Physical Separations and Chromatography

Size: px
Start display at page:

Download "Physical Separations and Chromatography"

Transcription

1 Lab #5A & B: Physical Separations and Chromatography Individual Objectives: At the end of these experiments you should be able to: Ø Distinguish between Rf and tr; chromatograph and chromatogram; adsorption and absorption; mobile and stationary phase Ø Apply these chromatographic parameters to a qualitative and quantitative interpretation of various chromatograms Ø Describe the potential application of chromatography in the agri-food industry Ø Gain confidence in sample handling and use of a column chromatography and TLC, Prelab Exercise: Ø Prepare a one page flow chart of the procedure you will use in lab each week. Ø Read about Chromatography, the various methods available, the experiment and its background. Ø Research the components found in spinach leaves. THIS LAB IS DONE OVER A TWO WEEK PERIOD Introduction: Chromatography is the separation of a mixture into its individual components. This is accomplished by distributing the components of the mixture into two different phases (solid & liquid; solid & gas). The stationary phase (solid or liquid) is fixed and does not move. The mobile phase (liquid or gas) flows continuously through the stationary phase, carrying the components of the mixture along with it. Some components of the mixture will move more slowly through the stationary phase than others due to factors such as the solubility of the components in the mobile phase (absorption) and the strength of the physical interactions (adsorption) with the stationary phase. Chromatography can therefore be used for the qualitative identification of individual components in a mixture. Some chromatographic analyses are also able to quantify these individual components. 1 P age

2 There are several different chromatographic methods available today. These include thin layer chromatography (TLC), paper chromatography, column chromatography, gas chromatography (GC) and high performance liquid chromatography (HPLC). Only the latter two involve sophisticated, expensive instruments. i) Column chromatography: Is a type of chromatography that allows for the separation of individual components from a mixture. In this case a stationary phase, typically silica or alumina, is held in a column (usually glass) and the mobile phase, typically an organic solvent, is allowed to pass down the column by the aid of gravity. The sample (mixture) is placed on top of the column and eluted (washed down the column) by the mobile phase. Individual components have different adsorptive affinities (attractions) for the stationary phase due to their polarities and other structural features and will move down the column at different rates. As the components separate out into bands, the individual bands can be collected from the bottom of the column and are called fractions. These fractions are saved and can be analyzed further for additional qualitative and quantitative information. ii) TLC: In thin layer and paper chromatography, the stationary phase is a flat surface, either a sheet of paper or a glass or plastic plate with a stationary coating. In TLC or paper chromatography the sample is dissolved in a solvent and then applied to the lower edge of the paper or the plate. The plate or paper is then immersed into a mobile phase. The solvent will move up the adsorbent layer by capillary action, carrying the components of the sample spot with it. Due to the relative solubilities of the components and the interactions of these components with the stationary phase will determine the rate in which these components move up the plate or paper. Once the mobile phase has nearly reached the top of the stationary phase the separation is halted. The location of the individual components in the mixture can then be identified by visual observation, by the use of uv light or by the addition of chemicals ( chemical development ). The individual components can then be identified by a comparison of the Rf values of the samples to those Rf values of known standards. The Rf values are the distance traveled by the individual component divided by the distance traveled by the mobile phase. The Rf values are constant for a given compound under set conditions and should be 2 Page

3 consistent. The equation for Rf values is given below. Refer below for a pictorial representation of this experimental datum. Rf = the distance traveled by the component the distance traveled by mobile phase Fig.1: Calculation of the Rf value iii) Gas chromatography - is a method used for the analysis of individual components in a mixture. Individual compounds may be tentatively identified and quantified using gas chromatography. In gas chromatography the sample (a mixture of compounds) is vaporized (in the case of a gas sample this is already accomplished) and carried through a column by an inert gas such as helium or nitrogen. This gas is known as the carrier gas. The column contains a solid of a finely packed material that is coated with a liquid of relatively low volatility. This packed material is known as the stationary phase. Each individual component of the sample will interact with the stationary phase differently. The amount of interaction (the affinity for the stationary phase) will affect the motion of the individual components through the column. As the sample moves through the column the affinity of the components for the stationary phase causes the sample to separate out into individual components. As these components pass through a detector, an electronic signal is produced for each component. The voltage of this signal is plotted as a function of time. The plot produced is known as the sample chromatogram. 3 P age

4 The time from the point of injection of a sample to the maximum inflection of the peak is known as the retention time. If instrument parameters stay constant, the retention time of individual components should remain constant. Therefore, if the retention time of ethylene is 1.10 minutes in run #1 it should be also be 1.10 minutes in run #2. Since retention time is a property of the individual components, it can be used to tentatively identify components of a sample. This can be accomplished by injecting a pure standard and comparing the retention time of this standard to the retention of the sample peaks. If the retention times are the same, the sample peak can be tentatively identified. Fig. 2: Calculation of tr for a peak on a gas chromatogram Gas chromatography can also be used for the quantifying the individual components in a mixture. Each component will result in a single peak. The area or the height of the peak can be measured and is assumed to be proportional to the concentration of the component. Peak height is measured from the baseline of the chromatogram to the peak apex. Peak area can be measured using an instrument called an integrator. Alternatively it can be measured and calculated manually using the method of triangulation, where the area of a triangle equals ½ base times height. See sketch below. 4 P age

5 A= ½ bh = ½ (2.11)(4.65) = 4.91 cm 2 Fig. 3: Triangulation of a chromatographic peak Standards of known concentrations can be run and plotted against their peak area or height. This standard curve can then be used to calculate the concentration of the sample. Fig. 4: Standard curve for ethanol 5 P age

