In The Lab

In the Lab

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 click 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 (see above) 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.

Special Equipment

Rotary Evaporator (Rotovap)

The rotary evaporator (or rotovap for short) represents a new addition to the organic chem labs here at Wake Forest. While the instrument looks pretty complicated, it really is pretty simple. The purpose of the rotovap is to remove low boiling organic chemicals, usually solvents, from a mixture of compounds. The rotary evaporator is the method of choice for solvent removal in the modern organic laboratory. The solvents or low boiling compounds are removed by a simple distillation. The rotovap is designed to be operated under a vacuum (to lower a compound’s boiling point) and to heat the sample at the same time. A cold finger is used to condense the vapors to a liquid, which are trapped in a separate flask.

Below is a photograph of a rotary evaporator. The lower red arrow indicates the bath temperature knob. The middle arrow indicates the point of flask attachment. The top arrow points to the rotation speed control knob.

 

 

 

 

 

 

Infrared Spectrometer

Infrared (IR) light or radiation has the correct energy to enhance stretching   and bending of bonds, which are vibrational transitions.  Because molecules   have many possible vibrational states, each with a slightly different energy   and thus a different frequency of radiation required to enhance the vibration,   many absorption bands appear in the infrared spectra of even the simplest molecules.    Today, infrared spectroscopy is typically used to identify the presence of functional   groups.

IR spectra can be obtained on solids, liquids, and gases.  Our organic students   typically analyze only liquid samples.  The department owns two Fourier transform   infrared (FT-IR) spectrometers that are located in Salem 104.  Both spectrometers   are controlled through a windows based program called OMNIC.

Galaxy FT-IR Genesis FT-IR

 

To use the IR, follow these simple steps:Preparing a sample:1.Obtain two salt plates from a desiccators.  To open the desiccators, slide off the lid.  Do not attempt to remove the lid by pulling straight up on it.  (Please keep the desiccators closed as much as possible.)2. Clean the plates with an organic solvent such as acetone.  Since the plates are made of salt (usually KBr), never wash the salt plates  with water.  Place the plates on a Kimwipe, cover the plate with acetone, and then gently wipe the plate with a Kimwipe.  Clean both sides.  (Handle plates only on the edges to avoid obtaining an IR spectrum of oils from your fingers.)
3. Once the acetone has evaporated from the plates, add one drop of your sample to one plate.  Place the second plate on top of the first to sandwich your sample between the two salt plates.4. If a salt plate sample holder is not installed inside the IR spectrometer, slide one into place.  (The holder will only fit in one of the two available tracks.)  Place your sample into the holder and close the IR compartment.
Placing samples inside IR: The holes in the sample holder and instrument side panels align to allow light to pass from the IR source through the sample, and then to the detector. GenesisGalaxy

Obtaining a spectrum using the OMNIC Software on the computer attached to the Genesis:

5.      If the software is not already open, open it by double-clicking the “OMNIC” icon on the desktop. If the software is already open, close any existing windows within the program.

6.      Use the dropdown menus to carry out the following tasks:

a.    Obtain the data.

For the Galaxy:
– Collect: Collect Background. Make sure the light path is clear (no sample or salt plates in IR) while obtaining the background. Click OK.
-When asked if you want to add the spectrum to a window, select NO. – Collect: Collect Sample Place your sample in the sample holder and select OK.
– When collection of data is complete, you will be prompted for a spectrum title. Enter your name and some type of sample identification and hit enter.
– You will be asked if you want to add the spectrum to a window. Click YES. Otherwise, your data will be discarded.

For the Genesis II:
Collect: Collect Sample The default is currently set to prompt the user to prepare for a background scan. Make sure the light path is clear while obtaining the background. Click OK when the pathway is clear.
– A “Prepare to Collect Sample” window will pop up after the background scans are complete. Place the sample in the sample holder and select OK.
– When collection of data is complete, you will be prompted for a spectrum title. Enter your name and some type of sample identification and hit enter.
When asked if you want to add the spectrum to a window, click YES. – To change the y-axis from absorbance to percent transmittance, Process: % Transmittance.

