...THIN LAYER CHROMATOGRAPHY (TLC) Thin Layer Chromatography is a simple, fast, multipurpose, sensitive, inexpensive analytical technique for the separation of substances. TLC has a mobile phase that is a liquid while the stationary phase is an active solid, known as the sorbent. Such sorbents are silica, cellulose, alumina, polyamides, ion-exchangers, and other numerous minor organic and inorganic sorbents. The considerable versatility of the sorbents depends on the type of substances being separated. TLC has been successfully applied to hydrophilic, lipophilic and inorganic separations. HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY (HPTLC) High Performance TLC can also be taken to be the conventional TLC with increased efficiency without decreasing the resolution and selectivity. To increase the efficiency of Thin Layer Chromatography, a couple of issues must be looked into. Efficiency is the kinetic variable determined primarily by the physical characteristics of the chromatographic systems, such as the size and uniformity of the adsorbent particles and the flow rate of the mobile phase. The efficiency of the system is slightly dependent on the nature of the solute. Efficiency is calculated as the theoretical plate number: N=(X/W) 2 Where, X is the distance species moved from the origin W is the zone width. N is the theoretical plate number. * High efficiency...
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...Separating and Determining the Colored Components of Capsicum fructescens Using Column and Thin Layer Chromatography Duran, K., Ednalino, M., Encarnacion, F., Fernandez, T., Fernando, S., Garcia, D. 2D-Pharmacy, Group 4, Department of Pharmacy, Faculty of Pharmacy, University of Santo Tomas, España Boulevard, 1015 Manila, Philippines ABSTRACT Chromatography was used to separate and determine the purity of the colored components present in a substance. Separation of the colored components of Capsicum fructescens (Red Siling Labuyo) using column chromatography, determining the purity of the components using thin layer chromatography (TLC), and measuring the retention factor (Rf) values of the colored components in TLC were the aims of the experiment. Dichloromethane:Hexane (DCM:Hexane) (1:1) was used to extract the pigments of the red siling labuyo. For the column chromatography, 0.5 mL of the extract was placed on top of the column containing silica gel. DCM-Hexane, DCM, and DCMmethanol was introduced in succession. Colored eluates were collected on separate test tubes and noted the number of drops for each. For the thin layer chromatography, the eluates were spotted on a pre-coated TLC plate and was placed on an equilibrated chamber, with DCM:Hexane as the solvent system. After visualizing the results on the TLC plate, the distance traveled by the pigments were computed for the Rf values. The results showed that the yellow pigment had the greatest distance traveled...
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...OF NAIROBI DEPARTMENT OF MECHANICAL ENGINEERING BULUMA MARK EUGINE F18/1494/2011 GROUP 4 EXPERIMENT 6: THIN LAYER CHROMATOGRAPHY. THE OBJECTIVE OF THE EXPERIMENT. 1. To separate the unknown amino acids mixture into its various components. 2. To identify the amino acids present in the unknown amino acid mixture. THE THEORY BEHIND THE EXPERIMENT. Chromatography is a method of separating a mixture into its components, by use of heterogeneous equilibrium established during the flow of the solvent called a mobile phase through a fixed (stationary) phase. The stationary phase can be either solid or liquid, while the mobile phase can either be a liquid or a gas. Therefore, chromatography can be classified as; solid- liquid, liquid- liquid, or gas- liquid. Experimentally, chromatography can be carried out in columns or in layers. The column chromatography uses a vertical tube packed with a medium/ adsorbent. The layer chromatography uses a thin layer embedded unto a plate unto which the samples are introduced. The thin film stationary phase may be: 1. A liquid (partition chromatography). Example is paper chromatography. 2. A finely divided adsorbent solid. (Adsorption chromatography). Example is Thin Layer Chromatography. INTRODUCTION TO THE EXPERIMENT. Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures.[1] Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material...
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...Types of Chromatography Adsorption Chromatography Adsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibriation between the mobile and stationary phase accounts for the separation of different solutes. Partition Chromatography This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibriates between the mobile phase and the stationary liquid. Ion Exchange Chromatography In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. Molecular Exclusion Chromatography Also known as gel permeation or gel filtration, this type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. The pores are normally small and exclude the larger solute molecules, but allows smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones. Affinity Chromatography This is the most selective type of chromatography employed...
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...In this lab, the use of gas chromatography was used to separate a mixture of components. These components then underwent infrared spectroscopy in order to identify their identity. For this lab, the unknown mixture had the code 130-O. This unknown was identified as t-butyl alcohol and cyclopentanone. These compounds were isolated by using the GC and determining when to change vials. The determination of when to change vials came when the first peak began to level of at the bottom. This was around 1.5 according to the GC screen. A source of error in this lab comes from the lack of a GC graph. Not acquiring a print out of the curve from the GC, causes inaccurate analysis of the data. A print out would have given the precise area of each curve...
