Lab 4- Paper Chromatography Flashcards
Explain the beginning of the lab.
Chromatography is an improv ant tool in chemistry and is used in almost every fact of the physical and biological sciences. In this experiments, we are going t separate and identify mixtures of chemical substances by paper chromatography. Most chromatographic techniques achieve separation because the various components of the chemical mixtures deferentially favor one of the two phases: the mobile phase (a pre-selected solvent) and a stationary phase (in this case paper). The chemical mixture is “spotted” onto the paper stationary phase and then placed into the solvent. As the solvent spreads through the paper, it creates a readily discernible solvent front followed by a trail of chemical components from the mixtures. A finished “chromatogram”is obtained once the solvent flow is stopped and the paper is allowed to dry. The chromatogram is the final picture of what has happened to the individual solutes with time. The “spots” on a paper chromatogram may be directly visible (as in the case with demes and food colors) or may require further treatment in order to achieve visualization (as in the case with certain ions or amino acids). UV light and chemical sprays (known as developing agents) are commonly used to visualize paper chromatography spots. The distance which components migrate may be quantitatively measured by calculating “Ratio to front” (Rf) values: Rf= distance component moved/ distance solvent moved.
What is the formula for ration to front values?
Rf= distance component moved/ distance solute moved (CS)
Define Capillary Action
Defined as the movement of water within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension.
Define Partition
Divide into parts.
What is the process of chromatography in this lab?
In paper chromatography, the solvent ascends the paper strip via capillary action and the components of the sample partition between the moving liquid phase and the stationary phase. Each component of the sample mixture experiences many equilibrio tío s between the moving and stationary phase.
Why does the separation of components occur?
Separation of the components occur because the components travel along the paper at different rates. Components that interact more strongly with the mobile (liquid) phase would travel up the paper strip at a faster rate than components that interact more with the stationary (paper) phase. The paper is removed and allowed to dry when the solvent front reaches a few centimeters from the top of the paper strip. As mentioned earlier, if the compounds in the mixture were colored we would see a vertical series of spots on the plate. However, most components are not colored and need to be detected by some other means (such as shining ultraviolet light on the paper, placing the paper ion the presence of iodine vapor, or a host of other chemical straining techniques).
In this experiment, we will prepare two paper chromatograms: the separation of dyes present in commercial food colors and the separation of some metal ion solutions.
What is the experimental procedure for PART A?
Technique: Separation of Dyes in Food Colors
Obtain a 4.5x4.5 inch piece of filter and draw a thin pencil line across the bottom of the paper (approximately 2cm from the bottom). Fold the paper in half so that the line you have drawn is bisected. In the same way, fold the papers in half a second and then a third time. The line will have been divided into 8 equal segments. Mark the approximate center of each of the five inner segments of the line with pencil. These marks will be for the four known food colors and the “unknown” liquid (which contains one or more of the colors).
Add one drop of each food color and the unknown to separate components (“wells”) of a multi well tissue culture plate (“well plate”). Leave several empty wells in between to prevent cross-contamination of the solutions. Spotting is accomplished by dipping a toothpick in the solution, draining and then touching the tip onto the spots marked on your paper chromatogram. Try not to allow the spots to get too large. You may add more samples by tying the paper and re-spotting on the same spot. Label each spot in pencil (usually with a letter or symbol) just below each spot.
Once the spots have dried, fold the paper into an accordion shape and place the paper, spotted end first, into an 800-mL beaker, to which 15mL of 0.1% NaCLl solution has been added. The level of the liquid in the beaker should not exceed the spotting lines on your paper strips. Do not allow the paper to come in contact with sides of the beaker.
As soon as you have placed the paper in the solution, tightly wrap plastic film over the top of the beaker. The solvent should begin to travel up the paper strips immediately. Let the chromatography continue until the solvent front has traveled to within 2 cm of the top of the paper strip. In the meantime, proceed to part B. When this chromatogram is finished, dry your chromatogram and record your observations in your data section. You will not need to calculate Rf values for part A. Sketch your chromatogram in your laboratory notebook. Be sure to identify which dyes are present in your unknown mixture.
Wash the well plate. Rinse it with distilled water and pat it dry with a paper towel before using it in part B.
What is the experimental procedure for part B?
Technique: Separation of Metal Ions
Obtain another 4.5x3.5 inch piece of filter paper and dry 600-mL beaker and place about 18mL of acetone (c3h6O) and 2 mL of 6 M Hydrochloric acid (HCl) into the beaker without getting any liquid on the sides of the beaker. Draw a pencil line across the bottom and fold the filter paper as in part A. Mark the approximate center of each of the inner 4 segments. Write “Co2+” under the first mark, “Ni2+” under the next, “Cu2+” under the third, and unknown under the fourth mark. Spot each mark with the appropriate solution as was done in Part A. Dry the paper, then ref old in accordion and place it in the 600-mL beaker so that the 2-cm pencil line is above the level of the liquid in the beaker. Be sure to not let the sides of the paper touch the walls of the beaker. Cover the beaker tightly with plastic film and allow the liquid to ascend within 2cm of the top of the paper (around 40mins).
When the liquid has reached the desired height, remove the paper from the beaker. Place the wet paper on a power towel and mark with a pencil the position to which the liquid reached (the solvent form). PARTIALLY dry the paper by gently waving it in the air.
CAUTION: Ammonia vapors and solutions are irritating to the nose, eyes, lungs, and skin. Perform the following procedure in the hood while wearing gloves.
Wearing gloves, hold the dry paper over the beaker containing a solution of ammonia (NH3) for 1-3 mins. Formation of a small amount of “smoke” is normal. Note the color, and circle with a pencils, any spots which become visible after contact with the ammonia vapors. Next, lightly spray the paper with a solution of dimethylglyoxime. If you do not observe a spot for Ni2+, expose the paper again to the ammonia vapor. Allow the paper tod ray and note the color of any spots which become visible or change appearance. Circle any new spots which appear. A colored spot should no be visible for each substance.
Using the cm scale on the inside cover of the manual, measure the vertical distance that the approximate center of each of these spots has traveled from the original mark on the 2cm-line. Your measurements should be to the closest tenth of a centimeter. Record results in the lab notebook.
Why do most chromatographic techniques achieve separation?
Because the various components of the chemical mixtures deferentially favor one of the two phases: A mobile phase (pre-selected solvent) and a stationary phase (in this case, paper)
What substances, known as developing agents, are commonly used to visualize paper chromatography spots?
UV Light and Chemical Sprays
Why does separation of components occur?
Because the components travel along the paper at different rates.
Components that interact more strongly with the mobile (liquid) phase would travel up the paper at a faster rate than components that interact more strongly with the stationary (paper) phase.
How are most components that are not colored detected?
Shining UV light onto the paper-Placing the paper in the presence of a iodine vapor- A host of other chemical staining techniques. UIO (UV-Iodine Vapor-Others)
Explain why all Rf values should be between 0 and 1.
Due to the fact that the solvent front is ALWAYS larger than the distance travelled by the solute, Rf values are always between 0 (an extreme where the solute remains at its origin) and 1 (the extreme solute is so soluble that it moves as fast as the solvent).
