Guidelines for Use of the Analytical HPLCs (Agilent 1100 Series)

Compiled by J ake Wiener. J us tice.

I. Sample Preparation Never use EtOAc as an eluant or to dissolve your sample when using chiral columns 1. For chiral columns: dissolve your sample (5 mg/1 mL solvent) in ethanol, isopropanol, and/or hexanes ONLY. Other solvents will destroy the chiral columns. 2. For the achiral columns: dissolve your sample (5 mg/1 mL solvent) in any solvent that will dissolve your sample. 3. Place this solution in a small sample vial, and place the vial in any position on the autosampling tray. II. Sign Up Rules Always sign the logbook before running a sample 1. If you insert runs ahead of other people's runs, let ALL of those people know so that they can plan accordingly. 2. Generally, ee runs have priority over racemic assay runs, though MacMillan users also have priority over other groups. If you bump a user from another group, let them know. III. Running a Sample 1. At Chemstation on the PC, go to the Online instance of either instrument 1 or 2, depending on where your sample is. 2. Select sequence table from the sequence pull-down menu. 3. Enter the information for your run, including the location of your sample, the name of your sample, the method you wish to run, and the datafile where you will store the data. Enter a 1 under the inj/vial column. 4. Methods are typically in the format of percent additive in hexane over time (e.g, 6EOH40 is 6% EtOH in hexanes for 40 minutes). You should check each method to make sure it involves the correct solvent percentages and time by choosing Load Method from the method pull-down menu. Do this also to modify an existing method for a new solvent composition or time, and then usesave as to give this modified method a new name. 5. If you wish automatically to switch columns during a sequence, use a line with a method labeled POSX where X = the position of the column you wish to use (see below). This line does not require a vial or sample name but you must still write 1 in the inj/vial column. 6. Before injecting a sample for a run of a certain polarity, the column must be equilibrated for 20 minutes with that solvent system. You can do this by inserting a line with no sample name, no datafile, 1 inj/vial, and no vial number with a method ending in E (for equilibration), as in 4IPAE, which is a 4% iPrOH equilibration run of 20 minutes (these methods do not print.) Alternately, you can select a wait time in the sequence parameters, as discussed below.

7. Follow steps 3, 4, 5, and 6 for all runs you wish to have in the sequence. 8. For chiral columns: the last line of a sequence is 10_IPA which stores the column under 10% isopropanol. All chiral columns must be stored under 10% IPA when they are not in use. Note: If you use multiple columns then you must store EACH column under 10_IPA. That is, before the POSX method, you need a 10_IPA. If you suspect that your sample may require additional time to come off the column, use 10% IPA for a longer amount of time. 9. For achiral columns: the last line of a sequence is 100HEX20 which stores the column under hexanes. All achiral columns must be stored under hexanes when they are not in use. Note: If you use multiple columns then you must store EACH column under its appropriate storage solvent. That is, before the POSX method, you need a 100HEX20 for the achiral columns. If you suspect that your compound may require additional time to come off the column, use a more polar method to wash and then store under 100HEX20. 10. Click OK. 11. Select sequence parameters from the sequence menu and fill in the appropriate information. Make sure that the box on the right side under shutdown and next to the post sequence cmnd/macro is checked. Then select the shutdown.macro from the pull down list of choices. If you want the machine to wait for a certain amount of time after loading a solvent system but before injecting (to equilibrate) then enter a wait time. Click OK. Then click save sequence as from the sequence menu. Name your sequence. 12. Check that the solvent bottles are full. 13. On the main chemstation screen, turn on the HPLC using the on button on the bottom right of the picture of the system. 14. Choose run sequence from the run control menu. 15. Check the pressure to make sure the system is functioning properly (see pressure limits below.) IV. Solvent Refilling Rules 1. After your runs finish, refill all solvent reservoir bottles with filtered, HPLC grade solvent. This kind of solvent is located in the fire cabinet in the wooden cabinet next to the HPLC. If you use the last of (or almost the last of) the solvent in the bottle, get a new bottle and filter the solvent using the filter tool located in the drawer under the HPLC (see the operator to learn how to filter the solvent.) Return bottles to the fire cabinet when done. 2. When you refill the hexane solvent reservoir bottle, make sure the computer thinks there are 150 mL less than there really are. If you fill the reservoir all the way up, enter into the computer the maximum volume (0.8 L.) 3. No EtOAc on the chiral columns. V. Pressure Limits Acceptable pressures are less than 55 bar for the SIL, SIL-RX, OD-H, and OJ columns.

Acceptable pressures are less than 35 bar for the AS and AD columns. If the pressure is too high, open the bypass valve (black finger tight valve near the top of the HPLC) and then tell the HPLC operator. VI. Using the Column Changer Two Options: 1. Use a normal sequence or method Press the left button on the front of the column changer to switch between columns. Set the Position number according to the column you want to run. Position 1 is a bypass line that is used to flush the system, especially after using the Silica column and before using the chiral columns. Position 1 = Bypass Position 2 = AD + G Position 3 = ODH + G Position 4 = OJ + G Position 5 = AS + G Position 6 = Silica 2. Use a sequence containing automated column switching Column selection is controlled by a method, with each column being selected by its own method. The column-selecting methods are given names corresponding to the Position number of each column, as above (pos1.m, pos2.m, etc.). The method to switch a column takes about 15 seconds to run and the column-selecting methods set the solvent flow rate to 0.00 mL/min. Insert a column-selecting method before the actual methods of a sequence that you wish to run on that column. For a column-selecting method in a sequence, set the vial as blank and inj/vial to 1. In a multi-column sequence, make sure to wash each column with 10_IPA before moving on to the next column. If you have set up a wait time in the sequence parameters, loading the method that switches columns will invoke that wait time, in addition to the wait time that will occur when the next solvent-method loads; thus you may wish to equlibrate as in section III, part 6 above and use 0 for the wait time. IMPORTANT: If you use the silica column with EtOAc, flush the system with some non-EtOAc solvent system for 30 minutes by using the pos1.m method (the bypass method), or by manually setting the changer to position 1 with the right button on the front of the changer, ESPECIALLY before using the chiral columns.