at 78°C and pure water boils at 100°C.

56.Discuss four heating techniques used in laboratory

Bunsen Burners

Bunsen burners are generally used to rapidly heat high-boiling liquids with low flammability (such as water). Bunsen Burners can reach temperatures of approximately 1500oC and can easily ignite most organic compounds. If an apparatus is improperly set up, or if there is a small gap that allows organic vapors to escape from an apparatus, these vapors can ignite with a burner. Therefore, it is generally recommended to use other heat sources to warm flammable organic liquids. Bunsen burners should never be used with highly flammable solvents such as diethyl ether.

Hotplates

Hotplates are perhaps the most versatile heat source in the laboratory and can be used to heat beakers, Erlenmeyer flasks, and various hot baths (water, sand, and oil baths).

Hotplates work by passing electricity through a heating element covered by a ceramic top. The hotplate surface can reach temperatures up to 350oC, which is hot enough to ignite many low-boiling solvents. Diethyl ether, pentane, hexane, low-boiling petroleum ether, and acetone should therefore never be heated in an open vessel with a hotplate.

Caution should be used when heating any flammable organic liquid in an open vessel on a hotplate, as organic vapors may spill out of containers and ignite upon contact with the heating element, which may be hotter than the ceramic surface.

A steam bath

A steam bath is a relatively safe way to heat flammable organic liquids. They are designed to heat beakers, Erlenmeyer flasks, and round-bottomed flasks…

Water baths.
Water bath are most commonly used to heat solutions to 100oC (boiling baths). Hot plate may be used in heating. They may also be used to heat to lower temperatures, although it can be difficult to maintain a constant temperature. Water baths can be covered with aluminum foil to prevent excessive evaporation, or to prevent excess moisture from entering open vessels. Cold water baths can also be used to cool apparatus in a quick manner
Sand baths.

Sand baths can be used to heat solutions to a wide variety of temperatures, from moderate to high temperatures (> 250oC). A vessel containing the substance to be heated should be buried in a sand bath as much as possible as the surface is often much cooler than the sand below. A metal spatula can be used to pile the sand up to at least the height of liquid inside the flask. Sand takes a long time to heat up, and a long time to cool down. To save time, a sand bath may be preheated while an apparatus is assembled as long as it is preheated a distance away from volatile organic liquids.

If the sand overheats and causes a liquid to boil uncontrollably, the flask can be partially lifted out of the sand, or the sand moved with a metal spatula away from contact with the liquid. Sand will remain warm even after turning off the hotplate, and therefore flasks have to be lifted out of the sand bath in order to cool.

Oil baths

Oil baths are much like water baths, but use silicone or mineral oils is used in order to enable temperatures hotter than the boiling point of water. Silicone oil baths can be heated to 250oC, while mineral oil baths can be heated to 300oC. Mineral oil is composed of mixtures of long-chain alkanes, and so is combustible. Direct contact with open flames should therefore be avoided.

Heating mantles
Heating mantles are a relatively safe way to heat flammable organic liquids in a round bottomed flask. The mantles are cup-shaped and designed for different sizes of round bottomed flask .If a mantle does not fit a round bottomed flask perfectly, sand can be added to ensure good thermal contact. Heating mantles take some time to warm up and also take some time to cool down. The mantle will remain warm even after turning off, and therefore flasks have to be removed from the mantle in order to cool.

57.Explain factors that govern the type of solvent used in soxhlet extraction

The thermal stability of the extracted material determines the boiling range of solvent to be used.
The polarity of the extracted material determines the polarity of the solvent.
Some plant materials decomposes at 70 -100o C and thus solvents of relatively low B. P. must be used.
1. Describe any four differences between batch extraction and continous extraction

Batch extraction is the simplest and most commonly used method, consists of extracting the solute from one immiscible layer in to other by shaking the two layers until equilibrium is attained, after which the layers are allowed to settle before sampling.

This is commonly used on the small scale in chemical laboratories.

The most commonly employed apparatus for performing a batch extraction is a separatory funnel.

The batch extractions may also be used with advantage when the distribution ratio is large.

b) Continuous extraction

continuous extraction, makes use of a continuous flow of immiscible solvent through the solution or a continuous countercurrent flow of both phases.

Continuous extractions are particularly applicable when the distribution ratio is relatively small.

Continuous extraction device operates on the same general principle, which consist of distilling the extracting solvent from a boiler flask and condensing it and passing it continuously through the solution being extracted.

The extracting liquid separates out and flows back into the receiving flask, where it is again evaporated and recycled while the extracted solute remains in the receiving flask. When the solvent cannot easily be distilled, a continuous supply of fresh solvent may be added from a reservoir.

Describe the process of electrophoresis as a separation techniques

Electrophoresis is the process of separating certain large molecules so they can be examined more easily. Electrophoresis is used mostly with colloidal or macromolecule particles–large particles made of more than one simple molecule structure–such as proteins or complicated nucleic acids.

Process

These molecules are separated via an electrical current that is usually sent through a gel. This gel, often silica based, is used to suspend the particles and hold the charge. Two electrodes are attached to the gel, and the current they produce is used to attract the molecules toward one part of the gel while repelling them from the other side. The gel provides a friction force that prevents all the molecules from moving through it at once, but the larger molecules can generally overcome the friction and separate anyway. The movement of the molecules through the gel creates a strata of different types of molecules.

Uses

There are a number of different factors at work in electrophoresis, and each one is important for defining the sort of molecules being examined. How fast they move, how strong the electrical current is, the precise qualities of the gel, the shape of the molecules, the size of the molecules, the temperature of the solution and other factors all tell scientists what kind of molecules they are looking at.

To hold the molecules in their positions, they are stained in different striations throughout the gels, which makes it look like a series of colored bands. This process is one of the most important steps in DNA analysis, allowing scientists to draw out DNA proteins and examine them closely to determine their specific characteristics.

Join the conversation