Asking: Pick your four concentrations in the range of 10-75 M and calculate the dilutions youll need to perform from the stock solution to prepare them.

This week in lab, we will be performing two tasks. First, we will see if we can separate a mixture of two compounds using chromatography. Second, we will determine the concentration of an unknown sample of p-nitrophenol, a compound that absorbs light in the blue part of the visible spectrum (and therefore appears yellow to us). Chromatography is the application of separating compounds based on some difference in properties. This might be polarity, charge, size, etc. Each chromatographic technique has two essential components - a stationary phase and a mobile phase. The stationary phase, as you might guess, stays in place. The mobile phase moves. Separation of a mixture occurs when the components of that mixture interact differently with the two phases. So, if one thing is more attracted to the stationary phase, it will take longer to move, and therefore be separated from the other components.

The stationary phase is often some kind of solid medium, like silica gel or a resin. They might be porous, so small molecules are trapped in the pores and retained longer, whereas large molecules are allowed to pass through the medium along with the mobile phase. The mobile phase is generally some kind of solvent (or a gas in some cases). If the mobile phase is highly polar, it will likely carry polar molecules through the stationary phase more quickly. If youve taken organic chemistry lab, youve likely encountered this with TLC or column chromatography. More polar solvents carry molecules through or across the silica gel stationary phase more quickly.

In our case, we will be using chromatography to separate molecules with different sizes, in this case blue dextran (MW ~2,000,000 g/mol) and Yellow 5 (MW = 534.3 g/mol). Our stationary phase will be Sephadex G-75, a resin that has pores which can encapsulate small molecules, but excludes large ones. We will use a mobile phase that consists of 20 mM sodium phosphate buffer with pH = 7.0. In the second part of the lab, well be determining the concentration of an unknown sample of p- nitrophenol. Well be using absorbance spectroscopy to determine the absorbance of our solutions, create a calibration or standard curve, then determine the unknown concentration. Spectroscopy is essentially light interacting with something, or more technically, the interaction between matter and electromagnetic radiation. In our case, it means we shine a light at something and measure how that light changes, specifically how much of it is absorbed by the compounds in solution. We did this last week when looking at the absorbance of Yellow 5 food dye. This week well look at p-nitrophenol, a compound that takes on a bright yellow color once deprotonated. There are many different types of spectroscopy, such as fluorescence, absorbance, impedance, and acoustic. MRI scanning is also a type of spectroscopy (which is very similar to NMR used for organic molecules). For the type well be doing, absorbance, a colored compound absorbs light of a certain wavelength. Generally, this is not one specific wavelength, but a range. However, there is a wavelength of greatest absorbance, which is termed the lambda max, or max. You will often report this value (in nm) and the absorbance at that value, as a reference. Again, well be constructing a calibration or standard curve to use a reference point to determine the unknown concentration. This involves measuring the absorbance of several samples with a known concentration, which will then be plotted to determine an equation of the data. You can then plug unknown absorbance values into your data to determine their concentration. Safety and Waste P-nitrophenol is an irritant - avoid exposure to solutions or the solid and wash with lots of water if you spill on yourself (not a bad idea for the person working with the solutions to wear gloves). Similar idea with chromates - avoid exposure. All waste from this experiment must be discarded in the appropriate waste bottle. It will be located on the front table. Procedure Part 1 Obtain a sample that contains a mixture of compounds. This is what youll try to separate. Pick up both a column, ring stand, and one of the suspensions of Sephadex G-75. Swirl the suspension gently to resuspend the resin, and then carefully pour this into the column. Filling it nearly to the top with the suspension should give you enough Sephadex once it is settled. Next, give it a couple minutes to settle, then add some of your elution buffer, the 20 mM sodium phosphate, and allow that to run through the column. If youre convinced the resin is fairly settled and packed down, you can continue. Run out the buffer until it is just near the top of the resin. Then carefully add your solution mixture youre aiming to separate. Let the liquid run down the top of the resin again, then add more elution buffer, only a bit at first. Drain again to the top of the resin.

Now you can add more buffer and let the mobile phase drain through the column. Youll want to be collecting fractions in test tubes as you go. Be sure not to miss the first compound, it may come off fairly quickly. Keep adding the buffer until youre confident youve collected both compounds. Record any relevant data about both compounds, including in what fraction they were collected, what color they appear, etc. Part 2 There will be both an unknown sample as well as a known, which has a concentration of 0.1 mM PNP. There will be several of the known, but please use them and return them for other groups. Using the known concentration, prepare four dilutions, in the range of 10-75 M, of this sample for the other points in your calibration curve. Youll want to have at least four points for this curve. Dilute with pH 10 buffer. You should prepare 4 mL of each of your dilutions - this can be done in test tubes. Once your standards are prepared, measure their absorbance in the Spec 200 spectrometers, similar to how we measured Yellow 5 last week. Be sure you properly calibrate the instrument. Measure and record each absorbance value at 405 nm, which is the lambda max for PNP. Record the absorbance of your unknown sample in a similar manner, mixing 1 mL of unknown with 3 mL of pH 10 buffer. I will have the unknown at the front of the room. Generate your calibration/standard curve and use this to determine the unknown concentration.