chromatography

CHROMATOGRAPHY : 

CHROMATOGRAPHY Group Members Tharani Shanmuga Nathan 01-200703-00360 Puvanaadevi Rajandren 01-200703-00961 Siva Malar Ganasen 01-200703-00217 Sharon Premela Daniel 01-200703-00207 Suhania David John 01-200703-00297 Praveena Vishwanaden 01-200703-00959

What is Chromatography : 

What is Chromatography Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components. Separate Analyze Identify Purify Quantify

Definition of Chromatography : 

Definition of Chromatography Detailed Definition: Chromatography is a laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary adsorbing medium through which they pass. Terminology: Differential – showing a difference, distinctive Affinity – natural attraction or force between things Mobile Medium – gas or liquid that carries the components (mobile phase) Stationary Medium – the part of the apparatus that does not move with the sample (stationary phase)

Definition of Chromatography : 

Definition of Chromatography Simplified Definition: Chromatography separates the components of a mixture by their distinctive attraction to the mobile phase and the stationary phase. Explanation: Compound is placed on stationary phase Mobile phase passes through the stationary phase Mobile phase solubilizes the components Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases

Uses for Chromatography : 

Uses for Chromatography Chromatography is used by scientists to: Analyze – examine a mixture, its components, and their relations to one another Identify – determine the identity of a mixture or components based on known components Purify – separate components in order to isolate one of interest for further study Quantify – determine the amount of the a mixture and/or the components present in the sample

Uses for Chromatography : 

Uses for Chromatography Real-life examples of uses for chromatography: Pharmaceutical Company – determine amount of each chemical found in new product Hospital – detect blood or alcohol levels in a patient’s blood stream Law Enforcement – to compare a sample found at a crime scene to samples from suspects Environmental Agency – determine the level of pollutants in the water supply Manufacturing Plant – to purify a chemical needed to make a product

Illustration of Chromatography : 

Illustration of Chromatography Separation Stationary Phase Mobile Phase MIXTURE COMPONENTS

Types of Chromatography : 

Types of Chromatography Column chromatography in chemistry is a method used to purify individual chemical compounds from mixtures of compounds. It is often used for preparative applications on scales from micrograms up to kilograms. Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase) Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)

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Column chromatography

Column chromatography : 

Column chromatography Is a method used to purify individual chemical compounds from mixtures of compounds. 2 methods are used :- Dry method Wet method

Stationary phase : 

Stationary phase Stationary phase is also known as adsorbent in column chromatography is a solid eg :- Silica gel alumina

Mobile phase : 

Mobile phase The mobile phase or eluent is either a pure solvent or a mixture of different solvents. It is chosen so that the retention factor value of the compound of interest id roughly around 0.2 – 0.3 in order to minimize the time and the amount of eluent to run the chromatography .

Column chromatography resolution calculation : 

Column chromatography resolution calculation Column chromatography is set up with peristaltic pumps flowing buffers and the solution sample through the top of the column. The samples that are eluted from the column pass through a detector such as spectrophotometer or mass spectrometer so that the concentration of separated samples in the samples solution mixture can be determined. The higher the resolution of the chromatogram the better the extend of separation of the samples the columns gives.

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Retention time the time from the start of signal detection by the detector to peak height of the elution concerntration profile of each different sample Curve width the width of the concerntration profile curve of the different samples in the chromatogram in units of time

Method of calculation : 

Method of calculation A simplified method of calculating chromatogram resolution is to use the plate model. The plate model assumes that the column can be divided into a certain number of sections or plates and the mass balance can be calculated for each individual plate.

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RESOLUTION (RS) RS = 2(tRB-tRA)/(WB+WA) tRB= retention time of solute B tRA= retention time of solute A WB = Gaussian curve width of solute B WA = Gaussian curve width of solute A

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Paper chromatography is an analytical chemistry technique for separating and identifying mixtures that are or can be colored, especially pigments. Paper chromatography

Practical requirement : 

Practical requirement Stationary phase and papers used; paper consist of alpha-cellulose-98-99%, beta –cellulose used Application of sample; The sample to be is dissolved in mobile phase and applied using capillary tube or using micro pipette. Mobile phase Pure solvents, buffer solutions or mixture of solvents are used- isopropanol:ammonia:water-7:2:1

Principles of Paper Chromatography : 

Principles of Paper Chromatography Capillary Action – the movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity. Solubility – the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents. Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and the stationary phase.

Paper Chromatography Experiment : 

Paper Chromatography Experiment What Color is that STABILO?