6 Lab #5 Part A: Carotenoids and Chlorophyll from Spinach Background: Photosynthesis is the process of converting energy in sunlight to chemical forms of energy that are made available for plant use. The process of photosynthesis takes place in the organelles called chloroplasts. These chloroplasts contain a number of pigments (colored compounds) that are broken down into two categories. These categories are chlorophylls and carotenoids. The chlorophylls are the green pigments that act as the principle photoreceptor molecule in the plant. These pigments are capable of absorbing certain visible light that is then converted to chemical energy by the plant. Chlorophyll a will absorb red and blue-violet light and chlorophyll b will absorb blue and red orange parts of the spectrum. The carotenoids will absorb blue-violet and blue green wavelengths, meaning that they will reflect wavelengths that are red, orange, and yellow. These plant pigments are soluble in a combined organic-aqueous extraction solvent hence a combination of polar acetone, nonpolar hexane and polar water is recommended for the extraction of the pigments. Unfortunately, water interferes with the subsequent separation by column chromatography. A drying step is required to remove the water; this is achieved using anhydrous Na2SO4, a common chemical used to remove water from organic solutions. 6 P age

7 Table 1. Safety Notes for Lab 5A: Extraction of Pigments SAFETY NOTES CHEMICALS HAZARD PRECAUTIONS Acetone Hexane Anhydrous sodium sulfate Alumina (aluminum oxide) Flammable liquid and vapor, irritant to skin, eyes and respiratory tract. Flammable liquid and vapor, irritant to skin, eyes and respiratory tract. Not expected to be a health hazard. Irritant to skin, eyes and respiratory tract. No open flames, use in fumehood when possible, Avoid contact with skin. Wash hands well after use. No open flames, use in fumehood when possible, Avoid contact with skin. Wash hands well after use. Wash hands well after use. Avoid contact with skin. Wash hands well after use. DISPOSAL Place acetone, hexane, and other organic waste in the organic waste container marked non- chlorinated in the fumehood. EQUIPMENT Specific Objective(s): In this part of the lab you will extract pigments from a food product and using column chromatography, separate the mixture into individual pigment fractions. 7 P age

8 This part of the lab data collection will be done in pairs; you may wish to bring colored pencils or markers for the sketches of the chromatographic separation. The lab report is done individually. Procedure: 1. Extraction of the chlorophylls and carotenoids: Before beginning this section, obtain approximately 20 ml of acetone, 20 ml of hexane, and 20 ml of the hexane:acetone mixture in small beakers. Label and cover with a watch glass. 1.1 Using a mortar and pestle crush a small amount of spinach (~ one leaf broken up). To the crushed sample add approx. 5 ml of acetone (graduated cylinder). Grind the sample thoroughly. Slightly tilt the mortar and remove the liquid using a disposable pasteur pipet, leaving all small particles of the leaf behind. 1.2 Use a graduated cylinder to place 2 ml of this solution into a capped centrifuge tube and add 2 ml of hexane (repipet in hood), cover and mix on the vortex mixer for 1 minute. 1.3 To the mixed sample add 2 ml of water (repipet) and vortex the sample for an additional minute. 1.4 Place samples in the benchtop centrifuge and centrifuge for 5 minutes. When placing samples in the centrifuge ensure that the samples are balanced. Set the speed of the centrifuge at a setting of 4. Balancing tubes for use in a centrifuge: Place a tube with the same mass of water (blank) or a tube with the same mass of someone else s sample exactly opposite your sample tube in the centrifuge. 1.5 Samples are now ready for drying. You will use only the upper, organic layer for the rest of the procedure, but first this upper layer has to be dried as described in Section 2 below. 8 P age

9 2. Drying of the extracted sample: A drying column will be provided to you by the Lab Instructor. It will be a pasteur pipette filled with anhydrous sodium sulfate. By letting your sample flow through this drying column, the water in the solvent will be removed. Before beginning Section 2, obtain one clean dry collection tube from the front bench along with a prepared drying column. 2.1 Using a thermometer clamp and a ring stand, adjust the height of the drying column so that the collection tube can be placed just below the column to collect the sample. (See example on the front bench.) 2.2 Transfer the upper layer of the sample that is in the centrifuge tube and place it on top of the column using a clean dry pasteur pipet. Once the sample has drained into the column, add 25 drops of hexane (using a pasteur pipet) to the top of the column. This step will extract all of the pigments from the drying agent. 2.3 When the hexane has passed through the drying column, remove the test tube containing the dried solvent-pigment mixture. 2.4 Evaporate the collected sample-hexane mixture to dryness using a nitrogen evaporator (in the hood). 2.5 Using an eppendorf pipet, add 0.5 ml of hexane to the dry residue on the bottom of the test tube; vortex the sample for 1 minute to assist with dissolution. The sample is now ready for column chromatography. This is called the crude extract. 3. Separation using Column Chromatography: (Be sure to make observations and a sketch) In this Section, the crude pigment sample will be separated into pigment fractions or bands based on the color and solubility of the different pigments. 9 P age

10 3.1 Before beginning Section 3, obtain approximately 5 clean dry disposable tubes from the front bench. Label the Tubes #1-5, including your name on the label. **** Note that it is extremely important that once the column chromatography procedure has been started that the chromatography column must not be allowed to run dry. Add additional solvent if necessary. **** 3.2 Prepare a chromatography column (a different pasteur pipette that will be filled with wetted alumina). Do not let this go dry until you have completed all the steps in Section 3 of the procedure. 3.3 Using the chromatography column you prepared, place the column in a thermometer clamp and place the collection tube labeled #1 under the column. 3.4 Put 2 ml of hexane (measure with graduated cylinder) through the chromatography column. Collect in Tube # Allow the hexane to drain down until it is just at the top of the packing material in the chromatography column. 3.6 Now promptly add about half (~0.25mL) of the dissolved pigment sample called crude extract from Section 2. Save the other half of the sample crude extract for thin layer chromatography analysis on another lab day. Label it with your names and lab group number and leave it with your lab instructor. 3.7 Just as the last of the crude extract solution begins to reach the top of the column packing material, add 1 ml of hexane (graduated cylinder). 3.8 Allow this to flow through the column. You should start to see a yellow band or fraction separating. Then add hexane, one ml at a time until enough has been added (3-4 ml) to separate and move the yellow band halfway down the chromatography column. 3.9 When the hexane has reached the top of the column packing material, add approx. 1 ml of 90:10 mixture hexane: acetone Just before the yellow band begins to elute off the column, change and collect into Tube # P age

