HPLC

Peptide Solubilization

1. Weigh and record weight of peptide to be solubilized (~5 mgs for analytical work and 5-100 mgs for preparative work).
2. Place peptide in 15 ml or 50 ml conical tube and record weight on tube. (Note: Peptide can be highly static and will easily stick to gloves as well as plastic. Use a clean metal spatula to place the peptide on the weigh paper. Gloves may need to be removed as well during this process.)
3. Calculate the proper amount of solvent to add to reach a specified concentration (typically 1-3mM). Solvents for dissolving peptide can be solvent A (0.1% TFA in filtered MilliQ H20/10% acetonitrile); a mixture of solvent A and solvent B (0.1% TFA in acetonitrile); 10% acetic acid, or DMSO. Consult with PI or lab head if unsure.
4. Be sure that material is fully solubilized before proceeding to the next step; it may take a few hours of shaking to accomplish this.
5. Filter entire dissolved peptide with 0.2 uM syringe filter prior to HPLC injection. Be aware of the hangup volume of the filter to ensure that you recover all the peptide from the unit. Alternatively, you can centrifuge the filter in an appropriate tube to recover all the liquid from the unit.
6. Store solubilized peptide at -20°C (lab freezer).

Start Up

1. Check your solvent levels.
2. Prepare your solvents: filter H20/10%AN/0.1% TF A before using it.
3. Turn on instrument in this order: UV detector, pumps, and computer.
4. For HPLC purification: Install appropriate column (analytical or preparative); have PI check for leaks once you've started the pumps.
   1. To remove the current column, unscrew the bottom and top screws to remove plumbing. Ensure that proper safety equipment including goggles and a lab coat is worn in case of buffer leaking during change.
   2. Quickly screw the top line into the new column. Then add the bottom line by screwing it into the bottom of the column.
   3. Add screws into the old column for storage.
   4. Be sure to check for leaks when running the pumps.
5. Make sure that the loop size corresponds to your volume that you are going to inject. Be sure to check for leaks when running the pumps.
6. Turn on computer. The username is "biolab" and the password is "Biology 13". This information can also be found on the front face of the computer.
7. Click on Varian workstation icon on the desktop.
8. Create a folder within the Star/Data/Fairman/ area on the computer. You will store your data here.

Clean the Column in Manual Mode:

1. Navigate to the "system control" panel in the software.
2. Switch the injection value to "inject" and navigate to the "Manual Control" section located on the "ProStar/Dynamax. 24" panel.
3. Start an isocratic gradient with 0% solvent B at a slow flow rate. (C18 Analytical: 0.2 ml/min; Semi-preparative: 1 ml/min). Keeping a slow flow rate prevents air bubbles from entering the system when the column is switched. Make sure there are no leaks at the loop or column connections.
4. Once no leaks are detected, wash the column running a gradient for 10 minutes from 0% solvent B to 100% solvent B. Then run at 100% solvent B for 5 minutes. Reverse the gradient (100% solvent B → 0% solvent B). Then equilibrate the column with appropriate concentration of solvent A.

Generate a Method

1. Navigate to the "Method Builder" section by clicking the second icon from left on the top bar of the software.
2. In the pop-up box check "Create a New Method File" and click "OK".
3. In the pop-up box "Configuring method" clicked "next" through all screens and then push the "Finish" button.
4. In the main method builder menu navigate to the "Pump and CIM Program" tab. In this tab you may add method steps to design an elution or run that fits your needs. Work with your PI to build an appropriate method.
   1. Note: Ensure that the wavelength is set to the correct value as this tends to reset.
5. Additionally, the maximum flow rate for the analytical column is 1.0 ml/min and 10.0 ml/min for the semi-preparative column.
6. Name and save the method file into your personal folder.

Run a Method

1. In order to select a method, click on furthest right side icon of the toolbar: view /edit method.
2. After selecting your method, click on inject single sample icon: In this window you can select your method as well as save your run in your folder
3. Injecting the sample is easy: first, make sure your valve is in the "load" position. Wash the loop with solvent A using the appropriate syringe. Insert the syringe needle in the injection valve then inject the sample, keep the needle in and turn the knob from the load position to the inject position. Make sure that the syringe is in as you switch the knob position.
4. Start your run with small amount (~1 mg) of peptide using the appropriate analytical column to check the purity of your peptide. You will develop the appropriate method/gradient using these results and then switch to the preparative column for collecting purified peptide.
5. Make sure to set the right wavelength (each peptide will be different depending on the presence of aromatic amino acids or chromophores attached to it). Recommended wavelengths, in the absence of specific chromophores, include 235 nm and 254 nm. Be sure to set this wavelength on the UV detector.
6. If you have more questions about this protocol please do not hesitate to ask.

