Basic Lab Skills

Filtration

Filtration is a technique used to separate a solid from a liquid and is used in many labs. Though filtration can be done simply with gravity, more often vacuum filtration is employed because it is much faster. Several points are worth remembering. First, be sure to clamp your filter flask down. These flasks are easily upended. Place a water trap between your filter flask and the aspirator. Also, be sure to use thick-walled vacuum tubing and not the thin walled tubing used for water lines.    You should also turn the water on full blast to achieve the maximum vacuum.
The Water Trap
Filtration Water Trap
1) Turn water on full blast when filtering. Otherwise please turn water off as we have found that after prolonged used the cup sinks will leak into the lab drawers, onto the floor, and through the ceiling of the basement labs!2) Close pinch clamp to filter. Open clamp to release vacuum.3) Clamp should be used to keep trap from tipping.Also notice that thick-walled tubing is used. Please keep the water trap assembled on the benchtop at all times. Do not disassemble the water trap in an attempt to clean up after an experiment. You may disassemble, clean, and reassemble the water trap as necessary, but it should be left assembled and ready for use all the times.
You will need to find a filtration flask (Erlenmeyer with side arm), vacuum adaptor, Buchner funnel, filter paper (5.5 cm diameter), thick-walled rubber tubing, and a spatula. Supplies
Filtration Supplies
Typical Apparatus Setup
Typical Apparatus Setup

1) Thick-walled rubber tubing is used.
2) Pinch clamp should be closed during filtration..3) Clamp filter flask securely.Once your setup is complete, place a piece of filter paper on the filter and wet it with the solvent that you will be pouring through filter. A mixture of a solid and a liquid can be separated by simply pouring through the filter. Solid that remains in your flask can be further transferred with the aid of a spatula or with a little more solvent.
After filtration, the solid or crystals that you collect can be washed with a small amount of a solvent in which they have a low solubility. Often, a solvent cooled in an ice bath is a good choice. After washing, the apparatus can be disassembled by breaking the vacuum. You may either open the pinch clamp or remove the vacuum tubing from the filter flask. If you turn off the water before releasing the vacuum, you will likely draw water into the water trap (if you remembered to use the trap!) or into your filter flask, which is undesirable.

 

Crystallization

Crystallization is a technique used to purify solid compounds. This success of this process is based upon the different solubility of the desired compound and impurities in a given solvent and also the fact that all compounds are more soluble in a hot solvent than  a cold one. In general, the crude product crystals is dissolved in a minimal amount of hot (boiling) solvent. A boiling stick or stone  can be added to ensure smooth boiling. At this stage, any material which does not dissolve can be removed by filtration. The hot solution  is allowed to slowly cool and crystals of the desired material form while the impurities remain in the solvent. The crystals are usually collected by filtration. While this technique does result in some product loss, the resulting crystals are much more pure. This procedure is outlined below for crystallization of benzoic acid from water.


Above on the left, the powdered benzoic acid is added to water and boiled. Be sure to add a boiling stick or chip to avoid boil overs. On the right the benzoic acid has dissolved and the excess solvent was boiled off and the solution was allowed to cool at room temperature.


    On the above left, the crystals have begun to form. After the crystals and solvent has reached room temperature, the flask can be transferred to an ice bath for further cooling. On the above right, the crystals and a wash solution to be used during the filtration step are in the ice bath.



The filtration apparatus is shown above. Be sure to note that the water is turned on all the way and the flask is clamped down. The crystals will be poured on to the filter. Be sure that before adding the crystals some liquid is added and filtered to ensure that the filter paper is secure. Wash the crystals with solvent and allow to dry. On the right above is the starting benzoic acid (hexagonal weigh boat), a powdery white substance, and the final crystals (watchglass), shimery white flakes.

 

Distillation

 


Please note: (from top) use of clips to hold apparatus together, position of the thermometer, and the clamp holding the round bottom flask in place.

Please note: from left: use of clamp to hold collection flask, white tubing is where water goes in, red tubing is where water goes out.

