give a swift flow rate-no fire-hoses, though! Keep the pressure on and change the test tubes once they become filled. Remember to replenish the solvent in the column frequently 22)Monitor the columns progress by tLC -this can get a little hectic, trying to run TLCS and collect your fractions, so in the beginning you might want to decrease the air pressure (or remove it entirely)when you are checking the progress of the column 23)When running a gradient elution column, use the initial solvent system until the higher Rf compounds have come off the column. Once they are safely in collection flasks crease the polarity of the eluent. Caution: Increase the polarity gradually. Drastic olarity changes can"crack"the silica gel -sending fissures through the column like in a bad earthquake movie. This cannot help your separation! Instead, increase the polarity by about 5%o for every 100 mL (or more)until you reach the desired solvent system. Then, stay with this eluent until your desired compound has eluted. At this point, you can either change eluents again or proceed to the next step 4)Once you have determined that all of the compounds you are interested in have eluted from the column, you are ready to wrap everything up. First, put a large Erlenmeyer flask underneath th e column. a nd use a green Keck clip to attached the compressed air source to the column. Allow the air to push all of the remaining solvent out of the column and then to dry the silica gel. (It's difficult to remove the silica from the column until it is completely dry. This will take at least one hour for large columns 25)While the column is drying, start to combine fractions. Using TLC, determine which test tubes contain your pure compound(s). Combine fractions of similar purity in large round-bottomed flasks and concentrate them on the rotavap. For longer duration columns, combine fractions while the column is still going to expedite the process 26)Once the solvent is completely removed, analyze the compounds by nMrgive a swift flow rate—no fire-hoses, though! Keep the pressure on and change the test tubes once they become filled. Remember to replenish the solvent in the column frequently. 22) Monitor the column's progress by TLC - this can get a little hectic, trying to run TLCs and collect your fractions, so in the beginning you might want to decrease the air pressure (or remove it entirely) when you are checking the progress of the column. 23) When running a gradient elution column, use the initial solvent system until the higher Rf compounds have come off the column. Once they are safely in collection flasks, increase the polarity of the eluent. Caution: Increase the polarity gradually. Drastic polarity changes can "crack" the silica gel - sending fissures through the column like in a bad earthquake movie. This cannot help your separation! Instead, increase the polarity by about 5% for every 100 mL (or more) until you reach the desired solvent system. Then, stay with this eluent until your desired compound has eluted. At this point, you can either change eluents again or proceed to the next step. 24) Once you have determined that all of the compounds you are interested in have eluted from the column, you are ready to wrap everything up. First, put a large Erlenmeyer flask underneath the column, and use a green Keck clip to attached the compressed air source to the column. Allow the air to push all of the remaining solvent out of the column and then to dry the silica gel. (It's difficult to remove the silica from the column until it is completely dry.) This will take at least one hour for large columns. 25) While the column is drying, start to combine fractions. Using TLC, determine which test tubes contain your pure compound(s). Combine fractions of similar purity in large round-bottomed flasks and concentrate them on the rotavap. For longer duration columns, combine fractions while the column is still going to expedite the process. 26) Once the solvent is completely removed, analyze the compounds by NMR. 67