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...Objective: Analyzing three of the fraction samples from the previous lab: “Separation, Extraction, Fractional Distillation & Gas Chromatography” to determine the contents purity Equations: Area of peak=1/2HxW % composition= (Area of peak/Total area of peak(s)) x 100 Properties: Diethyl ether 1,2-dimethoxy ethane Formula C4H10O3 ClCO(CH2)8COCl Melting Point −116.3 °C -58°C Boiling Point 62 - 64 °C 82 - 83°C Density 1.005 g/cm3 1.121 Physical Properties Clear, colorless liquidSweet, ether-like odor Clear, colorless liquidSharp, ether-like odor Hazards Extremely flammableIrritant Flammable liquid and vaporMay cause irritation to skin Procedure: 1. Use a syringe to obtain 0.5uL of fraction 1 2. Insert syringe as far as possible into the Gas Chromatography (GC) 3. Quickly press plunger to release fraction sample into GC 4. Press “Start” on the integrator 5. Remove syringe 6. At the end of process, approximately 3 minutes, press “Stop” on integrator 7. Press “shift” and “enter” keys to feed paper through integrator 8. Clean syringe with cleaning solvent 9. Repeat steps 1-8 using the other fraction samples 10. Determine the percent composition to each of the fractions Data & Results: Glass Beads: Run Time (min) Area (%) Fraction 1 Ether 0.7 70 1,2-dimethoxyethane 1.2 30 Fraction 6 Ether n/a n/a 1,2-dimethoxyethane 1.1 100 Fraction 10 Ether n/a n/a 1,2-dimethoxyethane 1.1 100 Raschig...
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...from spinach leaves (including chlorophyll a, chlorophyll b and beta carotene). The experiment also compared and contrasted two different methods of separation. The first part of the experiment explored column chromatography and the second part explored thin-layer chromatography. Introduction: (3 marks)* The three pigments that can be extracted from spinach (chlorophyll a, chlorophyll b and beta carotene) all differ based on their polarity, which arises from functional groups in their structure. The most effective way to separate pigments from spinach is based on their differences in their polarities and this lab explored two chromatographic methods to do so. Chromatography is the technique of separating organic mixtures by allowing them...
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...Chromatography is commonly used to separate mixtures to determine different chemical components. The main types of chromatography include gas, thin layer, paper and column chromatography. Generally used most, paper has a stationery phase and a mobile phase in a chromatographical experiment. The stationery phase is the paper itself absorbing the solvent, while the mobile phase is the solute moving up the strip of paper. Solvents are solids, liquids or gasses that dissolve the solute, in this instance it separates the pigments of the ink so you can see what colours make the solute. (ABC, 2018) Each solvent has a different structure based on their dielectric constant. The solvents being used in this experiment are acetone, acetic acid (5%) and...
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...(Unknown #212) Thin Layer Chromatography The main objective of this experiment was to use a Thin-layer chromatography analytic technique to analyze the relative polarity of given samples and to identify the components of a given unknown solution. TLC is usually done on a small plate coated with silica which is the stationary phase. The solvent is the mobile phase. Small sample of different compounds is placed across the plate and each plate is then put into a jar containing different solvents. Each solvent climbs up the plate with capillary action with different rate bringing up the sample to the top of the plate along with it. After the plate is allowed to dry, the relative distance the compounds traveled along the plate can be compared to that of the solvent using a ratio of Retention factor, Rf. A sample of five compounds and one unknown was placed on a small TLC plate. The original spot of each compound was represented with alphabetical letter which is ~ 1cm from the bottom of the TLC plate. Accordingly, A represented the acetanilide solution, B represented the benzophenone solution, C represented the mandelic acid solution, D represented benzyl solution, E represented Benzoic acid and X represented the unknown. This process was repeated four times making the total of five TLC plates samples. Five empty jars were obtained and a peace of large filter paper was inserted into each jar sideways. Approximately 10ml of solvent was added into each jar. Each jar contained...
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...An experimental method for determining pyrantel pamoate (PYR) via an isocratic reversed phase HPLC method with UV detection was previously optimized by Solich et al. In our experiment, we determine the PYR concentration in an old sample of a novel, anthelmintic raccoon bait. Chromatography was performed using a phenyl-hexyl column with a mobile phase of 0.5% triethylamine at pH 9.0 and acetonitrile 55 : 45 (v/v) at a flow rate of 1.0 ml min-1. For pyrantel determination, UV detection was performed at 290 nm. Analysis of the chromatographic study of the pyrantel base peaks showed that PYR makes up 2.05% of the raccoon bait by weight. Introduction Pyrantel pamoate is widely used in a variety of anthelmintic drugs, in the pharmaceutical and veterinary industries, that are designed to fight parasitic worm infections in both humans and animals. Raccoons...
