Cong T. Trinh:HPLC SOP

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HPLC Standard Operating Procedures

1. Sign in the log sheet.

2. Determine what mobile phase you plan to use in your experiment and then do the following:

a. The standard mobile phase is 10mN H2SO4
b. Make sure the mobile phase reservoir be full or have enough solvent for your run.
i. Run time (min) x 0.6 (mL/min) x # of samples + 250mL (safety volume)
c. Inspect the quality of your mobile phase solvent since very often particulates are formed.


3. Turn on the HPLC devices by pressing Power buttons on all of the modules (6 total)

4. Turn on the computer and connect the computer to the HPLC controller unit (CBM-20A) by clicking on Ezstart icon on the desktop.

  • You expect to hear a beep sound when the connection is successful. Otherwise, an error message window will pop up. Another way to check whether the connection is made is to look at the activated icon “Activate the SCL” on the toolbar.

5. Purge your mobile phase line by doing the following:

a. On the LC-20AB, turn the Drain knob counterclockwise ¼ - ½ and then press “purge”. After the purging is done (~5min), turn the “Drain” knob back to the original position. The knob should be snug tight, if it is not tight enough, you will notice a lower than expected pressure.
  • If your run uses a different column, you need first to remove the existing column, then to wash the line before installing the new column. At this point, you need to contact the administrator to perform the task.
b. On the LC-20AB, press “pump” to establish the constant flow in your line.

When the pump is on, the “pumps on/off” icon will be activated on the toolbar. The pump pressure will depend on the set flow rate. Typically, when samples are not run, you can set the flow rate at 0.05-0.1 ml/min to establish a constant flow.

6. Next bring the system up to standard operating conditions.

a. Click File -> Sequence → Open → Startup-> StartUp_F06_T50.seq
  • This sequence gradually raises the flow rate up to 0.6mL/min and the warms the oven to 50OC. This sequence takes 50 min to complete.

7.While the Start-Up sequence is running, you can write the sequence for the sample you are going to run. See bullet 10 for instructions. 8.Once the startup sequence has been completed, the data collecting equipment must be zeroed. In the Ezstart toolbar.

a. For the UV-Vis: Click Zero for Det. A
b. For the RID:
i. Click flow on/off (Det. B) The icon looks like a faucet. This action Clears the refrence cell. After ~5 minutes click on the icon again to close the refrence cell solonoid.
ii.Click on the “Balance Det. B” icon, this process takes ~1 min and is complete when the RID LCD display shows 0 for the balance.
iii.Click Zero Det. B button to zero the RID.

9.Next Purge the autosampler by pressing the “Purge” button on the SIL-20AC HT autosampler. After the purge is completed, press the “rinse” button to rinse the autosampler.

10.To create a sequence, click file → Sequence → Open → test sequence.seq. This sequence if to be used as a template for modification.

a. The values for each block should be as follows:
i.Rep: 1. This cell refers to how many times you want to run the same sample
ii.Vial: This cell refers to the position on the deep well plate.
1.The format is #-# (plate number, #1 is closest to the door, #2 is closest to the back of the autosampler)-(Posisition on the plate, A1=1; H12=96)ex. Plate 1 Location C4 = 1-27
iii.Sample ID, this cell is where you can give the sample a name.
iv.Method, Insert the method that is appropraite for the samples you are analyzing. If you are not sure which method to use, Rezex_RHM_F06T50_45min is a good place to start.
v.Filename, this is the filename output. Be as descriptive as you can with the file name.
b. Save the sequence (File → Sequence → Save As)

11. Before running your sequences, you should establish good base lines for both RID and UV-VIS chromatograms by clicking on “preview” on the toolbar.

12. After set-up is complete, now you can click “Sequence run” or “Single run” to start running your samples.

13. After your sequence run is completed, you can detect peaks of interest and export them to a different location designated as C:\HPLC Export Data\Your Name. This process involves in reanalyzing your sequence that contains a table to detect specific peaks. Contact your administrator to develop a specific method for your run.

Current administrator: Michael Wierzbicki

Contact: wierzbickimc@gmail.com

History of modification:

+ 11/15/06: first write SOP
+ 01/15/07: modify SOP to make it more detailed and clearer
+ 08/12/11: Modified for use in UTK Trinh Lab HPLC

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