 Biomass Carbohydrate Composition Part 1. Acid Hydrolysis Place the Wayboat onto the analytical balance and tar the balance. Grab your sample of unknown biomass that has already been pre-prepared for your lab. Weigh out about 0.2 grams of your unknown biomass onto the analytical balance. Transfer the biomass from the Wayboat to a 125 milliliter Erlenmeyer flask. Typehead 3 milliliters of the 72% sulfuric acid into the flask. Make sure you observe what is happening to the biomass when you add the acid to the flask. Swirl the flask around to fully saturate the biomass with the acid. Grab a clean 400 milliliter beaker and fill it halfway with hot tap water. Cover the flask with aluminum foil and submerge it in the hot water beaker for 30 minutes. While waiting on the hot water bath, fill 100 milliliter graduated cylinder with 84 milliliters of deionized water. After 30 minutes slowly add deionized water to the flask and then recover with the aluminum foil followed by swirling the flask to mix with the acid. Bring your flask to the lab's auto-clave machine. Turn the auto-clave on and place your flask onto the tray. You'll need to fill the auto-clave with water if it's not visible on the slit on the bottom. Then close the door. Set the temperature knob to 121 degrees celsius and then set the bottom knob to sterilize. To begin auto-claving set the timer to 15 minutes. The heat light will turn on and the auto-clave will begin to accumulate pressure. The whole process should take over an hour to finish. When the timer reaches 0, do not open the auto-clave. Only open the auto-clave when the pressure gauge is reading 0 psi. After the pressure gauge has reached 0 psi allow a few more minutes for your flask inside to cool down. Then you can open the door and retrieve the flask. Attach the vacuum hose and the buckner funnel to the filter flask. Remove the filter paper and place into the funnel and then wet with DI water to seat the filter into the funnel. Attach the other end of the vacuum hose to a sink aspirator or a vacuum nozzle and adjust to a light suction. Make sure to swirl the flask to reach the spend the biomass before you filter it. Quantitatively transfer the contents of the sample flask to the buckner funnel with DI water by continually washing until all the biomass has gone through the funnel. Take the filter from the filter flask and again quantitatively transfer to a 500 milliliter volumetric flask. A funnel may make things easier in this case. After transferring, cap the volumetric flask. Grab a DI water bottle and dilute to the mark of the volumetric flask. Then cap again and invert a few times to mix. Now grab a test tube that is larger than 10 milliliters and transfer about 10 milliliters of the filter to the test tube and seal it if there is no cap used parafilm. Grab a marker and label the test tube acid hydrolysis and store later for your analysis. Part 2. Hot Water Extraction. Begin by preparing a hot water bath by filling a large beaker with 200 milliliters of tap water and placing it on a hot plate. Turn the hot plate up to a maximum setting and quickly bring the water to a boil. Grab two wayboats and your unknown biomass and weigh out 75 milligrams of the biomass sample onto each wayboat and record the mass. Transfer your weighed biomass samples to suitable containers for boiling that can be either a test tube or other glass bottles. Pipet 5 milliliters of DI water into each test tube or bottle and swirl to mix thoroughly. Place the bottles into the boiling water so that the samples are fully submerged. Allow the samples to be heated by a rolling boil for about 5 minutes. After, remove the samples and allow them to cool for an additional minute. Transfer the samples to labeled tubes that can be centrifuged. In order to counterbalance the centrifuge properly, weigh out each sample test tube and create a counterbalance tube by weighing it out and filling it with DI water to the nearest .1 gram. Place the test tubes in each slot symmetrically so that the centrifuge remains counterbalanced. Set the centrifuge speed to 2500 rpms and the time to 3 minutes. Then close the lid and press start. Carefully decant the supernatant from the centrifuge tubes into clean bottles then pipet 5 milliliters of DI water into each bottle. Place the bottles filled with supernatant back into the boiling water bath for another 5 minutes and 1 minute to cool after you have removed them. You can save time at this step by keeping the beakers filled with water hot in between boiling periods. Transfer the re-boiled supernatant into 25 milliliter Erlemire flask or something similar that can be diluted with DI water to 25 milliliters. Transfer the diluted supernatant to new test tubes through gravity filtration by using a plastic funnel and Wattman No. 1 filter paper. Make sure to fold the filter paper over a few times so that it sits nicely in the funnel. Use a new piece of filter paper for each sample. The process of gravity filtration is rather slow so you can end it early after you've collected only a few milliliters of your sample. Cap and save the filtered samples for UV-Vis analysis later. Part 3, UV-Vis Spectrometry Sugars Analysis. Obtain and label 9 test tubes on a test tube rack. The standard 0, 50, 200, 400, 700, and the samples AH1, AH2, HW1, and HW2. Prepare the standard glucose solutions for constructing the beer's law curve by following Table 1 in the lab manual, which is also listed on this slide. Pipet 1000 microliters of each hot water extraction sample into the respective test tubes labeled HW1 and HW2. Prepare the acid hydrolysis samples in duplicate by following the table listed on this slide. Bring your test tube rack to the fume hood and pipet 5 milliliters of 18.3 molar sulfuric acid and 2 milliliters of 2.1% phenol into each test tube. When pipetting these solutions in the fume hood, make sure you're wearing both protective gloves and goggles. Allow all of your test tubes to remain in the fume hood for about 5 minutes while the reaction goes to completion. You should notice a yellow gold color of different shades depending on the sugar concentration you have in each test tube. Take your prepared test tube rack to the lab's UVViz spectrophotometer. Set the wavelength to 490 nanometers, per pressing the go to wavelength button and entering 490. Before reading the absorbance of your samples, you need to create a blank. Fill a cuvette with 1 milliliter of DI water and place it into the housing with the arrow or ribbed side of the housing facing you. With the blank cuvette inside, press the auto zero button. The machine will be properly calibrated if the absorbance reads zero with the blank inside. Before transferring any of your samples to cuvettes, use a vortex mixer to properly mix all of your samples. While in the fume hood, use an eppendorf pipet to pipet 1 milliliter of each sample into respective cuvettes. Do the same for the gluco standard solution test tubes. If not using disposable cuvettes, you need to wash them out with DI water and then coat the cuvette with the next solution before filling it and reading the absorbance. For each cuvette, allow the machine to reach a steady reading and then record the absorbance.