Chromatography - Paper Chromatography

We have seen in our previous post that any chromatographic technique consists of a stationary phase and a mobile phase. In the case of paper chromatography, which is one of the type of plane chromatography, paper acts as a stationary phase while the mobile phase generally consists of a mixture of water and an organic liquid. Lets understand the principle and types of paper chromatography.

Principle:

The sample is spotted on the filter paper at the base line or the spotting line. This filter paper is then placed in the suitable solvent. When the solvent rises up via the capillary action, the individual components in the sample get separated. Different components travel at different rates and the most soluble substance move further on the filter paper than the least soluble substance. The solvent travels the farthest on the filter paper and it leaves the line called as ‘solvent front’.

The Retention Factor or Rf Value:

Okay, the components got separated, but now, the question is, how to identify the components of the mixture? The answer to this is by calculating the retention factor or the Rf value. The distance traveled by a particular component is constant relative to the solvent as long as all other factors (like, the type of paper and the exact composition of the solvent) are kept constant .

Rf is the distance moved by the component divided by the distance traveled up the paper by the solvent as can be seen in the adjacent diagram.

For example, if the component traveled 5 cm (distance 'a' in the adjacent diagram) and the solvent traveled 15 cm (distance 'b' in the adjacent diagram), then the Rf value of that particular component will be 5/15 or (a/b) i.e., (distance traveled by paper/distance traveled by the solvent) = 0.3.

Nature of Paper:

The paper used in paper chromatography consists of a highly purified cellulose (homopolysaccharide of glucose). The paper exhibits weak ion exchange adsorptive properties. In recent times, various forms of modified paper are available wherein the paper has been impregnated with alumina, silica gel, ion exchange resins, etc. To get excellent results, the exact type of paper to be used is important. Generally, Whatmann filter papers are used for paper chromatography. Just to get an idea, this is the composition of Whatmann filter paper no.1:
a-cellulose – 98-99%b-cellulose – 0.3-1%Pentosans – 0.4-0.8%Ash – 0.07-0.1%Ether soluble matter – 0.015 – 0.1%

Types of Paper Chromatography:

1. Ascending Paper Chromatography

The filter paper is put into an airtight tank containing the solvent (solvent tank). The paper is supported on a frame or on the thread with the help of the clips. This is carried out in an air-tight container lined with paper and saturated with solvent so that there is a constant atmosphere and the separation takes place in constant temperature. Here, the solvent will ascend into the paper via the capillary action i.e., the solvent will travel in the upward direction of the chromatographic paper. The substance which is the most polar will be at the bottom of the tank while the least polar substance will be on the top end of the tank.

2. Descending Paper Chromatography

In descending type paper chromatography, the upper end of the filter paper is put into the solvent mixture contained in a narrow trough. This paper is mounted near the top of the container. This develops along with gravity which makes this technique little quick and easy. In contrast to the ascending technique, the substance which is the most polar will be at the top of the tank while the least polar substance will be on the bottom of the tank.

3. Radial Paper Chromatography

In this type of chromatography, there is a circular paper. The sample is placed at the center of the paper. This paper is tied parallel (in a horizontal way) to the petri-plate which contains the solvent (As can be seen in the 'side view' diagram). There is a wick of the paper and this wick is dipped inside the solvent. As a result, the solvent rises through the wick via capillary action and the components get separated in a circular zone.

4. Two-dimensional Chromatography

In this technique, a square or a rectangular paper is used. The first step is to perform an ascending type where the sample is placed at the bottom corner (say, right corner) of the paper saturated with and dipped in the solvent. After few hours, the paper is turned at a right angle, 90degree, clockwise, to the direction of first run with a different solvent. 

This was all about different techniques of paper chromatography. In the next post, we will discuss about anohter method of plane chromatography which is thin layer chromatogrpahy. 

Chromatography - Principles

Introduction:

What is chromatography?

Chromatography is a technique of separating or analyzing complex mixtures. Now, the question asked is, what is so special about chromatography?  Well, chromatography can separate the components from the mixture with acute precision. For example, some proteins, which vary by a single amino acid, can also be separated by the chromatography technique. Also, the conditions employed in chromatography are not severe and for this reason, chromatography can be used when one needs to separate delicate products.

