HPLC Protocol

 

Preparatory Work

 

1.     Check to see if there is enough solution.  If there is not enough solution, then refill the bottles with solution in the A or B bottles.  There is extra solution on the shelf above the HPLC machine.  If there is no extra solution then prepare the solution according to the recipe on the bottle.

2.     Check the waste container.  If the waste container is full replace the container and write the information that is on the old one on the new one with a new date and your name.  Keep the cap that is one the waste tube and place the new cap on the old waste container.  If the waste container is not full then proceed to step 3.

3.     Turning on the machine.  The switch is on the right side of the machine.  A window will pop up and click biocad.  Everything will pop up for you.

4.     Sparge the solutions with helium.  Go to the helium tank and turn the knob on top of the helium tank to turn on the tank.  Then at the HPLC machine there is a switch on the right side of the machine.  The switch turns on the helium.  Once the helium is on, there will be bubbles that are sent through the solutions.  The helium stays on for about 10 to 15 minutes.  Once the purging is over turn off the helium at the HPLC machine with the switch on the right hand side.  Then the helium must be turned off at the tank, otherwise the tank will empty.

5.     Washing the HPLC.  Wash with 100% solution B for 5 minutes and then wash with solution A for 5 minutes.  To start the washes type in 100% for B in the control window and click the A box and it will automatically correct the A box.  The flow rate should be 10 – 15 mL/min.  Click Set and then click start to start the flow.  To stop the flow rate, click stop.  Click the A box and type in 100% and click the B box so it automatically sets the percentage.  Click Set and then click start to start the flow.  Stop the flow.  Solution B comes before solution A because our procedure starts with solution A.

6.     Make the sample solution.  The sample solution will be made in a 15 mL test tube with lid.  2 mg of the sample will be weighed out and placed into the test tube.  For K3 and Ct a 80:20 water:AcN solution will be made in a separate test tube with lid.  For Dt an 60:40 water:AcN solution will be made in a separate test tube with lid.  The water:AcN solution will then be added to the test tube with the 2 mg sample in it.  The sample solution will then be vortexed until it is dissolved.

7.     Setting up the machine.  In the program, go to the file menu and click run method.  Select “no_pep” for k3 and ct samples; the gradients start at 25% and go to 50% of solution B over a period of 30 minutes.  For dt select “no_dt”; the gradient goes from 50% to 100% solution B over a period of 45 minutes.  Click okay.  Name the file.  Click okay.  The method is now running.

8.     Print out method.  Go to the control window and click window for the window menu.  In the window menu click method editor and a new window will appear.  In the new window click Display and in the display menu click sequence view.  Print that screen and place in your notebook if it is the first time using that method.

 

Working with the HPLC

 

1.     Putting the sample solution into the syringe.  Take out a syringe, .22-.4 µm filter, and the needle box from the right hand drawer.  The needle will need to be cleaned with AcN.  Take the syringe out of its package and place a needle on the syringe.  Then put some AcN into a small beaker.  Take the syringe and needle and draw some AcN into the needle and squirt back out into the sink.  Do this a few times.  Now the needle is ready for the sample.  There are two ways of getting the sample solution into the syringe.  One way is to take the back out of the syringe and pouring the sample solution into the syringe.  The back of the syringe is then placed back in the tube with a little bit of the sample solution coming out the needle.  Another way of getting the sample solution into the syringe is to carefully suck all of the sample solution into the syringe via needle.  After the sample solution is in the syringe, there will be air in the syringe with the sample solution.  Turn the syringe upside down with the needle in the air and gently push all the air out. Now the syringe is ready.

2.     Turn on the graphing machine.  There is a graphing machine on top of the HPLC that graphs what is coming out of the HPLC.  Take the marker out of the holder and take off the black lid and place back in the holder with the maker on the paper.  To turn on the graphing machine flip the two bottom switches, one is in its own box and the other is above a knob.

3.     Putting the sample solution into the HPLC.  The HPLC should have beeped at you by now, telling you that it is ready for the sample.  Take the needle off of the syringe while the needle is in the air.  A filter will go on to the syringe and then the needle will go on to the filter.  The needle of the syringe will go into the port-6.  The port-6 is the part of the HPLC that has a lot of tubing going in and out of the machine.  One of the tubes is a brown tube that is coiled into a circle a lot of times and that is right next to where the sample is going to go in at.  There should be a part that sticks out that does not have a tube going in or out, place the needle into that part until the needle cannot go in anymore.  Start pressing on the back of the syringe until all the sample is in the machine.  Then click okay.  Mark on the chart recorder where the injection was.  

4.     Adjusting the computer chart.  In the window with the chart click display.  In the display menu click real time.  Then again in the display menu click options.  In the chart range section make the length of the chart 10 minutes.  This will make the chart show the last 10 minutes of the run.

5.     Collecting peaks.  The HPLC should run for 5 minutes before any peaks show up.   Make sure the computer graph is in real time other wise the peaks will not show because the graph is showing an image from another run.  After the 5 minutes, some peaks might show up or might not.  There should be a graph on the screen that also shows what is coming out of the HPLC.  Most of the time the paper graph will be the one that is followed because it show what it happening before the one on the screen is.  If the marker on the paper graph starts to go up really fast then a peak is going through.  To collect the peak take a 15 mL or 50 mL test tube with lid and place under the tube that is going into the waste container until the peak starts to go down.  The peak should go to the top of the paper graph when you collect if but it is okay to collect the bigger peaks if it is a run that has not been done before.

6.     Print out computer graph.  In the graph window go to file and click print to print the graph.  Each run should have a graph printed.

7.     Put on to the lyophilizer.  See lyophilizer directions.

 

Clean Up

 

1.     Turn off the UV lamp.  Go the detector menu in the control screen and click Lamp Off to turn the lamp off.  If there is going to be another HPLC run after this then you do not have to turn the lamp off, just remember to after the last HPLC run.

2.     The syringe.  Rinse the needle with AcN and place the needle back in to the needle box.  Put the needle box into the right had drawer.  Throw away the syringe, filter, and packaging after their use is done.  

3.     Check waste.  If the waste container is full replace the container and write the information that is on the old one on the new one with a new date and your name.  Keep the cap that is one the waste tube and place the new cap on the old waste container.  If the waste container is not full then proceed to step 4.

4.     Turn off chart recorder.  Turn off both switches at the bottom of the chart recorder.  Take out the maker and place the black lid back on the marker.  Then place the marker back on the paper upside down.