Meet the Busby Family, which includes mom Danielle, dad Adam, 4-year old daughter Blayke, and 5 baby girls. 40 bottles a day, 420 diapers a week and feedings every 3 hours became the new normal for Danielle & Adam when they welcomed home the only all-female set of quintuplets. Below is a drawing of a developed TLC plate, Label each spot as either compound A or B. Briefly justify your answer. Show transcribed image text. Solvent polarity on Rf. Look over the photos showing TLC on the course website. (1.) TLC analysis of analgesics. The powdered side of the silica gel plate, not the shiny, plastic side is the side that is spotted. Lightly draw a pencil line about 1 cm from the end of a plate. Use a pencil, not a pen,. The TLC Tool The TLC Tool is a drawing tool that allows you to easily depict Thin Layer Chromatography plates within a ChemDraw document. The tool creates a rectangular plate with origin line, solvent front, and one or more lanes. The lanes can be populated with spots of different R f, size, shape, or color. Draw thin layer chromatography plates using the TLC Canvas widget. A powerful interface to quickly complete your work. Intuitive Controls. ChemDoodle’s drawing controls are made to clearly model the atoms and bonds they manage. Copious visual feedback is provided. There are also many options for customizing the drawing.

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Abstract

Thin-layer chromatography (TLC) is a widely used method for qualitative analysis to determine the number of components in a mixture, to determine the identity of two substances, or to monitor the progress of a reaction. The more accurate high-performance TLC (HPTLC) is better suited for quantitative analysis. Unfortunately, HPTLC requires expensive equipment that most high schools and colleges cannot afford. If digital photography is combined with regular TLC, it can perform highly improved qualitative analysis as well as make accurate quantitative analysis possible. This novel, digitally enhanced TLC (DE-TLC) is easy to use. A fluorescent TLC plate is illuminated with UV light and a picture of the plate is taken with a digital camera. Then, on a computer, using either TLC Analyzer, the public domain software written for this work, or common photo-editing software, one can quickly produce multi-spectral scans, densitograms, and calibration curves—output previously available only from more expensive equipment or complex procedures. Digitally enhanced TLC is a valuable tool that can be added to every chemist's TLC toolbox. Since this technique is less expensive than other quantitative chromatographic methods, DE-TLC is ideal for high school and college labs.

On this webpage you can download and learn how to use the software that enables DE-TLC: TLC Analyzer.

Introduction

Essentially, TLC Analyzer is a simulated TLC scanner. TLC scanners pan across an HPTLC plate with a beam of light emitted through an adjustable slit. In contrast, a digital image is made up of many rows and columns of 'dots' called pixels. Thus, a digital image is essentially a matrix of numbers, and TLC Analyzer 'virtually' pans across the matrix, combining moving averages to create a graph. For this purpose, TLC Analyzer is much easier to use and quicker than a photo-editing program.

Downloads

Instructions

Downloading the Files/Starting the Program:

Note: It is important to save ALL of the following files into the same directory to run TLC Analyzer properly.

1. Click on the link and save the self-extracting zip file in a directory of your choice (The file is named 'TLCA11.exe'). You may also right click and save the file if you wish.
2. Double-click on the 'TLCA11.exe' file. A DOS window will appear (do not close it until the extraction is complete). This will extract three files, two with '.exe' extensions, and one with a '.ctf' extension.
3. The MCR Installer allows a user to run TLC Analyzer without owning or installing Matlab. From Windows, double-click on the 'MCRInstaller.exe' file. An install window will pop up to guide you through the installation. Just follow the instructions on screen (you will mostly be clicking 'Next'). Choose the folder you wish to install the files by using the 'Browse' button (make sure it is the same folder as the recently extracted files). If there are multiple users for your computer, and you wish to install the files for all of them, choose 'anyone using this computer,' otherwise just choose 'just for you.'
4. Once the MCR Installer is finished, you are ready to run TLC Analyzer!

Starting the Program:

1. The 'TLC_Analyzer.exe' file is the file you will double click to start the program.
2. When you double click the 'TLC_Analzyer.exe' file, a black DOS window will appear. Do not close this window! It will run the program for you. Note: It might take a little while for the program to load, so do not close the black window unless it has been more than a few minutes and the main TLC Analyzer screen has not popped up yet.