11 3.11 Continue to add the 90:10 hexane:acetone mixture until the yellow band has been completely eluted off the column Once the entire yellow band has been removed add several mls of pure acetone to the column and at the same time change to Tube #3. Remember not to allow the column to drain dry!!! 3.13 When the green band starts to elute from the column, switch to Tube #4. Continue to add acetone to the column until the entire green band has eluted off the column When the eluent becomes clear, switch to Tube #5, stop adding solvent and allow the column to go dry Evaporate the solvent in the tubes containing the yellow band (Tube #2), the green band (Tube #4) and the original plant crude extract by using the nitrogen evaporator. The pigment fractions are now ready for TLC analysis (Lab 5; Part B) which will indicate if the pigment fractions (tubes 2 and 4) were pure pigments or still a mixture of pigments. Save them for use in Part B; record your names and lab group information on the test tubes. At the end of Part A you should store the following with the Lab Instructor. (Remember to properly label all samples.): Part of your unpurified spinach leaf extract ( crude extract ) Dry residue from the Yellow Band (Tube #2; purified yellow pigment) Dry residue in Green Band (Tube #4; purified green pigment) Ó 2019 M. Tate & N. Pitts 11 P age

Chromatography Extraction and purification of Chlorophyll CHM 220

Chromatography Extraction and purification of Chlorophyll CHM 220 INTRODUCTION Extraction and purification of naturally occurring molecules is of the most common methods of obtaining organic molecules. Locating and identifying molecules found in flora and fauna can provide

More information

Chromatography: Thin-Layer Chromatography (TLC) & Column Chromatography

Chromatography: Thin-Layer Chromatography (TLC) & Column Chromatography Chromatography: Thin-Layer Chromatography (TLC) & Column Chromatography Part 1, p. 184: Separation of spinach pigments by TLC. (4 th Ed. P. 180) Part 2, p. 192: Separation of Fluorene and Fluorenone by

More information

Chromatography Lab # 4

Chromatography Lab # 4 Chromatography Lab # 4 Chromatography is a method for separating mixtures based on differences in the speed at which they migrate over or through a stationary phase which means that a complex mixture will

More information

Experiment Nine Thin Layer Chromatography

Experiment Nine Thin Layer Chromatography Name: Lab Section: 09 Thin Layer Chromatography Experiment Nine Thin Layer Chromatography Introduction Objective Separation of compounds from a mixture is an incredibly important aspect of experimental

More information

LAB #6 Chromatography Techniques

LAB #6 Chromatography Techniques LAB #6 Chromatography Techniques Objectives: To learn how to story board a procedure Explain how a chromatograph of pigments is formed from both paper and thin layer chromatography. Isolate and identify

More information

Experiment 1: Thin Layer Chromatography

Experiment 1: Thin Layer Chromatography Experiment 1: Thin Layer Chromatography Part A: understanding R f values Part B: R f values & solvent polarity Part C: R f values & compound functionality Part D: identification of commercial food dye

More information

PURPOSE: To separate the pigments of spinach by Column Chromatography. To analyze Column Chromatography fractions by Thin Layer Chromatography.

PURPOSE: To separate the pigments of spinach by Column Chromatography. To analyze Column Chromatography fractions by Thin Layer Chromatography. LAB 1: CHROMATOGRAPHY OF SPINACH Thin Layer and Column Chromatography PURPOSE: To separate the pigments of spinach by Column Chromatography. To analyze Column Chromatography fractions by Thin Layer Chromatography.

More information

3. Separation of a Mixture into Pure Substances

3. Separation of a Mixture into Pure Substances 3. Separation of a Mixture into Pure Substances Paper Chromatography of Metal Cations What you will accomplish in this experiment This third experiment provides opportunities for you to learn and practice:

More information

Chromatography & instrumentation in Organic Chemistry

Chromatography & instrumentation in Organic Chemistry Chromatography & instrumentation in Organic Chemistry What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify

More information

Photosynthesis. Introduction

Photosynthesis. Introduction Photosynthesis Learning Objectives: Explain the importance of photosynthetic pigments for transformation of light energy into chemical bond and the advantage of having more than one pigment in the same

More information

CHEMICAL SEPARATION EXPERIMENT 2

CHEMICAL SEPARATION EXPERIMENT 2 CHEMICAL SEPARATION EXPERIMENT 2 INTRODUCTION The term analysis in chemistry usually refer to the quantitative and qualitative determination of the components of a sample. Qualitative refering to the identity

More information

Chromatography and Functional Group Analysis

Chromatography and Functional Group Analysis Chromatography Chromatography separates individual substances from a mixture. - to find out how many components there are - to match the components with known reference materials - to use additional analytical

More information

Abstract. Introduction

Abstract. Introduction Investigating the Techniques of Solid- Liquid Extraction by isolating lycopene from tomato paste and Column Chromatography &Thin-Layer Chromatography (TLC)by purifying lycopene Mengying Li Department of

More information

Name Period Date. Lab 10: Paper Chromatography

Name Period Date. Lab 10: Paper Chromatography Name Period Date Lab 10: Paper Chromatography Objectives Known and unknown solutions of the metal ions Fe +, Cu 2+ and Ni 2+ will be analyzed using paper chromatography. An unknown solution containing

More information

THIN LAYER CHROMATOGRAPHY

THIN LAYER CHROMATOGRAPHY THIN LAYER CHROMATOGRAPHY OBJECTIVE In this laboratory you will separate spinach pigments using thin layer chromatography (TLC). INTRODUCTION Mixtures of compounds are very common in Organic Chemistry.