The inventor of chromatography is Michel Tswett who was the Russian biochemist. In 1906, he separated chlorophyll from the plant pigments mixture. In this mixture of pigments, each band had a distinct color and so the procedure was named ‘chromatography’ where ‘chromo’ means colour while ‘graphy’ means to write.

Principle:

Before understanding the principle, first lets get familiarized with frequently used terms in chromatography.

• Analyte – Mixture whose components needs to be separated
• Mobile phase (carrier) – Solvent moving through the column
• Stationary phase (adsorbent) – Substance that stays fixed inside the column
• Eluent – Fluid that enters the column
• Eluate – Fluid that exits the column

In all chromatography, there are two phases – a stationary phase and a mobile phase.  The mobile phase can be either a liquid or a gas while the stationary phase can be either a solid or a liquid. The analyte is composed of different components that need to be separated. So, the mobile phase (solvent) is made to flow through the stationary phase (like, silica bed). The components of analyte show different degrees of adhesion to silica. The components that adhere to the stationary phase more strongly, travel slowly as compared to those components who have a weaker adhesion. Thus, we can say that, when an analyte or mixture of components is introduced into the column (narrow zone), then, the different components move at different rates in the direction of solvent (mobile phase) flow.

Now, lets ask ourselves certain questions. 

(a) What decides the separation of the the components in the analyte?
The basic principle behind the separation is that the affinities of the components of the analyte is different towards the mobile phase and the stationary phase. The smaller the affinity the component has for the stationary phase, the shorter the time it will spend in the column.

(b) What decides the affinity?
The affinity, in turn, is decided by two properties:

• Adsorption: Adsorption is the property as to how firmly a component sticks to the stationary phase. So, the higher the adsorption, the slower the molecule will move through the column.
• Solubility: Solubility is the property as to how well a component of the analyte can get dissolved in the mobile phase. The higher the solubility in the mobile phase, the faster the molecule will move through the column. Note: Adsorption and solubility of a molecule can be changed by choosing the appropriate stationary and mobile phases.

(c) Why different components have different affinity towards stationary and mobile phase? 

The answer to this question lies in ‘polarity’. The component that is polar in nature, will adsorb on the polar stationary phase (say silica), while the components which are non-polar in nature, will readily dissolve in the non-polar mobile phase without adhering to the stationary phase and will elute from the column as can be seen in the adjacent diagram.

There are two techniques of chromatography as:
A. Plane chromatography which includes:

1. Paper chromatography 
2. Thin layer chromatography

B. Column chromatography which  includes:

1. Adsorption chromatography
2. Partition chromatography
3. Gel permeation chromatography
4. Ion Exchange chromatography
5. Affinity chromatography

Keeping all these basics in mind, we will discuss in the next few posts, the above mentioned different types of chromatography.

Crop Production and Management (Part II)

In the previous post, we have discussed about crop and its types. In this post, we will have a look at the different agricultural practices. 

There are several practices that needs to be taken care of while cultivating a crop. These activities are referred to as agricultural practices. Such activities are as follows:

• Preparation of soil
• Sowing seeds
• Providing nutrients 
• Irrigation
• Protection from weeds
• Harvesting and storage

Of these, we will discuss first four practices in this post while harvesting, protection and storage will be discussed in the next post.

Preparation of soil:

This is the foremost important step before growing a crop. The preparation of soil involves

• Ploughing and 
• Leveling

A. Ploughing: The process of turning and loosening the soil is referred to as ‘ploughing’ or ‘tiling’. This tiling has its own advantages as follows:

1. Penetration of the roots deep into the soil
2. Allows the roots to breathe easily (provides good aeration)
3. Helps in mixing fertilizers uniformly
4. Helps in uprooting the weeds
5. Exposes soil pests to the natural predators
6. Helps in the growth of earthworms, bacteria, fungi which further increases the fertility of soil 

Instruments for Ploughing:

Plough: It is made up of wood. There is a triangular iron strip, called as ploughshare while the long log of wood is called the ploughshaft.

