GPC Operation

 

1.     Launch Breeze software.

·       If Windows background screen is showing, click on “Breeze” icon.  Close “Waters Breeze Help” display.

2.     Look at the Flow meter.  Confirm that the flow is 0.1 mL/min.  If it is not, find Dr. Saterbak.

3.     Look at the Pressure meter. Confirm that the pressure is <1000 psi.  If it is not, find Dr. Saterbak.

4.     Locate carousel.  It may be on the counter or in the chamber of the Autosampler labeled “Carousel”.

5.     Load 1 mL vials into the carousel beginning at position 1 and continuing to the left.

6.     Place carousel in Autosampler.

7.     Click on “Manage Breeze” button.

8.     Click on “Change Project/User” box.

9.     Select your group name from the list of projects.  Click OK.  The screen that appears next will have the “Find Data” button depressed and a set of tabs available.

10.  Click on “Sample Queue” button.

11.  On pull-down menu, go to File à New Sample Set à Using Sample Set Method.

12.  To run samples, select “BIOE 441 Samples”.   Click “Open”.

13.  The screen that appears next will contain the following chart.  Columns further to the right of Injection Volume exist but should not be changed. 

 

	Vial	Sample Name	Function	Method	Run Time (min)	Injection Volume (mL)
1			Clear Calibration	PLLA/PLGA method
2			Condition Column	Ramp from 0_1	30
3			Equilibration	Equilibrate at 0_75	60
4	1	X	Inject Broad Samples	PLLA/PLGA method	20	100
5	2	X	Inject Broad Samples	PLLA/PLGA method	20	100
6…	3 …	X	Inject Broad Samples	PLLA/PLGA method	20	100
Y	Z	X	Inject Broad Samples	PLLA/PLGA method	20	100

where  X is the Sample Name that must be entered,

            Y is the number of command lines, and

            Z is the number of total vials that are being tested.

 

14.  Enter your sample names, X.

15.  Confirm that the number of vials in the chart EXACTLY corresponds to the number of vials you are testing.   Injecting air into the column by running a “non-existent” sample can cause expensive damage to the column.

·       If there are too many sample vials, delete a row (from the bottom) by right clicking in the Function column and selecting the Delete Rows option.

·       If there are not enough rows, right click in the Function column on the first empty line.  A line duplicating the one above should be added.  Make sure that the information in the columns on Vial Number, Sample Name, Function, Method, Run Time and Injection Volume are correct.

16.  Check with Dr. Saterbak to confirm your sample protocol.

17.  On the pull-down menu, go to File à Save Sample Set Method to save the updated method set. 

18.  On the pull-down menu, go to Inject à Run Sample Set.

·       Type in a name for your sample set such as “BIOE 441 9_23_01”.

·       For the settings, select “Run and Process”.

·       Hit “Run”.

19.  The “Current Sample Set Method” icon (3 test tubes and green triangle) should go gray.  The “Abort Run” icon (3 test tubes and red square) should be available.

 

Typically, your samples will take 2-5 hrs to run.  They can run without monitoring during the lab session and overnight.  After the last sample, the pump will automatically ramp down to 0.1 mL/min.

 

When your samples are running, the data is graphically reported in a plot of mV versus minutes at the bottom of the computer screen.

 

 

On Monday of each week, Dr. Saterbak will run a set of polystyrene sample.  She will post the retention times for compounds with the following molecular weights (g/mol):

 

            4,000      20,000    50,000    100,000  1,000,000

           

Use this data to develop a mathematical relationship between retention time and molecular weight.

 

 

 

Method Parameter Settings

·     Run time of 20 min for samples.

·     Flow rate of 0.75 mL/min for samples.

·     Sample injection volume of 100 mL.

·     Gradient flow.

·     High-pressure limit of 1000 psi.

·     RI detector with sampling rate of 5/sec, filter time of 3, + polarity, mV units, sensitivity of 64.

·     Internal and external 1 temperatures set at 30 °C.

 

 

The GPC runs at 0.1 mL/min during times when samples are NOT running (e.g. overnight).

 

GPC Data Analysis

 

1.     Launch Breeze software.

·       If Windows background screen is showing, click on “Breeze” icon.  Close “Waters Breeze Help” display.

2.     Click on “Find Data” button.  Click on “Channels” tab.

·       If you are running samples and want to look at the results so far, click on the “Update” button.

·       All of the samples that you have run should be listed here, sorted by Date Acquired.

3.     Select a run to analyze by clicking once on the row of interest.  To select multiple rows, hold the control button down and click on the rows of interest. 

4.     With cursor on row(s) of interest, right click and select “Review.”

·       Selected rows should appear as list under “View Data” button.

·       Additional rows may be added to the “View Data” list by repeating Steps 2.-3.

5.     The GPC chromatogram for a particular sample can be seen by clicking on the number in the first column of the table. 

6.     To determine the Retention Time, hit the Integrate icon (graph with two peaks and a liquid drop) or go to the pull-down menu and select Process à Integrate.

·       The Retention Time (in minutes) should appear on the graph.  Record the Retention Time.

·       If the integration ends (marked with red triangles) are not in a technically sound location, contact Dr. Saterbak.  The integration ends can be adjusted.

·       The Retention Times should be between 8 min and 14 min.  Water and other small molecules exit the column around 15-18 min.  Peaks above and below the baseline in this area should be ignored.

·       If you want to clear the integration and repeat, go to the pull-down menu and select Edit à Clear Integration.  You can then integrate the refreshed curve.

7.     Repeat Steps 5. - 6. for each run of interest.

8.     To look at all your chromatograms at once, click on the Overlay icon (graph with 2 traces, each with 2 peaks) or go to the pull-down menu and select Plot à Overlay.

9.     Once data analysis is completed, go to the pull-down menu and select File à Save à Result.  If you ever see a Breeze box asking “Do you wish to save before switching,” answer “Yes.”  Occasionally you will have difficulty saving because of a partially collected chromatogram or other issues; just follow through with “OK”.