More information

Thin Layer Chromatography

Thin Layer Chromatography Thin Layer Chromatography Thin-layer chromatography involves the same principles as column chromatography, it also is a form of solid-liquid adsorption chromatography. In this case, however, the solid

More information

Experiment 1: The Borohydride Reduction of 9-Fluorenone to 9-Fluorenol

Experiment 1: The Borohydride Reduction of 9-Fluorenone to 9-Fluorenol Experiment 1: The Borohydride Reduction of 9-Fluorenone to 9-Fluorenol Background: In this week s experiment, a metal hydride will be used as a reducing agent. Metal hydrides can be quite reactive, and

More information

CfE Higher Chemistry. Unit 3: Chemistry in Society. Chemical Analysis as part of quality control

CfE Higher Chemistry. Unit 3: Chemistry in Society. Chemical Analysis as part of quality control CfE Higher Chemistry Unit 3: Chemistry in Society Chemical Analysis as part of quality control 06/12/2017 Composition and purity 06/12/2017 Learning Outcomes : I can explain the basic principle of how

More information

PROGRAMMING THE RINSE ROBOT INTRODUCTION

PROGRAMMING THE RINSE ROBOT INTRODUCTION PROGRAMMING THE RINSE ROBOT INTRODUCTION Description During the initial part of this exercise, students will attach a chemical group to a solid material inside a reaction tube. Not all of this chemical

More information

Chromatography. What is Chromatography?

Chromatography. What is Chromatography? 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. Mixture

More information

Laboratory Exercise: Chromatographic Separation

Laboratory Exercise: Chromatographic Separation CHEM 109 Introduction to Chemistry Revision 1.0 Laboratory Exercise: Chromatographic Separation As we have discussed, chromatographic separations employ a system with two phases of matter; a mobile phase

More information

Exp 1 Column Chromatography for the Isolation of Excedrin Components. Reading Assignment: Column Chromatography, TLC (Chapter 18)

Exp 1 Column Chromatography for the Isolation of Excedrin Components. Reading Assignment: Column Chromatography, TLC (Chapter 18) Exp 1 Column Chromatography for the Isolation of Excedrin Components Reading Assignment: Column Chromatography, TLC (Chapter 18) Column chromatography separation can be achieved if the compounds have different

More information

Exercise 4: Thin layer chromatography of organic compounds

Exercise 4: Thin layer chromatography of organic compounds Chemistry 162 Exercise 4: Thin layer chromatography of organic compounds Objective: Use thin layer chromatography to separate and characterize the polarity of a mixture of benzene derivatives. Introduction:

More information

Volumetric Analysis. Quantitative analysis answers the second question

Volumetric Analysis. Quantitative analysis answers the second question Volumetric Analysis Volumetric analysis is a form of quantitative analysis involving the measuring of volumes of reacting solutions, it involves the use of titrations. When buying food we often have two

More information

Paper Chromatography. Identifying the components of a mixture

Paper Chromatography. Identifying the components of a mixture Paper Chromatography Identifying the components of a mixture QUIZ TIME! Take out a blank piece of paper Put your name and your lab day/time at the top Paper Chromatography Identifying the components of

More information

PRINCIPLES AND APPLICATION OF CHROMATOGRAPHY. Dr. P. Jayachandra Reddy Mpharm PhD Principal & professor KTPC

PRINCIPLES AND APPLICATION OF CHROMATOGRAPHY. Dr. P. Jayachandra Reddy Mpharm PhD Principal & professor KTPC PRINCIPLES AND APPLICATION OF CHROMATOGRAPHY Dr. P. Jayachandra Reddy Mpharm PhD Principal & professor KTPC CHROMATOGRAPHY Laboratory technique for the Separation of mixtures Chroma -"color" and graphein

More information

Dr. Jonathan Gutow Fall Looking for PCBs in Water or Can PCBs Wash out of Landfills and Contaminate Ground Water?

Dr. Jonathan Gutow Fall Looking for PCBs in Water or Can PCBs Wash out of Landfills and Contaminate Ground Water? Chemistry 103 PCBs on Sediments Lab Dr. Jonathan Gutow Fall 2003 Looking for PCBs in Water or Can PCBs Wash out of Landfills and Contaminate Ground Water? by Jonathan Gutow, Spring 1999. Revised 4/01,

More information

not to be republished NCERT THE technique of chromatography is vastly used for the separation, Chromatography UNIT-5 EXPERIMENT 5.

not to be republished NCERT THE technique of chromatography is vastly used for the separation, Chromatography UNIT-5 EXPERIMENT 5. UNIT-5 Chromatography THE technique of chromatography is vastly used for the separation, purification and identification of compounds. According to IUPAC, chromatography is a physical method of separation

More information

DATES: LAB: Liquid Chromatography Separation of Grape Kool-Aid

DATES: LAB: Liquid Chromatography Separation of Grape Kool-Aid NAME: AP CHEMISTRY DATES: LAB: Liquid Chromatography Separation of Grape Kool-Aid PURPOSE There are a number of analytical techniques used to separate components of a mixture, or solution. They include

More information

Thin Layer Chromatography

Thin Layer Chromatography Experiment: Thin Layer Chromatography Chromatography is a technique widely used by organic chemists to separate and identify components in a mixture. There are many types of chromatography, but all involve

More information

EXPERIMENT #1 SEPARATION AND RECOVERY OF ORGANIC COMPOUNDS, THIN LAYER CHROMATOGRAPHY, COLUMN CHROMATOGRAPHY, CRYSTALLIZATION AND MELTING POINTS

EXPERIMENT #1 SEPARATION AND RECOVERY OF ORGANIC COMPOUNDS, THIN LAYER CHROMATOGRAPHY, COLUMN CHROMATOGRAPHY, CRYSTALLIZATION AND MELTING POINTS EXPERIMENT #1 SEPARATION AND RECOVERY OF ORGANIC COMPOUNDS, THIN LAYER CHROMATOGRAPHY, COLUMN CHROMATOGRAPHY, CRYSTALLIZATION AND MELTING POINTS Overview In the first few weeks of this semester you will

More information

Principles of Thin Layer Chromatography

Principles of Thin Layer Chromatography REVISED & UPDATED Edvo-Kit #113 Principles of Thin Layer Chromatography Experiment Objective: The objective of this experiment is to gain an understanding of the theory and methods of thin layer chromatography.