Please Note: If someone used the instrument before you and you forgot to close old windows before collecting your data, your spectrum will simply add to the one(s) already on the screen. To delete any old spectrum, click on it. The selected spectrum and its title will appear in red. Once you have selected the spectrum you wish to delete, use Edit: Clear to delete it permanently.

b. (Optional) Analyze: Find Peaks If too many peaks have been labeled, you may move the threshold line down by sliding the bar running the left side of the screen. The line may not appear to move, but you will notice fewer and fewer peaks labeled as you slide the threshold lower and lower. Click the Replace button to put this new window of labeled peaks into your active window.

c. File: Print

Please Note: If you have trouble printing 1) make sure you are printing to the Salem 104 printer. 2) Make sure the Salem 104 printer is installed. Each user who logs into the computer must install the printer. 3) make sure you are logged into the network.

Though it is typically unnecessary for the organic labs, you may save your spectrum if you wish.

Cleaning up:

7.  Clean your plates   with acetone or ethanol (NEVER water!) and place them back in the desiccators.    Throw away any used Kimwipes and place disposable pipets in a broken glass container.

Gas Chromatograph

Gas Chromatography (GC) is used to separate volatile components of a mixture.  A small amount of the sample to be analyzed is  drawn up into a syringe.  The syringe needle is placed into a hot injector port of the gas chromatograph, and the sample is injected. The injector is set to a temperature higher than the components’ boiling points.  So, components of the mixture evaporate into the gas phase inside the injector.  A carrier gas, such as helium, flows through the injector and pushes the gaseous components of the sample onto the GC column.  It is within the column that separation of the components takes place.  Molecules partition between the carrier gas (the mobile phase) and the high boiling liquid (the stationary phase) within the GC column.

Top View of Oven and Columns

 

Two columns will fit inside the oven of our GCs.  A heating element is used to raise the oven temperature, when desired, and thus raise the column temperature.  GC columns typically have a metal identification tag clipped onto the column that lists column length and diameter, what material is inside, and the maximum operating temperature.

After components of the mixture move through the GC column, they reach a detector.  Ideally, components of the mixture will reach the detector at varying times due to differences in the partitioning between mobile and stationary phases.  The detector sends a signal to the chart recorder which results in a peak on the chart paper.  The area of the peak is proportional to the number of molecules generating the signal.

 

To use the GC, follow these simple steps:

1.      Wash a syringe with acetone by filling the syringe completely and ejecting the waste acetone onto a paper towel.  Wash 2-3 times.

2.      Pull some of your sample into the syringe.  You will most likely need to remove air bubbles in the syringe by rapidly moving the plunger up and down while the needle is in the sample.  Usually 1-2 mL of sample is injected into the GC.  It is okay to have small air bubbles in the syringe.  However,  you do not want to inject mostly air or your peaks will be too small on the chart recorder.

3.      Make sure the chart recorder is on and set to the appropriate chart speed (Arrow A). Set the baseline using the zero on the chart recorder (Arrow B). With the pen in place, turn on the chart (Arrow D), make sure the pen is down (marking the paper) and the paper is moving.

  Arrow A Set chart speed in cm/min
Arrow B Set zero so that the baseline is ~ 1 cm from bottom (right edge) of chart paper
Arrow C Record (but do not adjust) full scale setting
Arrow D Switch to turn movement of chart paper on and off.

4.      Inject your sample onto either column A  or column B as instructed.  Hold the syringe level and push the needle completely into the injector.  Once you can no longer see the needle, quickly push the plunger and then pull the syringe out of the injection port.

   
Injection Notes:
A) The injectors are very hot, so be careful not to touch the silver disk.
B) The needle will pass through a rubber septum, so you should feel some resistance.  For some of our GC’s, the column does not align properly in the injector, so the needle hits the front of the metal column.  If you feel that you are  pushing against metal, pull the needle out of the injector and try again, perhaps at a slightly different angle.  The needle should completely disappear into the injector for proper injection of the sample onto the GC column.
C) Inject quickly for best results.  Do not hesitate to inject once the needle is properly positioned in the injection port.
D) Remove the syringe immediately after injection.  (Carrying out notes C and D helps to insure that all of the sample enters the GC column at approximately the same time.)