Distillation is probably the most common technique for purifying organic liquids. In simple distillation, a liquid is boiled and the vapors work through the apparatus until they reach the condenser where they are cooled and reliquify. Liquids are separated based upon their differences in boiling point. Two important things to note: 1) the tip of the thermometer must be correctly positioned slightly below the center of the condenser to accurately reflect the temperature of the vapors (see above left) and 2) the water supply should be connected to the lower port in the condenser and the drainage tube connected to the upper (in the picture on the right the right tube is connected to the water supply and the red tube is a drainage tube). Also be sure to use the thin-walled tubing and not the heavy walled vacuum tubing. Be very careful that your water lines do not come in direct contact with your hot plate, as the lines could melt resulting in a flood. Be sure to clamp both the round bottom boiling flask and the collection tube. Knocking over your collection tube at the end of the experiment if VERY frustrating. Below is a diagram of assembly: Generally, boiling stones will be added to the boiling flask to ensure even boiling. It is also wise to use some type of clamps to connect the various pieces of the distillation apparatus together. For low boiling liquids, enough heat may be provided simply by resting your flask on the hot plate (as shown above). You can also insulate your boiling flask and Claisen adaptor with aluminum foil. For higher boiling liquids it may be necessary to use an oil or sand bath to reach higher temperatures. The individual pieces of glassware needed for a simple distillation are diagrammed below.

Be sure to use the blue clips to attach the vacuum adaptor and the Claisen tube and the distillation adaptor.

 

Fractional Distillation

The setup for a fractional distillation is very similar to that for simple distillation. The only difference is the addition of a fractional distillation column, usually packed with some material of high surface area that produces a more efficient separation than the simple distillation. The same advice regarding the thermometer placement, clamping, and hook-up of the water tubes in the simple distillation also apply to the fractional distillation. As this apparatus is larger, practice additional caution to be sure that no glassware is broken or product loss. The choice whether to use the simple or the fractional setup will depend on the compounds that you are trying to separate. Obviously, the simple distillation setup is simpler and the distillation generally will be quicker than the fractional. However, the fractional setup is more efficient at separating liquids with fairly similar boiling points and at times is required.

 


On the left, note that the tube connected to the spigot is connected to the lower part of the condenser, while the drainage tube is connected to the higher part.  Also, note the position of the thermometer.

Please note the use of clamps to secure the round bottom flask and the collection flask.

 

Pictured on the left is the fractional distillation apparatus. A closer shot is on the right. Notice that the only difference between this apparatus and the simple distillation apparatus is that here, the fractional distillation column has been placed between the boiling flask and the distillation head. As in the simple distillation apparatus, not that the white tubing is connected to the water supply and the red tubing is the drainage tube. Also notice the position of the thermometer, blue clips, and clamps. A diagram of the apparatus is below.

 

 

 

 

 

 

Extraction

Extraction is a technique used to separate compounds based upon their different solubilities in two solvents that do not mix. Most commonly, one of the solvents will be water and the other will be an immiscible organic solvent (often methylene chloride, diethyl ether, or ethyl acetate). In general, very non-polar compounds will partion to the organic solvent and very polar compounds and salts will partion to the aqueous phase. Since the two solvents do not mix, they can be separated in a separatory funnel providing a very quick and easy way to separate compounds. Whether the water layer is on the top or bottom depends on the density of the other solvent (methylene chloride is heavier than water and goes to the bottom of the separtory funnel and diethyl ether and ethyl acetate are lighter than water and stay on the top). This technique when coupled with acid-base chemistry provides a very powerful method for separating organic acids, bases and neutral compounds from one another. By treating your organic layer with either an aqueous acid or base solution one can ensure that organic bases or acids are converted to their corresponding conjugate acids or bases respectively. These compounds will be charged and thus favor the water layer allowing them to be separated from other organic compounds. To see a general scheme for separating organic acids, bases and neutral compounds go here.

 

Note the use of the 2 inch ring to support the funnel.  Also, the top yellow and the bottom clear liquid are immisible.

Bottom arrow:  Note that the stopper is firmly held in place.  Top arrow: Turn the stopcock to vent the funnel. While the lower layer is drained from the funnel it is necessary to remove the stopper.

In the left panel, a separatory funnel is shown containing two immiscible liquids. This sample is actually the mixture of the organic compound benzil, which is yellow, in diethyl ether and water. Since diethyl ether is less dense than water, it is easy to see that the organic compound is dissolved in the organic solvent (like dissolves like). Notice that the separatory funnel is placed in a securely clamped 2 inch ring. To be sure that the compounds are completely partioned, the separatory funnel is shaken with a stopper on top and with the bottom stop-cock closed. The center panel illustrates the mixing/venting process. When mixing be sure that the separatory funnel is corked and that the stopcock is closed. Shake the flask and then with the bottom of the flask pointing upward and not toward yourself or your neighbor, open the stopcock to vent the pressure in the flask. Be sure to vent the separatory funnel often to avoid pressure build-up. Place the flask back in the ring stack and allow the layers to separate again. Place a receiving flask under the funnel, remove the stopper from the top and open the stopcock and allow the bottom layer to flow out of the funnel. Close the stopcock and you have now separated the two layers. One should always keep both layers until the desired compound is isolated and one should also carefully label the layers that they have separated. In general, you will have to “dry” the organic layer by placing it over magnesium sulfate or sodium sulfate.