More information

Prelab Reading Assignment: Laboratory Techniques in Organic Chemistry, 4 th Ed. Chapter 19

Prelab Reading Assignment: Laboratory Techniques in Organic Chemistry, 4 th Ed. Chapter 19 CHEM 213 Technique Experiments Experiment 5: Column Chromatography Number of labs - one Reactions performed None Chemicals used: Fluorene-fluorenone mixture, hexanes, methylene chloride, silica gel Supplies

More information

Practical 1 Procedure Iron by Titrimetry

Practical 1 Procedure Iron by Titrimetry Practical 1 Procedure Iron by Titrimetry Introduction This experiment quantifies Fe 2+, ferrous iron, by reacting it with ceric sulphate in a 1:1 reaction, i.e. one mole of ferrous iron reacts with one

More information

Experiment 6: Dehydration of 2-Methylcyclohexanol

Experiment 6: Dehydration of 2-Methylcyclohexanol Experiment 6: Dehydration of 2-Methylcyclohexanol Dehydration of 2-Methylcyclohexanol This week's reaction: A B - dehydration of a 2 alcohol to give a mixture of alkene isomers - H 3 PO 4 is a catalyst

More information

Separations: Chromatography of M&M and Ink Dyes

Separations: Chromatography of M&M and Ink Dyes Separations: Chromatography o M&M and Ink Dyes Almost all substances we come into contact with on a daily basis are impure; that is, they are mixtures. Similarly, compounds synthesized in the chemical

More information

Chromatography. writing in color

Chromatography. writing in color Chromatography writing in color Outlines of Lecture Chromatographic analysis» Principles and theory.» Definition.» Mechanism.» Types of chromatography.» Uses of Chromatography. In 1906 Mikhail Tswett used

More information

Chromatography. Investigation Photosynthetic Pigments. Do all leaves contain the same pigments?

Chromatography. Investigation Photosynthetic Pigments. Do all leaves contain the same pigments? Investigation Photosynthetic Pigments Materials For Group of 2 - Fresh spinach leaves - Wet erase marker - Chromatography paper - 2 Vials with caps - Scissors - Developer solution (Lighter fluid + Acetone,

More information

Exploration of Protein Folding

Exploration of Protein Folding Exploration of Protein Folding Question: What conditions affect the folding of a protein? Pre-lab reading Atkins & Jones (5 th ed.): Sections 5.1 5.5; 9.8 9.9; and Section 19.13 Safety and Waste Disposal

More information

Biology Unit 2, Structure of Life, Lab Activity 2-2

Biology Unit 2, Structure of Life, Lab Activity 2-2 Biology Unit 2, Structure of Life, Lab Activity 2-2 Photosynthesis is the process by which energy used by living systems is converted from electromagnetic radiation from the sun to chemical energy. This

More information

Cl 2(g) + NaCl + H 2 O. light. 2Cl. Once formed, the chlorine radical can react with the heptane as shown below: + Cl

Cl 2(g) + NaCl + H 2 O. light. 2Cl. Once formed, the chlorine radical can react with the heptane as shown below: + Cl Experiment Free Radical Chlorination of Heptane In today s experiment, you will be chlorinating n-heptane with chlorine atoms (radicals) to form monochlorination products of that alkane. You will analyze

More information

High Performance Liquid Chromatography

High Performance Liquid Chromatography High Performance Liquid Chromatography What is HPLC? It is a separation technique that involves: Injection of small volume of liquid sample Into a tube packed with a tiny particles (stationary phase).

More information

Ester Synthesis And Analysis: Aspirin and Oil of Wintergreen. Vanessa Jones November 19, 2015 Thursday 8:30 Lab Section Lab Partner: Melissa Blanco

Ester Synthesis And Analysis: Aspirin and Oil of Wintergreen. Vanessa Jones November 19, 2015 Thursday 8:30 Lab Section Lab Partner: Melissa Blanco Ester Synthesis And Analysis: Aspirin and Oil of Wintergreen Vanessa Jones November 19, 2015 Thursday 8:30 Lab Section Lab Partner: Melissa Blanco INTRODUCTION For this lab, students attempted to synthesize

More information

AP Biology Lab 4 PLANT PIGMENTS AND PHOTOSYNTHESIS

AP Biology Lab 4 PLANT PIGMENTS AND PHOTOSYNTHESIS AP Biology Laboratory Date: Name and Period: AP Biology Lab 4 PLANT PIGMENTS AND PHOTOSYNTHESIS OVERVIEW In this lab you will: 1. separate plant pigments using chromatography, and 2. measure the rate of

More information

Abstract: An minimalist overview of chromatography for the person who would conduct chromatographic experiments, but not design experiments.

Abstract: An minimalist overview of chromatography for the person who would conduct chromatographic experiments, but not design experiments. Chromatography Primer Abstract: An minimalist overview of chromatography for the person who would conduct chromatographic experiments, but not design experiments. At its heart, chromatography is a technique

More information

CHROMATOGRAPHY. The term "chromatography" is derived from the original use of this method for separating yellow and green plant pigments.

CHROMATOGRAPHY. The term chromatography is derived from the original use of this method for separating yellow and green plant pigments. CHROMATOGRAPHY The term "chromatography" is derived from the original use of this method for separating yellow and green plant pigments. THEORY OF CHROMATOGRAPHY: Separation of two sample components in

More information

Chemistry Gas Chromatography: Separation of Volatile Organics

Chemistry Gas Chromatography: Separation of Volatile Organics Chemistry 3200 Gas chromatography (GC) is an instrumental method for separating volatile compounds in a mixture. A small sample of the mixture is injected onto one end of a column housed in an oven. The

More information

Chemistry 151 Lab 4: Chromatography

Chemistry 151 Lab 4: Chromatography Chemistry 151 Lab 4: Chromatography Last updated Dec. 2013 Introduction Mixtures, both homo- and heterogeneous, can be separated (or resolved) into substances by physical means. Common examples of physical

More information

Expt 10: Friedel-Crafts Alkylation of p-xylene

Expt 10: Friedel-Crafts Alkylation of p-xylene Expt 10: Friedel-Crafts Alkylation of p-xylene INTRODUCTION The Friedel-Crafts alkylation reaction is one of the most useful methods for adding alkyl substituents to an aromatic ring. Mechanistically,

