1. By Susan McCullough With Thanks to Lori Olson at SRI International

2. Why Chromatography? California State Standard 6f Solutions Students know how molecules in a solution are separated or purified by the methods of chromatography

3. Please set up your Paper Chromatography 1. Using a pencil draw a line across the bottom of your filter (1.5 cm from the bottom). 2. Place a small dot (0.5 cm) using your Vis a Vis pen just above the line 3. Place 7 ml. of hot water into your 250 ml. beaker. 4. Fold your filter paper in half lengthwise 5.Place your filter paper into your beaker (spot side down) and cover with a watch glass 6.Remove your paper when the water reaches 0.5 cm from the top of the paper. 7. Mark the boundary of each color and the solvent in pencil and let dry

4. Uses of Chromatography http://www.buzzle.com/articles/chromatography-uses.html Drug Testing – Urine is analyzed for known drugs Firefighting-identifying chemicals found in fires Forensics- Separate and analyze substances found on the crime scene Pharmaceutical- Chemists make compounds and purify them.

5. Our Goal We will be separating the pigments of leaf extract using column chromatography. Leaves are green…..right……. Mikhail Semyonovich Tsvet, the inventor of column chromatography, (Russian botanist) used it to separate a variety of pigments from leaf extract.

6. What is Chromatography? http://upload.wikimedia.org/wikibooks/en/thumb/3/36/Paper_Chromatography _SH.png/220px-Paper_Chromatography_SH.png *Chromato = color *Graphy = writing Chromatography is the separation of a homogeneous mixture using a stationary and mobile phase.

7. Identify the following on your paper chromatography Stationary Phase- filter paper Mobile Phase- water Analyte/mixture- black pen Your Paper Chromatography http://www.triplescience.org.uk/tripsci/chemistry/effectiveteaching/images/c_fig11.gif

8. How does it work? An analyte (mixture) is placed on the stationary phase. The mobile phase passes through the stationary phase Components of the mixture are separated according to their affinity for the stationary and mobile phases.

9. Basic concept: “Like dissolves Like” This process only works if the mixture will dissolve in the mobile phase. (See paper chromatography of vis a vis pen in alcohol) Components of a mixture are separated according to their affinity for the mobile and stationary phases. Nonpolar components will stay with the nonpolar phase. Polar components will stay with the polar phase In your Paper Chromatography Paper = nonpolar Water = polar

10. Let’s Review Polarity http://www.green-planet-solar-energy.com/electronegativity-values.html Polar molecules have atoms that do not share electrons equally. Nonpolar molecules share electrons equally. EN difference –>2.0 is ionic –0.4-2.0 is polar –< 0.4 is nonpolar

11. On your Paper Chromatography The polar colors traveled with the water (polar). The nonpolar colors stayed behind stuck to the paper (nonpolar). List the colors in order from nonpolar to polar –Yellow, red, blue

12. Identifying Parts of a Mixture http://materialsworld.utep.edu/Modules/Concrete/Chromatograp hy/Retention_factor.png (picture) http://materialsworld.utep.edu/Modules/Concrete/Chromatograp hy/Retention_factor.png Parts of the analyte/mixture are identified by how far they travel on the stationary phase Rf- Retention Factor Rf = cm traveled by sample cm traveled by solvent Rf of blue sample = 2.5/10 = 0.25 Rf of red sample = 7.5/10 = 0.75

13. Kinds of Chromatography Paper Chromatography- Paper is the stationary phase. The mobile phase is a liquid. Thin Layer Chromatography- A glass plate covered with a layer of silica or aluminum oxide is the stationary phase. Liquid is used for the mobile phase Column Chromatography- a column of silica beads are used for the stationary phase. The mobile phase is a liquid or a gas.

14. Why Use Column Chromatography? http://www.chem.ucla.edu/~bacher/General/30BL/tips/column.jpg Column chromatography is used when a scientist wants to collect separated parts of a mixture. Column chromatography will allow a scientist to collect only his or her compound (purification).

15. How It Works http://www.m2c3.com/chemistry/VLI/M4_Topic2/la_16_07.jpg

16. How to separate clear compounds using modern technology. http://www.biologie.uni-hamburg.de/b-online/ge17/12b.jpg A sample is injected onto the column and solvent is pumped through the column. A UV light shines through the solvent as it exits the column. When compounds are detected, they are collected. Their absorbance of light is recorded.

17. Identification of Fractions A mass spectrometer determines if the fraction that we have collected has the right molecular mass. http://www.kitmondo.com/category.aspx?CatID=126&cat Name=Mass-Spectrometer&mfr=Thermo-Finnigan A Nuclear Magnetic Resonance (NMR) is examined to determine if the fraction that we have collected contains the correct molecular structure. http://www.labwrench.com/?equipment.view/equipmentNo/9327/Va rian/300-MHz-NMR/

18. How did you do ? __________is the separation of a mixture. A _________ phase is passed through a _________phase. Nonpolar components of a mixture will stay with the _________ phase Polar components will stay with the ________ phase. Rf = _________ factor is used to identify a compound _________ chromatography is used to collect fractions of a mixture.

19. Let’s Check _Chromatography__is the separation of a mixture. A _mobile_ phase is passed through a stationary_phase. Nonpolar components of a mixture will stay with the _nonpolar_ phase Polar components will stay with the polar_ phase. Rf = Retention_ factor is used to identify a compound Column chromatography is used to collect fractions of a mixture.