TLC (Thin Layer Chromatography) plates come as 20 cm x 20 cm glass plates coated with a 0.2 mm thick layer of a stationary phase, typically silica. The most convenient size plate to use for analytical purposes, however, is about 2 cm x 5 cm, so these larger plates must be cut to the smaller size. A diamond tipped glass cutter is used to score the larger plate, which is then broken along the score by applying pressure. To do this well and consistently takes some practice, but it can be easily mastered. The basic process is as follows:
- Place the 20 cm x 20 cm plate coated side down onto a clean, dry surface. We typically use a block of wood, approx. 10" x 10" x 0.5" for this purpose, since the edges of the wood block can be used to break the plate.
- Using a ruler and the diamond cutter, make a score that bisects the plate into two halves of equal area, 10 cm x 20 cm. (The key to cutting plates is using the diamond cutter properly. A light, but not necessarily deep, score must be made. The cutter should be held approximately normal to the surface of the plate and drawn across it somewhat rapidly with a moderate amount of pressure.)
- Place the plate on the block of wood so that the entire length of the score is hanging off of the wood by about 1/8". Press evenly on the suspended half of the plate. The plate should break evenly along the score.
- Repeat this process:
- Cut each 10 cm x 20 cm plate into two 10 cm x 10 cm plates (four total)
- Cut each 10 cm x 10 cm plate into two 5 cm x 10 cm plates (eight total).
- Score each 5 cm x 10 cm plate width-wise at 1.5-2 cm spacings.
- Bringing each of these scores to the edge of the wood block, break each 5 cm x 10 cm plate into five to seven individual plates.
- Run the shop-vac over the area to remove any glass shards or silica dust.
The individual TLC plates, as well as the original 20 cm x 20 cm plates, should be stored in a cool dry place.
- J. Touchstone Advances in Thin-Layer Chromatography; Wiley: New York, 1982.
- J. Touchstone, M. Dobbins Practice of Thin-Layer Chromatography; Wiley: New York, 1978.
- J. Gasparic, J. Churacek Laboratory Handbook of Paper and Thin-Layer Chromatography; Wiley: New York, 1978.
- A. Zlatkis, R. E. Kaiser High Performance Thin-Layer Chromatography; Elsevier: Amsterdam, 1977.
- Thin-Layer Chromatography; A Laboratory Handbook E. Stahl, Ed.; Springer-Verlag: New York, 1965.
The most typical way of visualizing a developed TLC plate is by examining it under UV light to observe fluorescence quenching of the appropriate chromophoric groups. Unfortunately, many many compounds have no such chromophoric groups. In this case, the plate must be visualized by other means, typically by treating with a vapor or solution which reacts with residues on the plate to produce a contrasting image.
The most common stain is iodine vapor, which is most useful for isolated C=C double bonds. Conjugated or aromatic systems show up much weaker. The other common stains are solutions of reactive (destructive) agents with which one treats the plate, followed by heating until spots appear. Since UV, iodine, and other stains do not necessarily detect the same species, a combination should be used. The first examination should always be under UV light, with pencil marks noting the findings, then in iodine and/or with a destructuve agent. Clear notation should be made in your notebook as to which visualization method(s) was/were used and the relevant results for each! Note that UV visualization cannot be performed after a destructive method has been used.
- Can use a few crystals in a jar, or
- A few crystals with silica gel in a jar, or
- 0.5 g iodine in 100 mL chloroform.
- 5 g phosphomolybdic acid
- 100 mL 95% ethanol
(Commercial stain is available from Aldrich but should be diluted.)
- 5g ammonium molybdate
- 0.1 g cerium(IV) ammonium nitrate
- 100 mL 95% ethanol
- 186 mL 95% ethanol
- 6.9 mL conc. sulfuric acid
- 2.1 mL acetic acid
- 5.1 mL p-anisaldehyde
See J. Am. Chem. Soc., 1973, 95, 2307.
Micropipettes are used primarily for applying samples to TLC plates for subsequent elution and visualization. A solution containing the compound(s) to be analyzed by TLC is drawn into the micropipette by capillary action. The micropipette is then tapped onto the surface of the TLC plate to release the solution. It is crucial that the application of sample be done in such a way that a very small, tight spot of analyte is deposited. Hence, micropipettes used for this purpose must be quite small in diameter. Although micropipettes are commercially available, they are rarely of small enough diameter for typical use in synthetic organic chemistry. As a result, it is usually necessary to prepare micropipettes manually by melting and stretching disposable pasteur pipettes as described below.