Overview of the Experiment : 

Overview of the Experiment Purpose: To introduce students to the principles and terminology of chromatography and demonstrate separation of the dyes in STABILO Colour Pens with paper chromatography. Time Required: Prep. time: 10 minutes Experiment time: 10 minutes

Materials List : 

Materials List 6 strips of filter paper Different colors of STABILO pens Pencil Ruler Scissors Small beakers Distilled water

Preparing the Chromatography Strips : 

Preparing the Chromatography Strips Cut 6 strips of filter paper Draw a line 2 cm above the bottom edge of the strip with the pencil Label each strip with its corresponding solution Place a spot from each pen on your starting line

Developing the Chromatograms : 

Developing the Chromatograms Place the strips in the beakers Make sure the solution does not come above your start line Keep the beakers covered Let strips develop until the ascending solution front is about 2 cm from the top of the strip Remove the strips and let them dry

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Distilled water 2 min 3 min 4 min 6 min 8 min 10 min

Observing the chromatograms : 

Observing the chromatograms Observe how some dyes are made up of more than one colour Observe how some spots of same colour are separated in water Observe when spots of different colours started to separate into different components

Rƒ value : 

Rƒ value Rƒ a = 1.8 = 0.3157 5.7 Rƒ b = 2.7 =0.4737 5.7 a b

Paper chromatography : 

Paper chromatography

Thin layer chromatography : 

Thin layer chromatography Thin layer chromatography (TLC) is a chromatography technique use to separate mixtures. Thin layer chromatography is performed on a sheet of glass, plastic or aluminium foil which is coated with a thin layer of adsobdent material, usually silica gel, aluminium oxide or cellulose. This layer of adsorbent is known as the stationary phase. After the sample has been applied on the plate, a solvent or a solvent mixture ( known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, saperation is achieved

Plate preparation : 

Plate preparation Pouring technique Dipping technique Spraying technique

Preparation and Activation of TLC plates : 

Preparation and Activation of TLC plates TLC plates are usually commercially available, with standard particle size ranges to improve reproducibility. Are prepared by mixing adsorbent like silica gel with a small amount of inert binder. Then the mixture is spread on a unreactive carrier sheet. The resultant plate is dried and activated by heating in an oven for 30 mins at 110 Celcius.

Techniques : 

Techniques Development of the TLC plate a purple spot separates into red and blue spots. Chromatogram of 10 essential oils coloured with vanillin reagent

Advantages of TLC : 

Advantages of TLC Simple method and the cost of equipment is low Rapid technique and not time consuming like column chromatography Separation of micro gram of the substances can be achieved Efficiency of separation Detection is easy and tedious Better choice between different stationary phases

Principles of TLC : 

Principles of TLC Principles of separation is adsorption 1 or more compounds are spotted on a thin layer of adsorbent coated on a chromatographic plate. The mobile phase solvent flows through because capillary action (against gravitational force) Separation of compound is based on the competition of the solute and mobile phase for binding places on the stationary phase The compenents with more affinity towards the stationary phase travels lower The components with less affinity towards the stationary phase travels faster ( resulting in higher Rf value )

Practical requirements : 

Practical requirements Silica gel – H Silica gel – G Silica gel – GF Cellulose powder Stationary phase - Glass plates which are specific dimension like 20cm 20cm can be used Glass phase

Application : 

Application Separation of mixtures of drugs of chemical or biological origin, plant extract Separation of charbohydrates, vitamins, antibiotics, proteins, glycocides, alkaloids and etc. Identification of drugs eg :- amoxicillin, levodopa, methyldopa To detect the presents of foreign substances in drugs To study the decomposition of products in drugs Detection of pesticides or insecticdes in food and water Identifying compounds present in a given substance

Journal : 

Journal Title chromatography paper strip sampling of entric adenoviruses type 40 and 41 Positive Stool Specimen Abstract Th enteric subgroup of adenoviruses type 40 and 41 are the 2nd more important cause of acute infantile gastroenteritis aftaer rotavirus

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Method Using sodium dodecyl sulphate SDS/EDTA pretreated chromatography paper strip was evaluated for the colletion, storing, and shipping of adenovirus 40/41 contaminated Stool Sample. Chromatography Paper Strips Adenovirus Sample loading on the chromatography Paper strips PCR Detection Biasafety test for Adenovirus

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Result Adenoviral DNA can be successfully detected in the filter strips by PCR after 4 months. Storage at -20 celcius, 4 celcius RT (20-25 celcius) At 37 celcius, no adenovirus infectivity was observed upon with the SDS/EDTA-pretreated strips

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