5.      Mark your injection time on the chart recorder. This can be done by adjusting the zero just after the sample is injected.  It is often convenient for one person to inject the  sample while a lab partner marks the injection time at the chart recorder.

6.      Clean your syringe immediately after injection.  Syringe needles often clog quickly and must be replaced if they are not cleaned after each use.

7.      Record the settings of your chart recorder during a run.  You need to know the chart speed and the full-scale setting.

8.      Record the settings of your GC during a run.  A knob on the bottom center of the GC can be turned to read column (or oven) temperature, detector temperature, and injector port temperature in °C.  The bridge current is displayed in mA.  Note that there are two scales on the display.  Be careful to read the appropriate scale!

  Arrow A Top scale is reported in milliamps and is used to read the bridge current.
Arrow B Bottom scale is reported in degrees Celsius and is used to read all temperatures.
Arrow C Typically the ONLY knob to be adjusted by students.  Knob is turned for corresponding reading on the scale above the knob.
Arrow D Increasing the Attenuator setting decreases the area of a peak on the chart             recorder.  This knob should only be adjusted with permission of instructor.  Always return the knob to its original setting if you are given permission to change it.

Analysis of the Gas Chromatograph

Report the retention time of each peak (in minutes), the identity of each component in the mixture, and the percent composition of the mixture.  To determine the percent composition, you will first need to find the area under each curve.

Area = (height) x (width at ½ height)

Mark retention time, height, half-height, and width at ½ height on your GC trace.  Show your calculations either in your final report or directly on the chromatograph.

You may assume that each component of the mixture causes the same response in the detector.  Therefore, the areas under the curves can be used to calculate percent composition of the mixture of alkenes. (This is a reasonable assumption when the components of the mixture are very similar in structure, as are 2-methyl-1-butene and 2-methyl-2-butene.)

% Component 1 = [(area under peak 1)/(total area)] x 100%

The sample used to obtain the GC trace that is shown above did not have any solvent in it. Student samples will have at least one solvent present, so you will see another peak in your GC traces that typically appears very soon (usually within a minute) after injection. It is normal for the solvent peak to go off scale.

UV-VIS Spectrometer

Ultraviolet (UV) and Visible (VIS) light can cause electronic transitions. When a molecule absorbs UV-VIS radiation, the absorbed energy excites an electron into an empty, higher energy orbital.  The absorbance of energy can be plotted against the wavelength to yield a UV-VIS spectrum.   UV-VIS spectroscopy has many uses including detection of eluting components in high performance liquid chromatography (HPLC), determination of the oxidation state of a metal center of a cofactor (such as a heme), or determination of the maximum absorbance of a compound prior to a photochemical reaction.  Most organic compounds that absorb UV-VIS radiation contain conjugated pi-bonds. Both the shape of the peak(s) and the wavelength of maximum absorbance (lmax) give information about the structure of the compound.

 

Ultraviolet radiation has wavelengths of 200-400 nm.  Visible light has wavelengths of 400-800 nm.  Plastic cuvettes  can be used to hold a sample if you wish to scan only the visible region.  Since plastic absorbs UV radiation, more expensive quartz cuvettes are used when ultraviolet scans are desired.

 

Our department has two Hewlett Packard UV-VIS instruments, the HP 8342A and the HP 8453. Both are located inSalem104.  The HP software ChemStations is used to operate the instruments.

 

8432 UV-VIS spectrometer 8453 UV-VIS spectrometer

 

 

To use the UV-VIS spectrometers:  
1. Open ChemStations by double-clicking the “HP UV/Vis Online” icon.2.  Press the “Cancel” button when prompted to enter a password. No login or password is needed.
3. Use the “Instrument” dropdown menu to turn on the lamp(s).  There is only one lamp on the 8432.  It serves as both the visible and UV light source.  There are two lamps in the 8453.  Turn on the deuterium lamp for scanning the UV region and the tungsten lamp for visible scans.4.  At the top right of the ChemStations window, make sure the Mode  is set to standard.5. In the Task window, select “Spectrum/Peaks” from the dropdown menu if it is not already selected.                  Choose the Setup icon in the Task window.  The data type should be set to Absorbance. For a visible spectrum, make the lower wavelength limit 400 nm and the upper 800 nm.  For scanning the ultraviolet region, make the limits 200 and 400 nm.6.  In the Sampling window, select Manual from the dropdown menu if it is not already selected.