Loading an Image:

In the File menu, select 'Open' and a dialog box will prompt you to choose a digital image to open. TLC Analyzer can read TIFF and JPEG images; however, TIFF is better for analysis because the image is not compressed (compression can introduce errors in the brightness values).

Specifying the Scan Area:

1. Choose a row to scan (the y-axis). To choose what ROW to scan, input a pixel row into the 'Scan Row' field.
2. Specify how far the scan will go across the image. To specify how far to scan across the image, enter the number of pixels you want to be CUT OFF the image in the 'Left Margin' and 'Right Margin' fields.
3. Specify how many pixels are to be averaged for each data point. To do this, choose an eyedropper size (in most photo-editing programs, the 'eyedropper' tool has many different sizes). 1x1 gives the 'noisiest' signal; the more averaging the smoother the curve. 3x3 or 5x5 is optimal for most purposes. If the eyedropper size is greater than 1x1, the scan row is the center row of the eyedropper. Use the axis labels on the image to help estimate the appropriate Scan Row value. If an eyedropper size larger than 1x1 is chosen, the scan row will be the middle row.
4. When you 'Plot Graph' (see below), a white line across the image shows what rows and columns TLC Analyzer is 'scanning.' Sometimes for a large image, if a small eyedropper size is chosen, the white line will not appear on the display because not all rows and columns are shown on the computer (the image is sampled for display). You can try changing the scan row a row or two, and that might make the white line appear. Even if the white line does not appear, TLC Analyzer still performs the scan.

Producing the Graph:

Note: More information about color in a digital image and optical density can be found in the article, 'Digitally-Enhanced Thin-Layer Chromatography: An Inexpensive, New Technique for Qualitative and Quantitative Analysis.'

1. Click 'Plot Graph' to have TLC Analyzer display a graph of brightness values versus scan distance in pixels.
   • The red, green, and blue values vary from 0-255, and the black and white 'K' values are a percentage from 0-100%, the darker the pixel the higher the percentage.
   • TLC Analyzer displays the highest and lowest values for K, red, green, and blue, in a box to the right of the graph.
   • OD, ODR, ODG, and ODB are the optical densities for K, red, green, and blue, respectively.
   • The maximum is to the left of the minimum.

Exporting to Microsoft Excel:

1. To export the brightness values to Microsoft Excel, go to the 'File' menu and select 'Export to Excel. Paste the image into Microsoft Word or into another program by right clicking and choosing 'Paste' or by pressing Ctrl+v. This way the graph, all the data, and the conditions are all in one image for later viewing. (It might be a good idea to just copy all these images into one word file for quick viewing purposes).
2. A file window will pop up. Input a name for the file and save it in the directory of your choice. Then go to Excel and open up the file. (Note that there will be a few more values than pixel length that was scanned because of the way TLC Analyzer implements the moving average).
3. The Excel file will have all data for red (R), green (G), blue (B), and black and white density (K). It also shows the minimum and maximum values for R, G, B, K, and the minimum and maximum optical densities (OD) for them.

Printing/Saving a Graph:

Method 1:

To print a graph, the easiest way to do this is to use a print screen command.

1. For PCs, after you hit 'Plot Graph,' hit Alt+Prnt Scrn and the TLC Analyzer window will be copied as an image.
2. Paste the image into Microsoft Word or into another program by right clicking and choosing 'Paste' or by pressing Ctrl+v. This way the graph, all the data, and the conditions are all in one image for later viewing. (It might be a good idea to just copy all these images into one word file for quick viewing purposes).

Method 2:

Alternatively, if only the graph is to be printed.

1. Choose the 'Graph Window' under the File menu and use the exact same process as described above for 'Method 1,' but for the graph window instead.
2. Alternatively, this image of the graph (by clicking 'Graph Window' can also be saved as an image file using the 'Save' command in the new window's menu (just make sure to save it as a valid image file, not as a Matlab file).

FAQs

Q: My image is not loading.
A: Try a smaller image file; you are probably trying to load a file that exceeds your memory capacity.