TLC

Thin Layer Chromatography (TLC) is a powerful technique to separate compounds based upon their polarity and interaction with silica and to assess the purify of a sample. To perform TLC, a solution of a compound or mixture of compounds is applied to a TLC plate by using a thin capillary tube. First, make a thin pencil mark on your TLC plate about one-quarter of an inch up from one end. Dip the capillary tube into the solution of compound and then touch the tube onto the line on your TLC plate. Now place they plate into a developing chamber (can be a simple beaker with some filter paper and aluminum foil cover). The developing chamber should have some developing solvent in it but the level of this solvent should not be above the pencil mark on your plate. Allow the solvent to move up the TLC plate and remove the plate when the solvent nearly reaches the top. Mark the distance as to where the solvent traveled. Now visualize your plate by first noting any spots you can visually see, then spots you can see with the aid of an ultraviolet light source, and then lastly by adding your plate to an iodine chamber. Once your spots have been visualized you can calculate the Rf value of each, the Rf value is also a physical constant of an organic molecule.

This procedure is shown below.

Pictured at the left is a TLC plate. The white matte surface pictured is the solid phase of this chromatography procedure. The solid line on the plate is drawn one centimeter from the bottom of the plate IN PENCIL. This is the line on which the the sample is applied. The labels from left to right read C, A, and U, represent caffeine, aspirin, and the unknown.

 

 

 

 

 

 

 

 

The chamber in which TLC takes place is filled to less than one centimeter of solvent (ethyl acetate and acetic acid). It is extremely important that the solvent in the developing chamber be filled to LESS THAN ONE CM because the solvent must be drawn upward through the sample in order to draw the sample along with it. If the sample is dipped in to the solvent, it will damage the results. A piece of filter paper is placed in the chamber to draw the solvent into the top of the chamber. Finally it is important to cover the chamberto be sure that the solvent does not evaporate.

 

 

 

 

The samples are applied to the TLC plate with a capillary tube. To draw sample into the tube, heat the one end of the tube on a hot plate and then dip the cool end into the sample. This will draw the sample up into the tube. Then spot a small amount of sample onto the TLC plate as pictured at the left. Note that the sample is applied on the 1cm line.

 

 

 

 

 

 

After all samples are loaded, the plate can be placed in to the chamber. Be sure that the solvent is below the line on which the samples were applied and that the plate is not touching the filter paper. Also, keep an eye on the plate. It only takes a few minutes for the solvent to travel up the plate. When the solvent reaches approximately one centimeter below the end of the plate, remove it from the chamber. Be sure that the plate does not touch the filter paper.

 

 

 

 

 

As soon as the plate is removed from the chamber, mark the solvent front for later calculations (top arrow). Allow the plate to dry. After the plate is dried, two methods can be used to visualize the location of the sample on the plate. Ultraviolet light can be used. When UV is used, the area of the plate surrounding the solvent will appear fluorescent, while the solvent does not. Another way to visualize the sample is using an iodine chamber. Placing a few pieces of iodine in a covered container and then adding the plate will turn some samples brown. In either case, using a pencil outline the location of the sample for later calculations (bottom arrow). These calculations are shown below.

 

 

 

Rf is calculated as shown.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Melting Point

Melting Point is used to evaluate the purity of product. A MelTemp apparatus is utilized in this procedure.

 

  In the picture on the right please note:  from top, position of the meltemp tube, the viewing area, and the heat control.

 

 

 

 

 

 

 

 

 

 

 

Column Chromatography

 

Column chromatography is frequently used by organic chemists to purify liquids   (and solids.) An impure sample is loaded onto a column of adsorbent, such as   silica gel or alumina. An organic solvent or a mixture of solvents (the eluent)   flows down through the column. Components of the sample separate from each other   by partitioning between the stationary packing material (silica or alumina)   and the mobile eluent. Molecules with different polarity partition to different   extents, and therefore move through the column at different rates. The eluent   is collected in fractions. Fractions are typically analyzed by thin-layer   chromatography to see if separation of the components was successful.

