Physical Separations and Chromatography
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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
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