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...Thin-Layer Chromatography PRELAB READING/VIDEO: See video on Blackboard Zubrick: Cleaning, Chapter 9 pp. 77-79; Chromatography, Chapter 26 pp. 218-221 TLC Chapter 27 pp. 222-235, Melting Point Chapter 12 pp.88-93 PURPOSE: The purposes of this experiment are to: (1) determine the optimal conditions for separating substances in a mixture using thin-layer chromatography (TLC), and (2) use thin-layer chromatography and infrared spectroscopy to identify an unknown solid. EXPERIMENTAL TECHNIQUES: thin layer chromatography, infrared spectroscopy. This is a 2 week experiment. INTRODUCTION Thin-layer chromatography (TLC) is a quick, inexpensive procedure that provides the chemist information on the purity of a sample, while using a minimal amount of that sample. Chemists often use TLC after running a chemical reaction to determine the purity of their product. In this lab, you’ll be (1) determining the best solvent to use for a TLC separation of four known compounds (shown below), (2) studying how experimental parameters (the type of development chamber, the presence of a filter paper “wick,” and how well the development chamber is sealed ) affect the results of a TLC experiment, (3) determining the reproducibility of Rf values, and (4) identifying an unknown compound using TLC. Before the lab, be sure to read the chapter(s) in Zubrick on thin layer chromatography! The four compounds you’ll be studying are shown below. CHM25701 Spring 2015 Thin Layer...
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...Q1. What is the method of multiplying micro- organisms by letting them reproduce in predetermined media under controlled laboratory conditions called? A. Culturing Q2. a population of cells or multicellular organisms growing in the absence of other species or types. A. pure (or axenic) culture Q3. what is used For the purpose of gelling the microbial culture A. Agar Q4. A cheap substitute for agar used for the isolation and maintenance of thermophiles. A. guar gum, Q5. bacterial culture ideal for preparation of an antimicrobial assay A. liquid culture Q6. Common liquid culture nutrient medium used A. liquid broth Q7. Another normal media used in bacteriology A. nutrient agar Q8. A standard carbon source added to nutrient medium A. glucose Q9. nitrogen is often provided A. peptones Q 10. What technique must be used to reduce the likelihood of bacterial contamination? A. Aseptic techniques Q11.Procedure by which Bacteria may be introduced to the media A.innoculation Q12. Culture media is sterilised in A. an autoclave Q13. Solid culture media in a Petri dish is known as . A. agar plate Q14. What is added to culture medium to make nucleic acid and certain types of lipids A.Phosphorus Q15. inoculum is transferred using A. loop Q16. method for transferring micro-organisms onto an agar plate is A. streak plate technique. Q17. used in industrial fermentation where a commercial product is being made...
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...Separation techniques Chromatography Chromatography is a separation technique used to separate the different components in a liquid mixture. It was introduced by a Russian Scientist Michael Tswett. Chromatography involves the sample being dissolved in a particular solvent called mobile phase. The mobile phase may be a gas or liquid. The mobile phase is then passed through another phase called stationary phase. The stationary phase may be a solid packed in a glass plate or a piece of chromatography paper. The various components of the mixture travel at different speeds, causing them to separate. There are different types of chromatographic techniques such as column chromatography, TLC, paper chromatography, and gas chromatography. Paper chromatography is one of the important chromatographic methods. Paper chromatography uses paper as the stationary phase and a liquid solvent as the mobile phase. In paper chromatography, the sample is placed on a spot on the paper and the paper is carefully dipped into a solvent. The solvent rises up the paper due to capillary action and the components of the mixture rise up at different rates and thus are separated from one another. Distillation Simple distillation is a method used for the separation of components of a mixture containing two miscible liquids that boil without decomposition and have sufficient difference in their boiling points. The distillation process involves heating a liquid to its boiling points, and transferring...
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...The first of these methods was loss of weight.1 This method, although widely used at the time of its inception, was not the most accurate or effective. This type of analysis was not very involved but had its drawbacks. These included using too much sample, approximately one to two grams, the lack of specifics in the results, and the fact that the atmosphere could alter the results. This is where gas chromatography and micro-solid phase extraction remedied the issue. Although gas chromatography (GS) and solid-phase microextraction (SPME) were not the first types of separation and analytical determinants for identifying and removing residual solvents, they are extremely more accurate and effective compared to the methods that preceded...
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...Ethnopharmacology and natural product drug discovery remains a noteworthy hope in the current discovery of new drugs. Many modern drugs have origin in traditional medicine and ethnopharmacology. Ethnopharmacology is the use of tradition plant medicines as source of new drugs. Traditional Indian Medicine - Ayurveda has a long history and is one of the great living traditions. Considerable research on pharmacognosy, chemistry, pharmacology and clinical therapeutics has been carried out on Ayurvedic medicinal plants. Several preclinical and clinical studies have examined cytoprotective, immunomodulatory and immunoadjuvant potential of Ayurvedic medicines. The ethnopharmacology knowledge, is holistic and systems approach supported by experiential base can serve as an innovative and powerful discovery engine for newer, safer and affordable medicines. The Process of Modern Drug Discovery using Ethnopharmacology The first stage should be the reported use of a natural occurring material for some purpose which can be related to a medicinal use. There must be an indication of a genuine effect and therefore, the material needs to be identified and characterized. It can be collected for test studies which comprises tests for relevant biological activity linked with isolation and structure determination of any chemical present that might be responsible. The active compounds are discovered by fractionation of the extract linked with testing for activity of each fraction until pure compounds...
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