003 Liquid Chromatography

Expt 003 -- Liquid Chromatography

Chromatography is a technique in which components of a mixture are separated from one another on the basis of differences in solubility and attraction for two phases -- a moving phase and a stationary phase. The components of food coloring are separated using water or ethanol as the mobile phase and silica gel as the stationary phase.

Background

The name chromatography comes from the early experiments in which this procedure was used by Tswett to separate colored pigments. The sample is dissolved in a small amount of a weak solvent and applied to the column. A stronger solvent for the sample flows over the sample carrying it along the column.
The liquid phase is called the mobile phase or eluting agent. The portions that are collected from the column are called fractions. Fractions are eluted from the column by the mobile phase.
The solid material in the column is called the stationary phase.
As the mobile phase moves through the column the solutes adsorb or react with the stationary phase. For a separation to take place, this reaction must be a rapid equilibrium with respect to the speed of the mobile phase. The mobile phase keeps changing the concentration of the dissolved solutes. This change shifts the equilibrium according to Le Chatelier's Principle.
Even small differences between solutes are enough to separate substances with liquid chromatograph if the column of stationary phase is long enough.
Separations are faster when the mobile phase moves more rapidly. Increasing the pressure on the mobile phase increases the flow rate of the mobile phase. As long as the equilibrium between the solutes and the stationary phase is much faster than the flow rate of the mobile phase, the separation will improve. In this experiment, pressure is applied with a syringe to increase the speed of the separation.
Pumps and steel columns are used in high performance liquid chromatography (HPLC) to allow pressures of 3000 to 10000 pounds per square inch (psi). The color or electrical properties of the mobile phase is measured continuously, and the data is collected and analyzed with computers. When a colored fraction is collected, the computer records a peak and calculates the amount of colored substance in the sample. Commercial HPLC systems offer automatized analyses in nearly every analytical laboratory.
In this experiment, elution begins in one solvent and changes to another solvent. This techniques is frequently used in analytical laboratories to improve and speed up analyses. Note the important differences that the eluting solvent makes.

Safety

Be extremely careful when placing the rubber tubing over the end of the Pasteur pipet. Cuts are possible. Avoid flames and ignition sources. Know the location of the fire equipment; review use of that equipment before beginning work.
Handle the dry silica gel carefully at a hood. The solid silica gel forms an aerosol that is damaging to lungs. The room must be well ventilated.
The silica may be dispensed as a slurry. Be sure to mix the slurry before withdrawing a sample. Avoid spilling the slurry by using a beaker with your pipet at all times.

Procedure

Place a glass Pasteur pipette in pipette holder.

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Use a stick to push a wisp of cotton the size of a pencil eraser tip to the bottom of the pipette to form a plug.

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Roll a small piece of paper so that it makes a funnel. Add sand to the pipette using the funnel. The layer of sand should be about 0.5 cm high. If there is too much sand, dump some out. Adjust the pipette until perpendicular, and tap the side of the pipette until the sand is level.

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At a hood, add 5 mL of water to 2 cm³ of silica gel in a small beaker. (Use caution; dry silica gel is a very fine powder which can be inhaled and cause lung irritation.) Stir the water and silica with the tip of a plastic transfer pipet. Once all of the silica is wet, return to the bench. Mix the solution further by drawing it up in the pipet and squirting it out quickly several times until the silica is suspended in the water.

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Without allowing the silica time to settle, draw it into the transfer pipette and transfer it to the glass pipette. Fill the glass pipette to half a centimeter from the top with this silica slurry.

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Place a collecting beaker below the glass pipette. Pull the plunger out as far as it will go in the syringe. Attach the syringe to the column.

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Compress the plunger to push water out the bottom of the column. A layer of silica should begin forming from the bottom. You are looking for the interface with the densely packed material-not the diffuse dispersion in the solution above.

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When the water level reaches the top of this layer, release pressure on the syringe plunger and remove the syringe.

Note: Whenever removing the syringe from the column, it is necessary to release the plunger slowly, so that liquid is not pulled up the column by suction. Try not to let the top of the silica go dry during any part of this experiment.
Use a plastic transfer pipette to put one drop of green food coloring on the top of the column. If any food coloring sticks to the sides of the pipette before reaching the silica, remove it using a cotton swab.

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Use the syringe to push the dye just below the surface of the silica. Remember not to let the top of the silica go dry. Add three drops of water. And push the liquid down as far as it will go without the top getting dry. Repeat this three drop addition of water two more times.

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The solution on top of the column should appear colorless for the last fraction before filling the column.

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Fill the pipette close to the top with water. Use the syringe to push out more water. Set up a 96-well plate to collect dye fractions. Begin collecting, whenever a color reaches the bottom of the column. Switch the wells every time they fill up. Also, switch the wells whenever one color stops coming out or another color begins to come out.

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After the yellow dye is all collected, fill the pipette close to the top with 95% ethanol. Continue collecting fractions. Note the solvent front.