Prepare a clean work surface free from all solvents or other flammables. Use a hood if possible. Light a Bunsen burner and adjust the flame so that it is blue throughout. Hold a Pasteur pipette (9") horizonatally by the ends and move its center into the flame. Rotate the pipette along its long axis to ensure even heating. When the glass has become suficiently melted, remove the pipette from the flame and swiftly and evenly pull the ends of the pipette in opposite directions by extending your arms. The resistance of the glass to your pulling should increase as it cools during the extension. You should end up with a single, thin, long (depends on the length of your arms) micropipette. If the glass breaks in the middle then you have heated it for too long. If you cannot extend your arms fully then you have not heated it enough. THE GLASS IS STILL HOT!!! Treat it accordingly. Lay the stretched pipette down on a clean surface and allow it to cool. Break the thin section of the pipettes into 8-10" lengths and store upright in a beaker or flask. It is best to pull many pipettes at once (usually 20-30).
Remember: Pump oil is cheap, vacuum pumps are not!!!
The oil in a vacuum pump serves two purposes. It keeps the parts from grinding against one another (lubrication), and it establishes the seal between the rotating parts of the vacuum pump mechanism. Degraded or contaminated pump oil will therefore lead to both increased pump wear as well as decreased pump performance. It is imperative that pump oil be changed often. For a heavily used vacuum pump, once a month is a good rule of thumb.
Note that a vacuum pump can maintain a pressure no lower than the vapor pressure of the oil (or other liquid) it contains. That is why it is necessary to trap out solvents (in a dry ice- or liquid nitrogen-cooled trap) before they reach the pump. (Question: Why can an aspirator pull to only approx. 20 torr?) If a trap "goes dry," and a significant amount of solvent makes its way into the pump oil, the oil should be changed immediately, not only to increase the performance of the vacuum pump, but also to prevent the vacuum pump from seizing! Any corrosive volatiles such as HCl or SO2 gas should be trapped with potassium hydroxide-filled traps in addition to the cold traps.
To change vacuum pump oil:
- Turn off and vent the pump (i.e. make sure a vacuum does not exist at the inlet of the pump).
- Drain the oil into a waste container, being careful not to drop the drain cap into the container, spill a significant amount of oil, or burn yourself on potentially hot oil. Note approximately how much oil drains from the pump. Welch pumps have an oil drain with a cap located somewhere on the main body of the pump. They are usually just screw caps, but the 1402 has a stopcock-like drain.
- When the oil has finished draining, replace the drain cap.
- Refill the pump to about 1/4 to 1/3 capacity with new pump oil (use a funnel!). The new pump oil should be golden yellow in color, not dark orange. It is poured into the pump through either the pump inlet or outlet, although the outlet is usually more convenient. On Welch pumps the outlet has an aluminum spray shield which is mushroom shaped. This can be easily screwed out. Do not forget to put it back on!
- Plug the vacuum pump inlet. Turn the vacuum pump on and count to ten. Turn the vacuum pump off.
- Redrain the new pump oil into the waste container.
- Refill the vacuum pump with new oil to the full mark. On Welch pumps there is a window in the pump body with two etched horizontal lines. Fill the pump oil to just above the lower line. Do not overfill!!!
- Replace the outlet cap, store the new and waste oil containers, and clean up any spilled oil. You're done.
Remember: Pump oil is cheap, vacuum pumps are not!!!
The stockroom in OC (and associated satellite sites) has the vast majority of supplies we need for conducting everyday research. This includes glassware, "disposable" items, and chemicals. Items must be checked out on the computer terminal by the door using the account(s) coded onto your CatCard. Please be aware of what common supplies cost. The more money we spend today, the less money we have for tomorrow. Note that some common laboratory supplies can be very inexpensive or very expensive depending on the type you buy. For example, pyrex testtubes are quite expensive, but borosilicate ones are not. The price of what you are purchasing from the stockroom is displayed as you check out. Note that it is not unheard of for the price in the stockroom to be higher that what we can obtain by directly ordering from a supplier, especially with chemicals.
We no longer have 24-hour access to the stockroom (by way of your OC/CSML keys). This privilege was taken away as losses continued to accrue. Please do not fail to check out items that you obtain from the stockroom, whether inadvertantly or in an attept to save the group money. The money that is saved by "sneaking" items from the stockroom is lost many fold if access to the stockroom is further restricted (through research downtime) and prices are raised to cover losses.