More information

The Basis for Paper Chromatography

The Basis for Paper Chromatography APTER 4 Polarity The Basis for Paper hromatography bjectives The objectives of this laboratory are to: Use paper chromatography to determine the number of components in certain mixtures. lassify these

More information

Thin Layer Chromatography

Thin Layer Chromatography Introduction Thin Layer Chromatography Chromatography is an effective and very useful method for separation and purification of organic compounds that can be used even for complex mixtures. Chromatography

More information

Dehydration of Alcohols-Gas Chromatography

Dehydration of Alcohols-Gas Chromatography Dehydration of Alcohols-Gas Chromatography OBJECTIVE In this lab, we will examine the phosphoric acid catalyzed dehydration of 2-methylcyclohexanol. Gas chromatography will be used to monitor the outcome

More information

2. Synthesis of Aspirin

2. Synthesis of Aspirin This is a two-part laboratory experiment. In part one, you will synthesize (make) the active ingredient in aspirin through a reaction involving a catalyst. The resulting product will then be purified through

More information

PHYSICAL CONSTANTS: MELTING POINTS, BOILING POINTS, DENSITY

PHYSICAL CONSTANTS: MELTING POINTS, BOILING POINTS, DENSITY CRYSTALLIZATION: PURIFICATION OF SOLIDS ANSWERS TO PROBLEMS: 1. (a) (b) (c) (d) A plot similar to line A in Figure 5.1 on page 559 will be obtained. The line will be slightly curved. All of the substance

More information

CHROMATOGRAPHY, AND MASS SPECTRAL. FRACTIONS OF Lasianthus

CHROMATOGRAPHY, AND MASS SPECTRAL. FRACTIONS OF Lasianthus f^ l^ltt^^p^^* V^ COLUMN CHROMATOGRAPHY, HPLC AND MASS SPECTRAL ANALYSIS OF SOME FRACTIONS OF Lasianthus lucldus Biume 8.1 Column Chromatography 8.2 HPLC 8.3 Preparatory TLC 8.4 Mass Spectral Analysis

More information

Institute for Chemical Education, Fun With Chemistry; Vol. 1, Sarquis, Mickey and Sarquis, Gerry, Ed.; University of Wisconsin Madison, 1991,

Institute for Chemical Education, Fun With Chemistry; Vol. 1, Sarquis, Mickey and Sarquis, Gerry, Ed.; University of Wisconsin Madison, 1991, EXPERIIMENT #7 LIIQUIID CHROMATOGRAPHY References: Bidlingmeyer, B. A.; Warren Jr., F. V. An Inexpensive Experiment for the Introduction of High Performance Liquid Chromatography J. Chem. Educ. 1984, 61,

More information

Chromatography- Separation of mixtures CHEM 212. What is solvent extraction and what is it commonly used for?

Chromatography- Separation of mixtures CHEM 212. What is solvent extraction and what is it commonly used for? Chromatography- Separation of mixtures CHEM 212 What is solvent extraction and what is it commonly used for? How does solvent extraction work? Write the partitioning coefficient for the following reaction:

More information

Chromatography. Chromatography is a combination of two words; * Chromo Meaning color * Graphy representation of something on paper (writing)

Chromatography. Chromatography is a combination of two words; * Chromo Meaning color * Graphy representation of something on paper (writing) Chromatography Chromatography is a combination of two words; * Chromo Meaning color * Graphy representation of something on paper (writing) Invention of Chromatography Mikhail Tswett invented chromatography

More information

Chromatography. Gas Chromatography

Chromatography. Gas Chromatography Chromatography Chromatography is essentially the separation of a mixture into its component parts for qualitative and quantitative analysis. The basis of separation is the partitioning of the analyte mixture

More information

CHEM 108M, Binder. Experiment 1 - Separation of Carvone and Limonene

CHEM 108M, Binder. Experiment 1 - Separation of Carvone and Limonene Experiment 1 - Separation of Carvone and Limonene Reading Assignment Mohrig Chapter 18 (Column Chromatography) Terpenes encompass a large family of organic compounds widespread in nature and occurring

More information

Photosynthesis Lab. Table 1: Comparison of the two parts of photosynthesis.

Photosynthesis Lab. Table 1: Comparison of the two parts of photosynthesis. Revised Fall 2016 Photosynthesis Lab **Lab coat, eye goggles and gloves (nitrile or latex) are required for this lab. You will not be allowed to participate without this equipment. Before coming to lab:

More information

Supernatant: The liquid layer lying above the solid layer after a precipitation reaction occurs.

Supernatant: The liquid layer lying above the solid layer after a precipitation reaction occurs. Limiting Reagent Introduction The quantities of substances involved in a chemical reaction represented by a balanced equation are often referred to as stoichiometric amounts. Solution stoichiometry is

More information

Chromatography What is it?

Chromatography What is it? Chromatography Most things that are colored are mixtures of different substances of various colors. In a mixture you have several different kinds of chemicals that are all next to each other but not reacting.

More information

Substances and Mixtures:Separating a Mixture into Its Components

Substances and Mixtures:Separating a Mixture into Its Components MiraCosta College Introductory Chemistry Laboratory Substances and Mixtures:Separating a Mixture into Its Components EXPERIMENTAL TASK To separate a mixture of calcium carbonate, iron and sodium chloride

More information

Experiment 1: Extraction and Thin Layer Chromatography

Experiment 1: Extraction and Thin Layer Chromatography Experiment 1: Extraction and Thin Layer Chromatography Introduction: Chromatography is a useful tool in chemistry and can be very helpful in determining the composition of an unknown sample. In chromatography

More information

Chromatographic Separation

Chromatographic Separation What is? is the ability to separate molecules using partitioning characteristics of molecule to remain in a stationary phase versus a mobile phase. Once a molecule is separated from the mixture, it can

More information

GRIGNARD REACTION Synthesis of Benzoic Acid

GRIGNARD REACTION Synthesis of Benzoic Acid 1 GRIGNARD REACTION Synthesis of Benzoic Acid In the 1920 s, the first survey of the acceleration of chemical transformations by ultrasound was published. Since then, many more applications of ultrasound

