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 (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.
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 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.
|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.
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.