Liquid nitrogen is obtained from the corral adjacent to the north side of CSML. Do not bring individual open top dewar flasks to fill. Fill only the large 4-5 L storage flasks. If this is too heavy for you to carry comfortably when full then take a cart.
Nitrogen, Argon, Helium, and Hydrogen tanks can be obtained from the corral adjacent to the north side of CSML. Your CSML/OC front door key should allow you access to this area. Tank the tank cart from the lab to transport. Identify a full tank, tear off the lower third of the tag, fill out your name and account number, then place the tag in the slotted box. When the tank is empty, tear off the second third of the tag so that only the Empty portion remains and return the tank to the corral. NEVER EVER TRANSPORT A TANK WITHOUT THE SAFETY CAP ON!!!
Almost all of the solvents we use inthe lab are obtained from the stockroom. Some are purchased in 4 L "Winchesters," some in 5 gallon cans, and some from 55 gallon drums (transported in red safety colvent cans).
- Hexanes
- Ethyl Acetate
- Methylene Chloride (Dichloromethane)
- Ethanol
- Methanol
- Chloroform
- Acetone
- Ethyl Ether (anhydrous)
- Ethyl Ether (tech)
- Toluene
There are several common disposable items that are used by everyone in the lab. We obtain most of these on a annual or semi-annual basis and store them in the lab. Currently these supplies are stored under the HPLC equipment in CSML 622. Please sign out any supplies that you take from this area. An incomplete list of these supplies follows.
- Kimwipes
- Paper Towels
- Pasteur Pipettes
- Gloves
- Septa
- Polyethylene (yellow) caps
This is where we learn how regrease vacuum lines.
Another paragraph.
The equipment and glassware in the lab was purchased at great expense. Even small items such as hotplate stirrers cost more than you think. Replacement is rarely possible. Accordingly, keep everything clean and in good working order. Do not abuse equipment--use it only as it was intended to be used. Chemicals are very corrosive and should be cleaned off of equipment they come in contact with immediately! This includes, especially, chemicals you weigh out on the balances.
In the unavoidable circumstance where a piece of equipment (e.g. heat gun, hotplate stirrer, vacuum pump, etc.) or non-common glassware item (e.g. chromatography columns, vacuum lines, distillation equipment, etc.), it must be taken to the appropriate shop for repair. For repair of electronic equipment, the electronic shop supervisor is Ted Weigt (x1-2830). For glassware, the head glassblower is Charlie Amling (x1-6365).
To obtain a repair for an item:
- Make sure the item is no longer under warranty with the manufacturer!
- Bring the item and a standard requsition form down to the Electronics Shop (CSML 222) or the Glass Shop (OC 128). Identify yourself, that you are in the Chemistry Department, and that you are in the McGrath group.
- Describe the problem and your expectation for repair. For both glassware and electronic equipment a diagnosis/estimate is in order, since often it is less expensive to buy a new item than to have it repaired.
- Fill out the requisition form and bring it to Richard in the business office.
- Let Charlie or Ted know that you would like them to call you when the repair is completed.
- If you do not hear from them within one week, call to check on the repair. Pick up the item when it is done.
Any common piece of equipment should be kept as clean as possible. In the case of a rotovap this is especially true as research samples come in contact with the surfaces that are most often soiled. It is inexcusable to have a sample be contaminated by reducing its volume on a dirty rotovap! In addition, a rotovap has moving parts, and dirt, grit, and compound residues can result in excessive and accelerated wear.
Always keep bump traps clean always. Since one should NEVER use grease on a rotovap bump trap, cleanliness not only insures that your sample will not be contaminated, but also that the joints between your flask and the bump trap and the bump trap and the rotovap will not seize.
To clean the inside of the rotovap condenser, first turn on the vacuum (but not the rotation). Remove the bump trap and replace it with a glass tube that has a 24/40 standard taper outer joint on one end and is bent approximately 90 degrees (end of tube pointing up). Add acetone to the inside of this tube so that the surface of the acetone does not quite reach the top of the bend in the tube. Cover the open end of the tub with your hand and wait a minute or so for the vacuum to create a negative pressure in the rotovap. Quickly remove your hand from the end of the tub. The acetone with be swept up into the inside of the condenser. Repeat this process if necessary.
Email comments to mcgrath@u.arizona.edu
Last updated on August 2005 by DVM