Packing a (silica gel) column:
  1. Use a piece of wire to add a plug of cotton to the bottom of the column. There should be enough cotton that the sand and silica will not fall out of the column. However, too much cotton or cotton packed too tightly will prevent the eluent from dripping at an acceptable rate.
  2. Clamp the column to a ring stand and add enough sand to fill the curved portion of the column.
  3. Place a pinch clamp on the tubing, then fill the column 1/4 to 1/3 full with the initial eluent. (The composition of eluent is often changed as the separation proceeds.)
  4. Prepare a slurry of silica in the initial eluent by pouring dry silica into a beaker of eluent. (Add a volume of silica gel, such as 20 mL, to approximately double the volume of eluent, 40 mL.) CAUTION: keep the dry silica in your hood and be careful not to inhale the lightweight substance.

Step 1

  1. Quickly but carefully pour the slurry into the column. Stir and pour immediately to maximize the amount of silica that goes into the column instead of remaining behind in the beaker. You may find a clean spatula or glass rod helpful in transferring the silica.
  2. Remove the pinch clamp to allow solvent to drip into a clean flask. Tap on the side of the column with a rubber stopper or tubing to help the silica settle uniformly.
  3. Use a Pasteur pipet to rinse any silica that is sticking to the sides of the column. Allow the silica to settle while eluent continues to drip into the flask.
  4. Once the silica has settled, carefully add sand to the top of the column. Sand is heavier than silica. If the silica has not settled, the sand may sink into the silica instead of forming a layer on top of it. (You may need to rinse down sand that sticks to the side of the column.
Column after Step 3 Step 5

Column after Step 9

Loading a sample onto the column:
  1. Drain eluent from the column until no solvent remains above the surface of the sand.
  2. Using a long Pasteur pipet, carefully add your sample to the column.
  3. Drain eluent from the column until no sample remains above the surface of the sand.
  4. Use ~ 1 mL of eluent to rinse your container and pipet. Add this milliliter of sample to the sand. Drain eluent from the column until no liquid remains above the surface of the sand.
  5. Repeat step 12 two or three times to completely transfer your sample           onto the silica gel. If you do not do and repeat step 12, your sample           will remain in the sand instead of on the silica. Sample remaining in           the sand will dissolve in the eluent that you add in step 14, ruining           the possibility of good separation of components.
Step 10
Eluting the sample:
  1. Once you have rinsed your sample onto the silica, carefully add eluent to the top of the column. To avoid disturbing the top of the column, it’s a good idea to carefully pipet an inch or two of solvent onto the column instead of pouring solvent directly onto the sand.
  2. Add more eluent as necessary. The eluent collected prior to the elution of sample can be recycled. The composition of the eluent can be changed as the column progresses. If the eluent composition is to be changed, ALWAYS start with least polar solvent/mixture and change to the more polar solvent/mixture.
Column after Step 13 Components a, b, and c separate as column progresses. Fractions can be collected in test tubes,           vials, beakers, or Erlenmeyer flasks.
Analyzing the fractions:
  1. Analyze the fractions by thin-layer chromatography to determine a) if the fraction contains more than one component and b) if fractions    can be combined without affecting the purity of those fractions.
initial TLC
TLC of fractions
Other Comments:
  • The success of your separation will be dependant on how well you pack and  load the column. It is important to have level sand and silica. It is also important to carefully and evenly add your sample to the packed column.
  • Do not allow the silica to dry out as the column progresses. Cracks will form within the silica column if it dries, and compounds can fall down the cracks instead of partitioning between mobile and stationary phases.
  • Compounds pass through sand quickly and do not stick to it. Sand is used  at the bottom of the column to help ensure a level silica gel line. The bottom of the column is typically cone shaped. If no sand were present at the bottom of the column, molecules traveling down the center of the column would encounter less silica gel than molecules traveling down the edge, closer to the glass.     As a result, a particular component would elute as a broader band which is undesirable.
  • Sand is used at the top of the column to aid even loading of the sample. Sample diffuses evenly through the sand. Once the pinch clamp is removed from the bottom of the column, sample loads evenly onto the silica. Without sand, the sample would be added directly to the silica and would stick where ever it is added, not evenly across the surface of the silica.