Run, Collect, and Acquire Data

1. Activate the target method. Navigate to the "system control" panel in the software and select the open folder icon located to the right of the method name.
2. Inject the sample into the system.
   1. With the injection valve set to "load" insert the Hamilton syringe filled with sample into the appropriate slot. After the needle is fully inserted, slowly inject the sample into the loop.
   2. Slowly switch the injection value to "inject". This step will put the sample onto the column itself. Note: The syringe should remain in the injection valve when the valve is on the "inject" function. The syringe can be removed right after the valve is switched to "load".
   3. Immediately return to the "ProStar/Dynamax softward on select the "Run" key to start the run. Note: Ensure the gradient that appears on the screen matches the method you are running.
3. Collecting Fractions. Depending on the type of run (analytical versus semi-preparative), fractions may need to be collected. Initial analytical run chromatograms are essential to planning and outlining peaks and fraction collection for purification runs. For instance, analytical runs can provide a %B of the target peak to help orient the purification run.
   1. Obtain 10 25 ml glass tubes. Number (1-10) and arrange in order in a rack.
   2. Watch the chromatogram on the running screen and the UV fluctuations in preparation for peak collection.
   3. When the target peak arrives, collect it in one or multiple tubes and make note of when each fraction collection begins. Note: With each main peak, typically there are shoulders on each end of the peak. These can occur for several reasons. For purest sample quality, aim to avoid these shoulders or smaller peaks surrounding the main peak. After MS analysis, the shoulders and main peak may be combined depending on the result.
4. Following the run, transfer contents of glass conical into 50 ml conical tubes.
5. Place tubes in the -80 freezer to prepare for lyophilization. Note: To lyophilize the samples place a KimWipe over the open 50ml conical and secure with a rubber band. The sample can be placed in the proper lyophilization container and attached to the machine. Depending on the sample volume freeze drying it can take 50 hours.

Save Chromatograms

1. To alter the chromatogram save pathway and redirect the chromatogram save location from the default, follow these steps. If the pathway is set on "020420 kracp" (for example) follow procedure in step 2 below.
   1. Navigate to and open the "view/edit Chromatograms" function located in the top left corner of the screen. It is the third icon from the left.
   2. Navigate to and open the target folder or location where you would like to save the chromatogram file. Inside the folder, select a pre-existing file.
   3. Select "Add to List".
   4. Select "Open File(s)".
   5. The save pathway should not be altered.
   6. Note: Any new run will save under the selected file name along with a number.
2. Follow the method in step 2 to rename the file if needed. Additionally, this pathway alteration appears to remain only for one run. Otherwise follow the procedure below to find and rename the chromatogram.
3. Often the save pathway defaults to a "020420 kracp" chromatogram (for example) and saving location. If this is the case follow these steps to access and move your chromatogram file.
   1. To locate and open the chromatogram file following the elution run, navigate to the "computer" folder through the "start menu" on the bottom left side of the screen.
   2. Click on "my computer"
   3. Open the "local disk".
   4. Navigate to and open the "star" folder.
   5. Navigate to and open the "data" folder.
   6. Navigate to and open the "Biochemistry superlab 2023" folder.
   7. Inside the folder will be files titled "020420 kracp holo 50um 25ul run" where "

" denotes a run number. Use the top bar to sort the files by date. The most recent run under this file name will be your chromatogram file.

1. 1. Right click and select "rename" from the drop down list. Rename the trace with your desired name.
   2. Right click and select "copy"
   3. Navigate to and open the target folder or location where you would like to save the trace file. Right click in the folder and select "paste"

Export Chromatograms

1. View/edit your chromatogram
2. Open AIA/Import/Export window
   1. Select Varian to AJA option
   2. Navigate to your data folder and open appropriate run file
   3. Save as a cdf file
   4. Then select AIA to text option; choose the various options that you wish to save, but be sure to save the chromatogram
   5. Save as a txt file
3. Insert thumb drive in USB port
4. From Start menu, navigate to My Computer, find your text file in your folder and transfer to thumb drive.
5. You can import the raw data into Excel (or any other graphing program. If using Excel, first open Excel, then select the Open File option. Be sure to select the appropriate delimited column option, which is typically a comma or a space. This will make sure that each column of data are properly loaded into Excel
6. The first column of data represents your actual chromatogram. You can plot this as a line graph with the data points representing the y-axis values. Since the x-axis is in seconds, you can simply plot the single column against an x-axis scale starting at 1.

Clean and Shut Down System

1. Run system cleaning. See method.
2. Make sure all pumps are stopped. Close the "Varian Star Chromatography Workstation" software and turn off the PC.
3. Switch off solvent delivery module A, solvent delivery module B, and UV-VIS detector module.
4. Empty waste container into appropriate, marked container.