1. Lab 2 Understanding Chemical Bonds Using Chromatography LJ2
High Performance Liquid Chromatography
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures.

2. Lab 2 Understanding Chemical Bonds Using Chromatography LJ2 In this lab we will apply the technology of chromatography to separate the pigments found within the cells of various types of plants. We will also investigate the best conditions required to separate these pigments. Our ultimate goal is to use chromatography to better understand the nature of chemical bonding. Lab Journal Setup Title on the appropriate page of LJ2 (remember we only use the front of each page) Under the title, date the left-hand margin where you will begin today’s entry (8/24/18) Partner’s or partners’ name(s) (can be added later) Complete the Table of Contents (can be completed later) Lab number and full lab title, and eventually pages and dates Beginning page (leave space for final page) Beginning date (leave space for final date) Copy and consider the objective Objective This lab is meant to allow us to better understand several things… the fundamental definitions of hydrophobic, amphipathic and hydrophilic compounds the behavior of hydrophobic, amphipathic and hydrophilic compounds the important science of chromatography Title the first section “Background Notes” Notes demonstrate your mental engagement

3. Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures. The mixture is dissolved in a solvent called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate.
or Solution

4. Chromatography is used by scientists to:
Analyze – examine a mixture, its components, and their relations to one another
Identify – determine the identity of a mixture or components based on known components
Purify – separate components in order to isolate one of interest for further study
Quantify – determine the amount of the a mixture and/or the components present in the sample
Chromatogram derived from spectrophotometer

5. High Performance Liquid Chromatography

6. Real-life examples of uses for chromatography:
Pharmaceutical Company – determine amount of each chemical found in new product
Hospital – detect blood or alcohol levels in a patient’s blood stream
Law Enforcement – to compare a sample found at a crime scene to samples from suspects
Environmental Agency – determine the level of pollutants in the water supply
Manufacturing Plant – to purify a chemical needed to make a product
Gel Electrophoresis
A modification of the chromatography technique widely used in forensic science
Used in many fields of biology, and used extensively in biomedical research

7. How does chromatography work? Real simple
You have a mixture
dissolved sample in solvent = mobile phase
The mixture is moved through a substance that does not move
stationary phase
The components of the mobile phase move at different speeds through the stationary phase
Differential speeds of movement are brought about in different ways, depending upon the chromatography technique used
This differential movement separates the components of the mobile phase

8. How does chromatography work?
Chromatography separates compounds within a mixture by using the differential affinities of the compounds for a mobile medium and for a stationary adsorbing medium through which they pass.
Terminology:
Differential Affinity – showing a different attraction/force between things
Mobile Medium – gas or liquid that carries the compounds in the mixture (mobile phase/solvent)
Stationary Medium – the part of the apparatus that does not move with the compounds (stationary phase)
Steps of Chromatography:
Mixture to be separated is placed on stationary phase
The compounds of the mixture have a differential affinity for the SP
Some are attracted to it more and some less
2. The MP is passed over the mixture
The compounds of the mixture have a differential affinity for the MP
Some are more soluble in it and some less
3. Compounds that are more soluble in the MP and have less of an attraction for the SP will be drawn with the MP further as it passes over the SP
This will separate the compounds of the mixture across the stationary phase

9. Different types of chromatography:
Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase)
Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase)
Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase)
Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)

10. Why are there differential affinities?
What determines different movement across the stationary phase, and ultimately, separation of components in the mixture?
Intermolecular forces between the compounds of the mixture and the MP vs the SP help to determine movement across the SP
Are the components of the mixture more soluble in the MP solvent, or are they attracted to some degree to the SP?
Size and sometimes shape help to determine movement of the compounds of the mixture across the SP
Intermolecular Bonds
Hydrogen bonds (polar)
Salt Bridges (ionic attraction)
Mixed bonds (ion and dipole)
Hydrophobic bonds
Dispersion forces
Liquid chromatography

11. Paper Chromatography Dip the edge of a paper towel in water and what do you see? The water “climbs” up the towel, seemingly in defiance of gravity The stationary page is chromatography paper, a special porous paper made with polar cellulose as well as non polar fibers The mobile phase solvent depends upon your sample and components of interest The mobile phase “climbs” up the paper due in part to capillary action The “differential affinity” is between the attraction to the paper versus the attraction to the climbing solvent
Capillary Action – the movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity.
Solubility – the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents.
Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and the stationary phase.

12. Rf spot A = 15 = 0.60 25
Rf spot B = 12 = 0.60 20

13. What do we recognize in these 3 molecules?
They are miscible with water…
What do we recognize in these molecules?
They are virtually immiscible with water, but as a mixture they are miscible with the three molecules on the left
The mixture is called petroleum ether

14. In this lab you will… Create three chromatography chambers
In each you will run two strips (for a total of 6 strips)
The chambers will have the following solvents
50% isopropanol/50% water (Chamber 1)
0% isopropanol/100% water (Chamber 2)
90% petroleum ether/10% acetone (Chamber 3)
Samples
Chamber 1 Two types of ink pens
Water soluble and water insoluble
Chamber 2 Two types of ink pen
Chamber 3 Two different mixtures of leaf pigment

15. How would we characterize these solvents?
50% isopropanol/50% water (Chamber 1)
0% isopropanol/100% water (Chamber 2)
90% petroleum ether/10% acetone (Chamber 3)

16. How would we characterize these solutes?
Which would be the best mobile phase (among those above)?