Hoe: It is a tool with a long handle and a broad metal blade at one end and is mainly used for removing weeds. Some use it to loosen soil as well.

Cultivator: It is a large farm machine driven by a tractor which is used to loosen the soil in the modern agricultural method.

B. Leveling:

Leveling is the process where the big mud pieces called crumbs are broken down with the help of leveller. Leveller is used to level the soil for preventing it from getting eroded by the wind and also helps in retaining the moisture. Leveling is also important for properly sowing the seeds and for irrigation purposes. 

Sowing Seeds:

Sowing means, putting the seeds in the soil. Before doing so, it is necessary to take care of the quality of the seeds. The following are the criteria for selecting good and healthy seeds:

• Seed should be healthy meaning, it should be free from infections (by bacteria, viruses, insects, nematodes etc.)
• Seeds should have a good percentage of germination
• Seeds should be sown at the right depth in the soil
• Seeds should be sown at the correct distance from each other so that there is no overcrowding (if sowing is done very near to each other) or wastage of space (if sowing is done very far from each other)
• Seeds should undergo fungicide treatment to prevent seed-borne diseases.
• Seeds should have enough amount of water for its germination as seeds do not germinate in dry soil.

Note: Damaged seeds are hollow and can be identified by soaking in water. Being hollow (light-weighted), they will float, while the healthy seeds will shrink.

There are two main methods of sowing as:

A. Broadcasting: Broadcasting further can be done by three ways:

• By hand
• By traditional method - The traditional method uses a tool which is funnel-shaped. The seed passes through the funnel and the funnel end pierces into the soil thereby placing the seeds there.
• By a seed drill - Seed drill is the modern day equipment is where the seeds are sown with the help of tractors. Seed drills are considered to be better as the seeds are sown at equal distance and depth. After sowing, the seeds are covered with the soil thereby preventing the damage done by the birds. Also, it saves time and labour.

B. Transplanting:  In this case, the young plants are developed in the nursery bed and the seedlings are transferred to the field ensuring that there is proper spacing. It is mainly done in case of paddy crop and bananas.

Providing nutrients:

Continuously growing the same plant on the field makes the soil poor in nutrients. So, the soil needs to be replenished with the nutrients. This is done by two ways:

• Natural methods
• Adding manure and fertilizers

A. Natural Methods: These include the cropping patterns which allows the soil to retain the nutrients some of which are discussed below: 

1. Mixed cropping: It is also called as multiple cropping. This is the practice where two different crops or more are grown simultaneously in the same field. The crops are chosen in such a way that the products and waste material from one crop helps in the growth of the other. This type of cropping leads to improving the fertility of the soil thereby increasing the crop yield. Generally, one crop is of long duration while the other is of short duration. One crop requires more nutrients and water while the other requires lesser nutrients or water. As a result, there is a reduction in the competition between the crops for light, nutrients and water. If one crop fails to grow (due to untimely rain or no rains or shortage of nutrients), then the other crop can cover the risk of this complete failure.
   Example: groundnut and cotton; wheat and gram
2. Crop rotation: Here, different crops are grown alternately on the same land. For example, legumes are alternately grown with wheat. In one season, legumes are grown as fodder which also helps in replenishing the soil with nitrogen. After this, the wheat crop is then grown. It is a good method of replenishing the soil naturally.
3. Intercropping: this is the process of growing two or more crops together in proximity on the same land. As a result, two or more crops are managed at the same time in a definite pattern. This increases the productivity per area and soil erosion is reduced.
4. Field fallow: In this process, the field is left uncultivated for a season or two. This is help in regeneration of the lost nutrients. But it results in the wasting the land for a season and due to high demand these days, this method is no longer used.

B. Adding manures and fertilizers: Manures and fertilizers are the substances which are added to the soil in the form of nutrients in order to make the soil more fertile for the healthy growth of the plants.

Manure is natural organic substances which are produced by the decomposition of cow dung and other animal wastes and plant residues while the fertilizers are chemical inorganic substances which are rich in specific nutrients.

