Beckman Coulter HPLC Modules

HPLC Operation Protocol

 Flip the on switch to the power box (located to the left of the auto-sampler) to start up the HPLC and turn on the computer.

Log on to the computer using administrator as the username and 57108 as your password.

Once the computer has logged on double click the 32 Karate software program from the desktop.

In the next window that opens up you will need to select the type of injector you will be performing (either auto or manual injector)

After you have selected the type of injector method you will be directed to another window in which a smaller Instrument wizard- auto injector window will popup.  Just click ok on the popup. 

To control the HPLC you will click the control tab at the top of the screen and then click Direct Control. 

If the HPLC has not been used for approx. one week the pumps will need to be primed. 

To be the prime process you need to open the spin valve (spin it all the way to the left) if this is not done, the increased pressure will blow the lines.  Once the spin valve is open, you need to open the Direct control window In order to prime the pumps you must first attach the appropriate syringe to the     

            lower lour lock.  Then switch the lower knob from operate to prime lines (this will

            allow fluid from the machine to drain from the HPLC into the syringe.  Then

            switch the knob from the prime line setting to prime pump and slowly push the

            fluid from the syringe back into the HPLC (be sure not to pump the air bubble

            into the machine).  Once this has been completed the knob can be switched back

            to operate and the syringe can be removed.  Any waste that has been left in the

            syringe can be disposed in the waste funnel located in the back of the machine.             

            The steps listed above also need to be performed on the upper lour lock.  Each

            lour lock will need to be primed approx. 2-3 syringe fulls determined by the

            length of time the HPLC has not bee used for. 

Once the machine has been primed and the pressures on each pump are at their appropriate values you are ready to turn on the UV light.  Operation of the UV light can be maintained at the Direct Control window.  Click the light bulb and click yes to turn on the light.  Remember if you want to change the wavelength of the light you have to change it at the Direct Control window and instrument setup. 

Allow the light to run a couple of minutes. 

Once all is ready you are ready to load your sample and begin the run. 

Running a single sample 

Running a sequence of samples

To run a sequce: click File à Sequence à Sequence Wizard

Click Next

1)      Enter a sample ID (example: .1 dilutions 35nm 4252006)

2)      Enter a Data file (enter the sample thing as Sample ID) Then click the blue arrow and select Increment Number

3)      Enter the number of tubes to be run in the number of unknown runs in the sequence.

4)      Click Finish

Here is where you are to label each tube individually and by which name you will be able to find the data later on.  The run # corresponds to tube placed in the auto-sampler.  The tubes need to be labeled both at the Sample ID and Filename Data entry.  USE THE SAME ENTRY FOR BOTH.  Example: Sample ID: Rutin .1 dilution 350nm 4252006

                                                                     Filename:   Rutin .1 dilution 350nm 4252006

Once all tubes are labeled and inserted into the auto-sampler click the green arrow.    

How to change the wavelength of light used on the detector?

In order to change the wavelength of light used on the detector the desired wavelength must be input in both the Direct Control window as well as the instrument setup window (found under the method tab).  Don’t forget to apply these settings and save the method or else the desired wavelength will no be used.   

What is in pump A?

In pump A there is Acetonitrile, which can be poured directly into pump A tank. 

What is in pump B?

In pump B there is a mixture of HPLC water and formic acid.  The mixture in pump B needs to be made at a ph = 3.  To make this mixture only a small amount of formic acid is necessary for a large amount of HPLC water, be sure to check the ph using the ph meter. 

Caution!!!!!

The pumps cannot be allowed to run till empty this will cause damage to the HPLC machine. 

Seven flavanoid samples were used to create the standard curve.  The standards used in the HPLC were a mixture of the flavanoids (powder) and 10mL of ethanol.  The amount of flavanoid were based on their molecular weights and used to make a series of 1mm solutions.  From the 1mm solutions the dilutions were made.  The dilutions made were of .5, .33, and .1.  The 7 1mm flavanoid samples that were used in the HPLC were kept in a fridge at ~4º C.    

How to view past runs?

To view past runs: click File à Data à Open

When performing a run label each entry so that it can be found easily later. 

Example: Rutin .1 dilution 350nm 4252006

You cannot view past data while another run is progress. 

Flavanoids:

Naringenin                   

Quercetin                    

Quercetin 3B  

Rutin

Kampferol

Hesperidin       

Genistein