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Q: TLC Analyzer opened in a window that is larger than my screen size. I can't see the entire window.
A: Maximize the window and TLC Analyzer will fit the screen exactly.

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Q: The screen labels in TLC Analyzer are cut off or running into each other.
A: If you haven't already, maximize the window. If that doesn't work, try running the program on a computer with a larger screen or monitor.

Q: A couple warnings appear in the DOS window while TLC Analyzer is loading.
A: These are harmless and you can ignore them.

Additional Chromatography Resources

Students and teachers looking for beginning projects in chromatography may be interested in these resources I authored:

How to Contact the Author

If you still need help after reading the instructions for TLC Analyzer, the Journal of Chemical Education article, and the supplemental material (which does contain tips), please contact me by email. I am an undergraduate student at MIT, and since I am a full time student, I may not be able to respond immediately. Please keep in mind that these techniques do take quite a bit of practice in order to obtain good results, so keep trying and be sure to read my error analysis chart in the supplemental material.

My email is amber at sciencebuddies.org

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How To Read A Tlc Plate

TLC is a simple, quick, and inexpensive procedure that gives the chemist a quick answer as to how many components are in a mixture. TLC is also used to support the identity of a compound in a mixture when the R_f of a compound is compared with the R_f of a known compound (preferably both run on the same TLC plate).

A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina). A small amount of the mixture to be analyzed is spotted near the bottom of this plate. The TLC plate is then placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid. This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary action.

As the solvent moves past the spot that was applied, an equilibrium is established for each component of the mixture between the molecules of that component which are adsorbed on the solid and the molecules which are in solution. In principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others. When the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried, and the separated components of the mixture are visualized. If the compounds are colored, visualization is straightforward. Usually the compounds are not colored, so a UV lamp is used to visualize the plates. (The plate itself contains a fluorescent dye which glows everywhere except where an organic compound is on the plate.)

How To Run a TLC Plate

TLC Solvents Choice

When you need to determine the best solvent or mixture of solvents (a 'solvent system') to develop a TLC plate or chromatography column loaded with an unknown mixture, vary the polarity of the solvent in several trial runs: a process of trial and error. Carefully observe and record the results of the chromatography in each solvent system. You will find that as you increase the polarity of the solvent system, all the components of the mixture move faster (and vice versa with lowering the polarity). The ideal solvent system is simply the system that gives the best separation.

TLC elution patterns usually carry over to column chromatography elution patterns. Since TLC is a much faster procedure than column chromatography, TLC is often used to determine the best solvent system for column chromatography. For instance, in determining the solvent system for a flash chromatography procedure, the ideal system is the one that moves the desired component of the mixture to a TLC R_f of 0.25-0.35 and will separate this component from its nearest neighbor by difference in TLC R_f values of at least 0.20. Therefore a mixture is analyzed by TLC to determine the ideal solvent(s) for a flash chromatography procedure.

Beginners often do not know where to start: What solvents should they pull off the shelf to use to elute a TLC plate? Because of toxicity, cost, and flammability concerns, the common solvents are hexanes (or petroleum ethers/ligroin) and ethyl acetate (an ester). Diethyl ether can be used, but it is very flammable and volatile. Alcohols (methanol, ethanol) can be used. Acetic acid (a carboxylic acid) can be used, usually as a small percentage component of the system, since it is corrosive, non-volatile, very polar, and has irritating vapors. Acetone (a ketone) can be used. Methylene chloride or and chloroform (halogenated hydrocarbons) are good solvents, but are toxic and should be avoided whenever possible. If two solvents are equal in performance and toxicity, the more volatile solvent is preferred in chromatography because it will be easier to remove from the desired compound after isolation from a column chromatography procedure.

Ask the lab instructor what solvents are available and advisable. Then, mix a non-polar solvent (hexanes, a mixture of 6-carbon alkanes) with a polar solvent (ethyl acetate or acetone) in varying percent combinations to make solvent systems of greater and lesser polarity. The charts below should help you in your solvent selection. You can also download this pdf chart of elution order.