The following differences will further clearly explain the fertilizers and manures:

Manures	Fertilizers
Natural organic substance	Inorganic chemical Substance
Made by the decomposition of animal and plant wastes in open pits by the microbes	Made in factories (like, NPK, Super-phosphate, Potash)
Less rich in specific nutrients but replenishes the soil with all the nutrients in a balanced way	Rich in specific nutrients, which are required by particular crops
Provides humus to the soil thereby making it fertile	Does not provide humus to the soil and adds nothing to the fertility of the soil
Required in large quantities	Required in small quantity
Maintains the soil texture, makes it porous and promotes water retention	Does not maintain the soil texture or improve water retention properties of the soil
Non-polluting and maintains the ecosystem	Pollutes water as the salts present in the fertilizers contaminates the ground water table
Difficult to transport, store and apply	Easy to handle, transport, store and apply
If applied in large quantities, they do not harm the plants	If applied in large quantities, they do harm the plants

 Manures are again divided into three different types:

1. Farm yard manure – This type of manure consists of dung, urine, leaves and other farmyard wastes.
2. Compost – This type of manure is obtained by the decomposition of dead plants and animal wastes, sewage waste etc. All the organic material is buried in a pit with alternative layers of soil and is allowed to rot.
3. Green manure – This type of manure is formed by the decomposition of the leguminous plants like sunhemp and guar. These plants are grown and are sown back into the same soil.

Manures are however, considered better than fertilizers because of the following reasons:

• Improves the texture of the soil as being organic in nature, adds to the fertility of the soil.
• Makes the soil porous which makes the exchange of the gases easier
• Increases the water retention capacity of the soil
• Increases the number of friendly organisms like bacteria, earthworms etc.

Irrigation:

Irrigation is referred to as the supply of water to plants at different intervals. Irrigation is important because:

1. Water gets absorbed by the roots of the plants and along with water, several nutrients and fertilizers also get absorbed which are then transported to various parts of the plants.
2. Water is necessary for the germination of the seeds.
3. Water protects the crop from hot-air currents as well as from the frost.

Methods of irrigation:

The methods of irrigation are distributed as:

• Traditional methods of irrigation
• Modern methods of irrigation

Traditional method of irrigation involves:

Chain pump: It consists of two large wheels of which the bottom wheel is half immersed in water source and both the wheels are connected by an endless chain. When the wheel turns, the connected bucket dips into the water source and collects the water. The chain then lifts them to the upper wheel, where water from the bucket is transferred to the pool where it gets collected. The chain then again carries the empty buckets back down to be refilled and this cycle continues.

Moat system of irrigation – It is also called as pulley system. It is a manual irrigation method where water is directly taken out from the wells with the help of pulley and is used for irrigation.

Dhekli system of irrigation – Here, a rope and a bucket are connected to pole to obtain water from the well. The rope and bucket is connected to one end of a heavy stick at one end and a heavy counter weight at the other end.

Rahat system of irrigation: By this method, water is drawn out of the well with the help of animals. Animals like cow, buffalo, ox are connected to the wheel and when the animals move, the wheel draws water from the well.

Modern Methods of Irrigation: These methods are more sophisticated and require less efforts as the pumps are used to lift water.  These use biogas, diesel, electricity and solar energy for lifting water. They are more efficient and use water economically. Such modern methods include:

Furrow Irrigation: Here, the water is allowed to pass into the field through a furrow or channels made between two rows of the crop.

Basin Irrigation: Here, the filed is just filled with water as in the case of paddy crop.

Sprinkler System: This consists of a network of perpendicular pipes having rotating nozzles at their top. These pipes are joined to the main pipeline at regular intervals. Water when flows through the main pipe under pressure, it escapes from the rotating nozzles. As a result, water gets sprinkled on the crop (similar to how rains occur). This system works better on the uneven land where sufficient water is not available or where the soil cannot retain water for a long time.

Drip System: As the name suggests, this system provides water drop by drop at the position of the roots (at the root zone). It is also called trickle-irrigation or micro-irrigation. This is the best technique to water fruit plants, gardens and trees as water is not wasted at all and is very efficient.