More information

solution, Concentrated HNO 3

solution, Concentrated HNO 3 Exercise 21 Aim: To detect the presence of proteins. Principle: Proteins respond to some colour reactions due to the presence of one or more radicals or groups of the complex protein molecule. All proteins

More information

Principles of Gas- Chromatography (GC)

Principles of Gas- Chromatography (GC) Principles of Gas- Chromatography (GC) Mohammed N. Sabir January 2017 10-Jan-17 1 GC is a chromatographic technique utilizes gas as the mobile phase which is usually an inert gas (Hydrogen, Helium, Nitrogen

More information

CHEM51LC PROJECT DETERMINATION OF DIASTEREOSELCTIVITY USING THERMODYNAMIC VERSUS KINETIC CONTROLLED REDUCTION PROCEDURES: A REDUCTION

CHEM51LC PROJECT DETERMINATION OF DIASTEREOSELCTIVITY USING THERMODYNAMIC VERSUS KINETIC CONTROLLED REDUCTION PROCEDURES: A REDUCTION CHEM51LC PROJECT DETERMINATION OF DIASTEREOSELCTIVITY USING THERMODYNAMIC VERSUS KINETIC CONTROLLED REDUCTION PROCEDURES: A REDUCTION of 4-tert-BUTYLCYCLOHEXANONE REACTION: Oxidation of an Alcohol, Reductions

More information

DEHYDRATION OF ALCOHOLS-GAS CHROMATOGRAPHY

DEHYDRATION OF ALCOHOLS-GAS CHROMATOGRAPHY DEHYDRATION OF ALCOHOLS-GAS CHROMATOGRAPHY OBJECTIVE In this lab, one will examine the phosphoric acid catalyzed dehydration of 2-methylcyclohexanol. Gas chromatography will be used to monitor the outcome

More information

Experiment 5 Reactions of Hydrocarbons

Experiment 5 Reactions of Hydrocarbons Experiment 5 Reactions of ydrocarbons ydrocarbons are compounds that only contain carbon and hydrogen. ydrocarbons can be classified further by the type of bonds they contain. If a hydrocarbon contains

More information

#22 Visible Spectrum of Chlorophyll from Spinach

#22 Visible Spectrum of Chlorophyll from Spinach #22 Visible Spectrum of Chlorophyll from Spinach Purpose: Chlorophyll is extracted from spinach. From a spectrum of the solution produced, the ratio of chlorophyll a and b present is estimated. Introduction:

More information

CHEMISTRY Organic Chemistry Laboratory II Spring 2019 Lab #3: Friedel-Crafts Acylation

CHEMISTRY Organic Chemistry Laboratory II Spring 2019 Lab #3: Friedel-Crafts Acylation CHEMISTRY 244 - Organic Chemistry Laboratory II Spring 2019 Lab #3: Friedel-Crafts Acylation Purpose: In this lab you will predict and experimentally test the directing effects of substituent groups in

More information

Chemistry 3200 High Performance Liquid Chromatography: Quantitative Determination of Headache Tablets

Chemistry 3200 High Performance Liquid Chromatography: Quantitative Determination of Headache Tablets Chemistry 3200 High Performance Liquid Chromatography: Quantitative Determination of Headache Tablets Liquid chromatography was developed by Tswett in early 1900 s and was shown to be a powerful separation

More information

EXPERIMENT 17. Oxidation-Reduction Reactions INTRODUCTION

EXPERIMENT 17. Oxidation-Reduction Reactions INTRODUCTION EXPERIMENT 17 Oxidation-Reduction Reactions INTRODUCTION Oxidizing agents are compounds or ions that contain an element capable of achieving a lower oxidation state by gaining electrons The stronger the

More information

High Pressure/Performance Liquid Chromatography (HPLC)

High Pressure/Performance Liquid Chromatography (HPLC) High Pressure/Performance Liquid Chromatography (HPLC) High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the

More information

Chromatographic Methods of Analysis Section 2: Planar Chromatography. Prof. Tarek A. Fayed

Chromatographic Methods of Analysis Section 2: Planar Chromatography. Prof. Tarek A. Fayed Chromatographic Methods of Analysis Section 2: Planar Chromatography Prof. Tarek A. Fayed Planar chromatography includes two types: 1- Thin Layer Chromatography (TLC). 2- Paper Chromatography (PC). Thin

More information

Photosynthesis. Photosynthesis is the conversion of light energy to chemical energy and its subsequent use in the synthesis of organic molecules.

Photosynthesis. Photosynthesis is the conversion of light energy to chemical energy and its subsequent use in the synthesis of organic molecules. Photosynthesis Photosynthesis is the conversion of light energy to chemical energy and its subsequent use in the synthesis of organic molecules. In its simplest form the process can be expressed as: Carbon

More information

Supernatant: The liquid layer lying above the solid layer after a precipitation reaction occurs.

Supernatant: The liquid layer lying above the solid layer after a precipitation reaction occurs. Limiting Reagent Introduction The quantities of substances involved in a chemical reaction represented by a balanced equation are often referred to as stoichiometric amounts. Solution stoichiometry is

More information

Is There a Chemist in You?

Is There a Chemist in You? Young Scholars of Utica http://www.utica.edu/academic/yslpp/ Workshop January 8, 2011 Hosts: Professor Myriam Cotten, Lydia Rono, and Akritee Shrestha Is There a Chemist in You? 1 Schedule Morning Why

More information

Paper Chromatography Lab. Prepared for: Mrs. Freeman

Paper Chromatography Lab. Prepared for: Mrs. Freeman Paper Chromatography Lab Prepared for: Mrs. Freeman September 6, 2013 1 Introduction The separation of mixtures is an important part of chemistry. One such method of separation is called chromatography,

More information

CHAPTER 6 GAS CHROMATOGRAPHY

CHAPTER 6 GAS CHROMATOGRAPHY CHAPTER 6 GAS CHROMATOGRAPHY Expected Outcomes Explain the principles of gas chromatography Able to state the function of each components of GC instrumentation Able to state the applications of GC 6.1

More information

Liquid storage: Holds the solvent which is going to act as the mobile phase. Pump: Pushes the solvent through to the column at high pressure.