ChemStations Main Window

 

7.Fill a cuvette with solvent and place the cuvette into the sample holder.  Lock the cuvette into place using the lever on the side of the holder.8. Obtain a background spectrum by clicking the word Blank in the Sampling window.  If a “Last Blank Spectrum” window appears, simply close it.9. Remove the cuvette from the sample holder and fill the cuvette with your sample.  Return the cuvette to the sample holder.10. Obtain a spectrum by clicking Sample in the Sampling window.11.  The spectrum should appear in the display.  If it is satisfactory (absorbance <1 for most intense peak), then select the “Overlaid Sample Spectra” window and print your spectrum.  
Error Messages: If you see a red error message bar at the bottom of the ChemStations window, read the text and correct the problem.To export the data to Excel:12.  Select the spectrum itself by clicking on the curve. (Data point boxes should appear on the curve once it is selected.)  Use the File dropdown menu and choose “Export Selected Spectrum as” then “CSV format.”  Save your spectrum to a floppy disk. What’s the advantage? You can easily determine lmax, even for a shoulder, by viewing the data points in Excel. You can also format the graph in Excel and easily insert it into a Word document.When you are done:13.  If others are waiting for the instrument, click the “Clear” icon on the toolbar to clear your spectrum from the window.  If no one is waiting to use the instrument immediately, turn off the lamp(s) and close the HP software.14.  Dispose of any waste and clean your cuvette.

Organic Glassware

The following items can be found in your organic kits: (view entire kit)

West  Condenser (1)

A condenser is typically used to cool hot vapor, allowing the vapor to  condense as a liquid. A water hose is connected to the bottom of the condenser.   Water exits through a hose connected to the top of the condenser.  Note that the water flows through a sealed tube, and therefore, does NOT come  in direct contact with the vapor being condensed.

Claisen Adapter (1)

 

The Claisen adapter can be placed on top of a round bottom flask to convert   one opening into two. This is desirable if, for instance, you wish to add a   reagent slowly through an addition funnel (which requires one ground glass joint)   and cool hot vapors with a condenser (which requires another.)
Distillation   Adapter (1)

As the name implies, the distillation adapter is used during distillations.   As shown, the thermometer adapter connects at the top, a condenser connects   angled downward on the left, and a round bottom flask (simple distillation)   or a fractionating column (fractional distillation) connects to the bottom.
Distilling   Column (1)

We simply add ceramic “saddles” or stainless steel wire to a West Condenser to convert it into a fractionating column to be used in a fractional distillation. It is desirable for hot vapor to ascend slowly through the column. Therefore, the column is not cooled, and it is sometimes wrapped  with insulation.

Pennyhead   Stoppers (2)

Glass stoppers are typically used on round bottom flasks. All of the glassware in the kits have 19/22 standard taper ground glass joints. (The joints are 19 mm wide and 22 mm long.)
25 mL Round Bottom Flask (1)

50 mL Round Bottom Flask (1)

100 mL Round Bottom Flask (1)

250 mL Round   Bottom 2-Neck Flask (1)


 500 mL Round   Bottom Flask (1)


Separatory   Funnel with a teflon stopcock

Immisible liquids can be separated in a separatory funnel. Extractions are often carried out in a separatory funnel. The stopcock is opened to allow a layer to drain into an appropriate container. (To avoid a vacuum within the funnel, it should NOT be capped while you are draining.)
 Thermometer  Adapter (1)

Vacuum   adapter (1)

What does the marking on the ground glass joint mean?
 19/22 Joints follow a Standard Taper and are 19 mm wide by 22 mm high.

Organic Equipment   Check-in sheet (copies provided on check-in and check-out days)