Interactions Between the Compound and the Adsorbent

The strength with which an organic compound binds to an adsorbent depends on the strength of the following types of interactions: ion-dipole, dipole-dipole, hydrogen bonding, dipole induced dipole, and van der Waals forces. With silica gel, the dominant interactive forces between the adsorbent and the materials to be separated are of the dipole-dipole type. Highly polar molecules interact fairly strongly with the polar SiOH groups at the surface of these adsorbents, and will tend to stick or adsorb onto the fine particles of the adsorbent while weakly polar molecules are held less tightly. Weakly polar molecules generally tend to move through the adsorbent more rapidly than the polar species. Roughly, the compounds follow the elution order given above.

The R_f value

The retention factor, or R_f, is defined as the distance traveled by the compound divided by the distance traveled by the solvent.

For example, if a compound travels 2.1 cm and the solvent front travels 2.8 cm, the R_f is 0.75:

The R_f for a compound is a constant from one experiment to the next only if the chromatography conditions below are also constant:

• solvent system
• adsorbent
• thickness of the adsorbent
• amount of material spotted
• temperature

Since these factors are difficult to keep constant from experiment to experiment, relative R_f values are generally considered. 'Relative R_f' means that the values are reported relative to a standard, or it means that you compare the R_f values of compounds run on the same plate at the same time.

The larger an R_f of a compound, the larger the distance it travels on the TLC plate. When comparing two different compounds run under identical chromatography conditions, the compound with the larger R_f is less polar because it interacts less strongly with the polar adsorbent on the TLC plate. Conversely, if you know the structures of the compounds in a mixture, you can predict that a compound of low polarity will have a larger R_f value than a polar compound run on the same plate.

The R_f can provide corroborative evidence as to the identity of a compound. If the identity of a compound is suspected but not yet proven, an authentic sample of the compound, or standard, is spotted and run on a TLC plate side by side (or on top of each other) with the compound in question. If two substances have the same R_f value, they are likely (but not necessarily) the same compound. If they have different R_f values, they are definitely different compounds. Note that this identity check must be performed on a single plate, because it is difficult to duplicate all the factors which influence R_f exactly from experiment to experiment.

Troubleshooting TLC

All of the above (including the procedure page) might sound like TLC is quite an easy procedure. But what about the first time you run a TLC, and see spots everywhere and blurred, streaked spots? As with any technique, with practice you get better. Examples of common problems encountered in TLC:

• The compound runs as a streak rather than a spot: The sample was overloaded. Run the TLC again after diluting your sample. Or, your sample might just contain many components, creating many spots which run together and appear as a streak. Perhaps, the experiment did not go as well as expected.
• The sample runs as a smear or a upward crescent: Compounds which possess strongly acidic or basic groups (amines or carboxylic acids) sometimes show up on a TLC plate with this behavior. Add a few drops of ammonium hydroxide (amines) or acetic acid (carboxylic acids) to the eluting solvent to obtain clearer plates.
• The sample runs as a downward crescent: Likely, the adsorbent was disturbed during the spotting, causing the crescent shape.
• The plate solvent front runs crookedly: Either the adsorbent has flaked off the sides of the plate or the sides of the plate are touching the sides of the container (or the paper used to saturate the container) as the plate develops. Crooked plates make it harder to measure R_f values accurately.
• Many random spots are seen on the plate: Make sure that you do not accidentally drop any organic compound on the plate. If get a TLC plate and leave it laying on your workbench as you do the experiment, you might drop or splash an organic compound on the plate.
• You see a blur of blue spots on the plate as it develops: Perhaps you used an ink pen instead of a pencil to mark the origin?
• No spots are seen on the plate: You might not have spotted enough compound, perhaps because the solution of the compound is too dilute. Try concentrating the solution, or spot it several times in one place, allowing the solvent to dry between applications. Some compounds do not show up under UV light; try another method of visualizing the plate (such as staining or exposing to iodine vapor). Or, perhaps you do not have any compound because your experiment did not go as well as planned. If the solvent level in the developing jar is deeper than the origin (spotting line) of the TLC plate, the solvent will dissolve the compounds into the solvent reservoir instead of allowing them to move up the plate by capillary action. Thus, you will not see spots after the plate is developed. These photos show how the yellow compound is running into the solvent when lifted from the developing jar.

TLC Technique Quiz

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See how well you understand TLC by taking the online TLC Technique Quiz!