Importance of Irrigation:

All crop plants require water at some or the other stage of development. And it is necessary to provide right amount of water at the right time.

• Excess of water may cause waterlogging in the soil which in turn, will inhibit the germination of the seeds as seeds will not get sufficient amount of air to respire.
• Waterlogging also causes poor growth of the roots.
• The fully matured crop, when irrigated, gets damaged. The plants which are not able to resist the strong winds and excessive water, fall down. This falling down of the crop due to untimely irrigation is called lodging and this affects the yield of the crop. 

This is in reference to the CBSE class VIII Biology. 

Crop Production and Management (Part I)

We will divide the topic in three different sections for a better understanding as:

·         Crop and its types

·         Agricultural practices - Preparing the soil, sowing seeds, providing nutrients and irrigation

·         Agricultural Practices - Protection, harvest and storage 

In this post, we will discuss about crop and its types. A part of agricultural practices are discussed here.

Crop and its Types:

Before going into the details of the crops, lets first understand, what is meant by agriculture and a crop?

Agriculture Definition: Agriculture largely means the art of cultivating a piece of land, or in simple terms, planting and growing food plants on it along with rearing of animals (i.e., animal husbandry).

Crop definition: When same type of plant is grown on a large scale (i.e., commercially), then that is referred to as a crop. Crops are grown on a large scale to obtain food.

Classification of Crops:

The crops can be classified into 4 main categories depending on the source of nutrients:

·         Vegetables/fruits/spices – Main source of vitamins and minerals in the diet.

·         Cereals – A starchy grain used for food and source of carbohydrate in the diet.
Example: Wheat, barley, maize

·         Pulses - Part of legume family and are high in protein and fiber and low in fats.
Example: Lentil, pea, gram

·         Oilseed crops – A seed or crop mainly grown for oil and are sources of fats in the diet.
Example: Sunflower, Canola, Olive, Soyabean

Crops can be classified as Kharif and Rabi crops based on which season they are grown.

There is also a third category of crop called as zaid crop. These crops are also known as summer crops. These crops are grown in the short duration between Kharif and Rabi crops, mainly from March to June. They are grown on irrigated lands and so need not wait for monsoons.
Examples: Cucumber, muskmelon, sugarcane, watermelon.

Differences between Kharif and Rabi crops:

	Kharif Crops	Rabi Crops
Season of growing	Rainy season (June to September)	Winter season (October to March)
Sowing time	June/July	October/November
Harvesting time	September/October	March/ April
Alternate name	Monsoon Crops	Winter crops
Dependency on monsoons	Depended on south-western monsoons	Independent of monsoons
Examples	Rice(Paddy), maize, soyabean, groundnut, cotton, pulses,	Wheat, barley, gram, pea, mustard, potato

This post is in reference to the CBSE, class VIII Biology. 

Spectrophotometry - Atomic Absorption Spectrophotometry

Atomic absorption spectroscopy (AAS) is similar to flame photometry with the difference that it measures the absorption of a beam of monochromatic light by the atoms in the flame. This technique was first introduced by Alan Walsh in Australia in 1954. We will discuss the principle, instrumentation and applications one by one.

Principle:

The basic principle behind the AAS is that the free atoms normally remain in the ground state which are capable of absorbing the energy of their own specific resonance wavelength. If light of the resonance wavelength is passed through the flame containing the atoms (in sample), then part of the light will be absorbed. The atoms absorb UV or visible light and make the transitions to higher energy levels. The absorption will be directly proportional to the number of atoms in the ground state in the flame.

Instrumentation:
The major difference in the instrumentation of AAS and flame spectrophotometry is the presence of a radiation source (a particular resonance wavelength cannot be isolated from the continuous source using a prism or diffraction gratings). So, for this purpose, a hollow cathode lamp is used.

Light Source: (Hollow Cathode Discharge Lamp): It contains a tungsten anode and cathode (as can be seen in the diagram on the right) is a hollow cylindrical tube which is lined by the element to be determined. These are sealed in the glass tube filled with an inert gas like neon or argon at a low pressure. At the end of the cylinder is a window, made up of quartz or pyrex, transparent to the emitted radiation. Each element in question will thus emit monochromatic radiation characteristic of the emission spectrum of that particular element involved. So, each element has its own unique lamp which must be used for the analysis.