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Optional

If time permits, add a second sample. This time elute with ethanol first and water second. Use the same column and begin as soon as the dye from the first sample is completely washed of the column.
Use a plastic transfer pipette to put one drop of green food coloring on the top of the column. If any food coloring sticks to the sides of the pipette before reaching the silica, remove it using a cotton swab.
Use the syringe to push the dye just below the surface of the silica. Remember not to let the top of the silica go dry. Add three drops of 95% ethanol. Push the liquid down as far as it will go without the top getting dry. Repeat this three drop addition of 95% ethanol two more times.
Fill the pipette close to the top with 95% ethanol. Use the syringe to push out more liquid. Set up a 96-well plate to collect dye fractions. Begin collecting, whenever a color reaches the bottom of the column. Switch the wells every time they fill up. Also, switch the wells whenever one color stops coming out or another color begins to come out.

!!!Click here to See Movie. Movie is speeded up 5 times by time lapse techniques.
Switch to water when the first blue fraction has been eluted.
Wash the column with water until no color remains. Return the used column to the instructor.

!!!Click here to See Movie. Movie is speeded up 5 times by time lapse techniques.
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Questions

Explain the relationship between the pigments observed from the food coloring and the green color.
Explain the differences observed when water and ethanol are used as solvents.
Describe the role played by the syringe.
Discuss the advantages and problems of each elution solvent system.

Handout Makeup

Name ___________________________ Class _______

Teacher __________________________

SmallScale 003 Liquid Chromatography

Watch the movies and record your observations.

Describe the elution when water is added first.
Describe the elution when alcohol is added first.

Curriculum-

Separation is usually taught early in a chemistry course, when the notions of mixtures, compounds, and elements are introduced. This experiments fits in early with these discussions. It has many applications, and can be used wisely nearly anytime during an introductory chemistry course. Because the ideas are so powerful, and the treatments of the theory usually cursory for general student audiences, the earlier, the better for chromatography.

Safety-

Be extremely careful when placing the rubber tubing over the end of the Pasteur pipet. Cuts are possible. Avoid flames and ignition sources. Know the location of the fire equipment; review use of that equipment before beginning work.
Handle the dry silica gel carefully at a hood. The solid silica gel forms an aerosol that is damaging to lungs. The room must be well ventilated.
Teachers may wish to dispense the silica gel as a slurry in the hood. Be sure to tell students to mix the slurry before withdrawing a sample.

Time-

Teacher Preparation: 15 minutes

Class Time: 50 minutes (One separation requires about 40 minutes. The second separation on the same column adds about 10 minutes.)

Materials-

Schilling green food coloring (The dye components are on the label. Note a green dye, which is more water soluble and less alcohol soluble than the blue dye, was isolated. Only one blue dye was listed.)
95% ethanol (optional but recommended)
5 g silica gel type G (May wish to prepare slurry by adding 5 mL of water to 2 cm† of silica gel per group.)
uniform sand
cotton
15-cm disposable Pasteur pipets
plastic transfer pipets
3-cm† plastic syringe (without needle)
tubing which will fit snugly over the syringe and the Pasteur pipet. Test the tubing to be sure it is a tight fit.
Pipette Holder-- Take a number 4 rubber stopper with a hole and Cut one fourth of a 1-hole # 4 rubber stopper with a razor blade lengthwise so that the hole is opened.
stick to push cotton down (25 cm wooden BBQ skewer works well or use long stem plastic transfer pipet)

Disposal-

Use wooden skewers to remove most of the silica gel from the columns. Allow the silica gel to dry, and save it for reuse in this experiment. Discard the remaining silica gel, sand, and cotton with ordinary solid trash. All of the liquids used may be discarded safely at the sink.

Lab Hints-

Pressure on the syringe plunger must be released slowly before removing the syringe to add additional solvent, or the packing material will be disturbed. Warn or demonstrate before students begin.
You may wish to dispense the silica gel as a slurry. See safety instructions.

Answers-

Q1. Explain the relationship between the pigments observed from the food coloring and the green color. A1. The blue and yellow pigments together make a green solution.
Q2. Explain the differences observed when water and ethanol are used as solvents. A2. The order of elution is reversed. It suggests that the blue dye is more soluble in ethanol than is the yellow dye, and vice versa for water.
Q3. Describe the role played by the syringe. A3. The added pressure from the syringe increases the flow rate making the experiment go at a more convenient rate.
Q4. Discuss the advantages and problems of each elution solvent system. A4. The water separated two components completely. Adding the alcohol during the procedure sped the elution of the blue. Adding alcohol first did not completely separate the main blue and yellow components. A third green component which was not observed previously with the water was separated.

Handout Ans.-

See Q4 above for a brief description of fractions collected. Yellow elutes first in water. Blue elutes first in alcohol. A small green band elutes last from the column started with alcohol and finished with water.

CoopLearn-

Consider having the students work in groups of two. Different groups may be assigned different dyes. Since preparing the columns is the slow step, the experiment is only shortened by about 10 minutes if some groups are assigned water first and others are assigned alcohol first.

Key Words 1-

solvent, solute, chromatography, liquid chromatography, stationary phase, mobile phase, Rf, separation, pigment, chromatogram, eluant, fraction

Elements-

Si C