Liquid storage: Holds the solvent which is going to act as the mobile phase. Pump: Pushes the solvent through to the column at high pressure. High performance liquid chromatography (HPLC) is a much more sensitive and useful technique than paper and thin layer chromatography. The instrument used for HPLC is called a high performance liquid chromatograph.

More information

SEPARATION TECHNIQUES

SEPARATION TECHNIQUES SEPARATION TECHNIQUES If a substance does not dissolve in a solvent, we say that it is insoluble. For example, sand does not dissolve in water it is insoluble. Filtration is a method for separating an

More information

CK-12 FOUNDATION. Separating Mixtures. Say Thanks to the Authors Click (No sign in required)

CK-12 FOUNDATION. Separating Mixtures. Say Thanks to the Authors Click   (No sign in required) CK-12 FOUNDATION Separating Mixtures Say Thanks to the Authors Click http://www.ck12.org/saythanks (No sign in required) Forsythe Robinson To access a customizable version of this book, as well as other

More information

Chromatography: Candy Coating and Marker Colors Student Advanced Version

Chromatography: Candy Coating and Marker Colors Student Advanced Version Chromatography: Candy Coating and Marker Colors Student Advanced Version In this lab you will separate a mixture of unknown composition using several common household items. You will then perform a more

More information

Rule 2. Rule 1. Rule 4. Rule 3. Rule 5. Rule 6. Rule 7. Rule 8

Rule 2. Rule 1. Rule 4. Rule 3. Rule 5. Rule 6. Rule 7. Rule 8 Rule 1 Follow the directions in your course reader, of your teaching assistant and of your instructor. They are usually much more experienced doing chemistry. Rule 3 When in doubt, ask. This will make

More information

BY PRACTICAL SKILLS IN BIOLOGY

BY PRACTICAL SKILLS IN BIOLOGY BY1101 - PRACTICAL SKILLS IN BIOLOGY Practical 3 Molecular Techniques 3 Protein Purification by Gel-Filtration and Thurs 18 and Fri 19 October 2012 CONTINUING OUR INVESTIGATION OF CHROMATOGRAPHY Some molecular

More information

BIOL 221 Concepts of Botany

BIOL 221 Concepts of Botany BIOL 221 Concepts of Botany Topic 13: Photosynthesis A. Introduction Through photosynthesis, the abundant energy from the sun is collected and converted into chemical forms by photosynthetic organisms

More information

Introduction to Chromatography

Introduction to Chromatography Introduction to Chromatography Dr. Sana Mustafa Assistant Professor Department of Chemistry, Federal Urdu University of Arts, Science & Technology, Karachi. What is Chromatography? Derived from the Greek

More information

Minneapolis Community and Technical College. Separation of Components of a Mixture

Minneapolis Community and Technical College. Separation of Components of a Mixture Minneapolis Community and Technical College Chemistry Department Chem1020 Separation of Components of a Mixture Objectives: To separate a mixture into its component pure substances. To calculate the composition

More information

To explore solubilities and reactivities of different metal ions. To identify ions present in unknown solutions using separation methods.

To explore solubilities and reactivities of different metal ions. To identify ions present in unknown solutions using separation methods. Qualitative Analysis PURPOSE To develop a separation scheme and confirmatory tests for Fe 3+, Ba 2+, and Ag + cations, and to use it to identify the ions in a sample of unknown composition. GOALS To explore

More information

CHEMISTRY Organic Chemistry Laboratory II Spring 2019 Lab #1: Oxidation of Alcohols to Ketones - Borneol Oxidation (2 weeks)

CHEMISTRY Organic Chemistry Laboratory II Spring 2019 Lab #1: Oxidation of Alcohols to Ketones - Borneol Oxidation (2 weeks) CHEMISTRY 244 - Organic Chemistry Laboratory II Spring 2019 Lab #1: Oxidation of Alcohols to Ketones - Borneol Oxidation (2 weeks) Purpose. In this lab you will learn about oxidation reactions in organic

More information

Coordination Complexes

Coordination Complexes Coordination Complexes Experiment 9 Part I (Day 1) Synthesis and Analysis of Coordination Complexes Coordination complexes are formed between a metal ion (Lewis acid) and ligands (Lewis base). The splitting

More information

LAB FOUR PLANT PIGMENTS AND

LAB FOUR PLANT PIGMENTS AND LAB FOUR PLANT PIGMENTS AND PHOTOSYNTHESIS OVERVIEW In this lab you will: 1. separate plant pigments using chromatography, and 2. measure the rate of photosynthesis in isolated chloroplasts using the dye

More information

Experiment 6 Alcohols and Phenols

Experiment 6 Alcohols and Phenols Experiment 6 Alcohols and Phenols Alcohols are organic molecules that contain a hydroxyl (-) group. Phenols are molecules that contain an group that is directly attached to a benzene ring. Alcohols can

More information

METHOD 3665 SULFURIC ACID/PERMANGANATE CLEANUP

METHOD 3665 SULFURIC ACID/PERMANGANATE CLEANUP METHOD 3665 SULFURIC ACID/PERMANGANATE CLEANUP 1.0 SCOPE AND APPLICATION 1.1 This method is suitable for the rigorous cleanup of sample extracts prior to analysis for polychlorinated biphenyls. This method

More information

LAB 7 Photosynthesis

LAB 7 Photosynthesis LAB 7 Photosynthesis Introduction In order to survive, organisms require a source of energy and molecular building blocks to construct all of their biological molecules. The ultimate source of energy for

More information

Analytical Technologies in Biotechnology Prof. Dr. Ashwani K. Sharma Department of Biotechnology Indian Institute of Technology, Roorkee

Analytical Technologies in Biotechnology Prof. Dr. Ashwani K. Sharma Department of Biotechnology Indian Institute of Technology, Roorkee Analytical Technologies in Biotechnology Prof. Dr. Ashwani K. Sharma Department of Biotechnology Indian Institute of Technology, Roorkee Module - 3 Chromatographic Method Lecture - 1 Introduction and Basic

More information