Nebulizer: It creates a fine spray of the sample for the introduction in the flame. The aerosol and the fuel and oxidant are mixed thoroughly for the introduction into the flame.

Atomizer: The elements which needs to be analysed needs to be in the atomic state. Here comes the role of atomizer. It breaks down the molecules into the atoms by exposing the analyte to high temperatures in a flame of graphite furnace (as explained in previous post, here).

Monochromator: A monochromator is used to select the specific wavelength of light which is absorbed by the sample and to exclude other wavelengths. The selection of the specific wavelength allows the determination of the element.

Detector: The light selected by the monochromator is directed onto the detector that typically is a photomultiplier tube that converts the light signal to electrical signal proportional to the light intensity.

Applications of Atomic Absorption Spectrometry

• It is highly sensitive technique and can measure upto parts per billion of a gram (ugdm-3)
• It is used to detect the presence of metals as impurity or in alloys.
• The minute levels of the metals could be detected in biological samples like copper in the brain tissues.
• The quantity of elements can be determined be agricultural and food products.
• It can also be used to determine the impurity in the environmental water sources like in the ocean water, river and stream water, waste water, sludge and suspensions.

Spectrophotometry - Flame Photometry

Flame spectrophotometry is a technique in which the intensity of the radiations emitted by a chemical into the flame is determined.  This basic concept of working of flame spectrometer is that, a flame, through its heat, can raise the atoms from a lower energy state to a higher energy state and when it comes back to its ground state, there is emission which is in the form of radiations. And determination of these radiations is by flame spectrophotometer.

Flame photometry can be applied in two ways as emission flame photometry or simple flame photometry and atomic absorption spectrophotometry. We will discuss the principle, instrumentation and applications of the two one by one.

Lets start with emission flame photometry or simply, flame photometry.

Emission Flame Photometry:

Principle:

Here, the solution containing the metallic salt (to be analyzed) is placed into the flame, whereby the solvent is evaporated, leaving behind only the solid. The solid is then dissociated by vaporization. The volatilization of the molecules in the solid produces free atoms which then, due to heat, excites to a higher energy level.  The emission spectrum is produced when the atoms return back to the ground state (as a result of radiation). This is the basic principle of the emission flame photometry.

Instrumentation:

Below is the basic representation of the components which are involved in flame photometry.

Nebulizers: Before the samples get into the flame, they must be converted to a fine spray, i.e., they must be nebulized. This is necessary as the large drops will not be able to stay in the hottest area of the flame for a long time and hence, will be difficult to volatize and excite.

Atomizers or Flames: It converts the sample or the analyte to free atoms. The atomizers can be flame atomizers or graphite rod atomizers.

Flame atomizers: To create flame, we need to mix an oxidant gas and a fuel gas. Generally, air-acetylene flame or nitrous oxide-acetylene flame is used (in the above diagram, this type is depicted). 

Graphite rod atomizers: These uses graphite rod instead of the flame.  The graphite rod is a small cavity in which the sample can be pipetted. These tubes are heated using a high current power supply such that the temperature can raise as high as 2500 degree Celsius. As a result, the sample is vaporized or atomized.

Monochromators: A monochromator is used to select a specific wavelength of light which can be absorbed by the sample while excluding other wavelengths. Generally, a simple filter is used. However, in sophisticated instruments, the prisms or diffraction gratings are used.

Detectors:  The light selected by a monochromator is directed into a detector, which is generally a photomultiplier. It converts the light signal into the electrical signal which is proportional to the intensity of the light.

Applications of flame photometry:

• It is used to determine even the small quantities of metals like lead, calcium, mercury etc.
• So, it is used in the determination of sodium, potassium, calcium, lithium etc. in the biological samples (like serum, interstitial fluids etc.).
• It is used in the determination of lead in the petrol.
• It is used in determination of calcium and magnesium in the cement.

In the next post, we will discuss about atomic absorption spectrometry.