Waters GPC User Guide and Tutorial for Using the GPC in the Reynolds Research Group 2 nd Edition: April 2012

Transcription

1 Waters GPC User Guide and Tutorial for Using the GPC in the Reynolds Research Group 2 nd Edition: April 2012 Georgia Institute of Technology School of Chemistry & Biochemistry School of Materials Science and Engineering Written by: James Deininger 1

2 Table of Contents 1. Basics of Chromatography Introduction The GPC System Experimental Procedure Sample Preparation Software Setup Sample Injection Data Processing

3 1. Basics of Chromatography 1.1. Introduction Liquid chromatography is a way to separate two or more components in a mixture. Therefore, when performing this technique all the components must be soluble in a liquid. The separation of components can be based on ionic strength, polarity, hydrophobicity, or size, as in the case of Gel Permutation Chromatography (GPC). For more detailed information on chromatography refer to the Waters Plastics & Polymers Characterization manual near the GPC The GPC System A typical chromatographic system can be seen in figure 1 below. As mentioned previously the solvent that is used in the system must be able to solubilize your entire sample, or you will not obtain reliable results. Currently, the Georgia Tech chromatographic system runs on THF, and will soon be able to run on o-dichlorobenzene. The solvent is pumped through the system with a Waters Isocratitic 1515 Liquid Chromatography pump. During separation, the solvent is pumped through the system at a constant flow rate of 0.30 ml per minute. This is the optimal flow rate in which the HPLC column, specifically the size-exclusion (SEC) chromatography column, is the most efficient. In general, this flow rate is predetermined for every column by the manufacture. When your sample is separated by the HPLC SEC column each component is analyzed by a detector. The Georgia Tech chromatographic system has two detectors with which components are analyzed, an RI and UV-Vis detector. The RI detector measures the 3

4 Refractive Index of each component, while the UV-Vis detector measures the absorbance of light by each component. Once your sample is detected the data gets sent to a computer where the data can be worked up. However, before you can obtain any molecular weight distributions (MWD) a calibration curve must be constructed. A calibration curve is generated by injecting narrow or broad PDI based polystyrene standards. In general, one should use 12 or more different standards that range from 200 to 1X10 6 Daltons to obtain a liner curve. Once a calibration curve is generated the computer program can now determine MWDs based off the PS standards. For our purposes the calibration curves are created and saved by the GPC operators. If you would like to know more about calibration lease refer to the Waters GPC Manuals Sample Preparation 2. Experimental Procedure Sample preparation is very important to obtain reliable results. The polymer that you are working with must solubilize in the mobile phase (in this case either THF or o- dichlorobenzene). If your polymer is not allowed enough time to settle into a relaxed conformation, your GPC results will not be accurate. Follow the sample preparation guidelines below: Comment [E1]: Should there be an Instrument Setup section? You mention later on that the lamp should be turned off, but no mention of turning it on. Is the instrument always on? Pictures of the various on-switches will be useful here as well Use the chart below to determine the molarity of the solution that you will need to prepare. In most cases about 1mg/mL will work. Molecular Weight Distribution Weight Percent of Polymer to Solution Less 10K 0.16 % 0.20% 10K 50 K 0.13% % 50K-100K 0.10% % (about 1mg/mL) 1,000K % Once you create your solution you must give your polymer time to reach a state of relaxed confirmation. This can take several minutes to several days. If you see particulates in your solution it needs more time to completely dissolve. If after a couple of days your polymer 4

5 does not dissolve then you may need to look into other methods to solubilize the sample, such as stirring, sonication, or heat. However, all of these alternative methods have the ability to damage your polymer and affect the molecular weight distribution. (Note: if you heat your sample it must be soluble at room temperature, or you will clog the GPC column) Comment [BR2]: If you have any references this would be good to mention here. Your presentation at group meeting was quite nice, and would be great to see it incorporated at the end of this document in some fashion Once you believe your polymer is in a relaxed conformation you must filter the solution with a 0.45 micron filter (if you use a smaller sized filter you risk mechanical damage to the polymer). By filtering the sample you will remove any particles that did not dissolve, sometimes even high molecular weight polymer depending on the solubility of your sample. Under no circumstances should you inject a sample that has not been filtered! If you are caught doing this you will lose your GPC privileges. Comment [E3]: Nylon or PTFE? 2.2. Software Setup On the Windows tool bar click on the Breeze icon (The username is Breeze). Once in the Breeze program click on the Run Samples Button which will bring you to the screen below. Usually the flow rat of the system is 0.1 ml/min when it is not in use. Click on the Flow Rate icon (which looks like a faucet) and input the following parameters: Flow A: 0.3 Ramp For: 3 min 5

6 Once this is complete you can now click on the equilibrate/monitor button, shown below: Select 03MLFlow, and press equilibrate. Comment [E4]: Brief one sentence description on what this is Allow the system to equilibrate for at least 20 min. At this point the baseline should be flat (although it may look like it is fluctuating due to the size of the Y axis. If the Y axis units of the W2489 ChA 254 nm are below 1, the baseline is okay). After the system equilibrates move the waste line (which has purple tape) from the THF bottle, to the waste bottle. Comment [E5]:?? 2.3. Sample Injection Now it is time to inject your sample. First clean the injection port 3 times with THF. After this, click on the Inject Sample icon. Comment [BR6]: Does it matter how fast the sample is injected? For sample name enter the name of your sample. Select Inject Broad Samples even if Inject Broad Samples is visible you must reselect it. Enter the values shown in the picture below for Vial, Injection Volume, and Run Time. Finally, click on the inject button. 6

7 Once you click the Inject button a window will pop up prompting you to inject your sample. This is the point at which you want to manually inject your sample into the Injection Port. Take the needle and draw 20 micro liters of your solution. Make sure that there are no air bubbles in your syringe this can alter your results, and also harm the column. Inject your sample into the port, and pull the Injection Port down to inject your sample. Once the sample is injected pull the Injection Port back to the Inject position. This begins the experimental run of your polymer sample Data Processing Once the run is complete click on the Find Data button on the left hand of the screen. Comment [E7]: What do you suggest is the best way to remove any that are present? Comment [BR8]: You had an entire discussion in your group meeting presentation about how it is advisable if you were analyzing a series of polymers to perform them all at the same time. This is again something good to delve into more depth at the end of the document. Comment [BR9]: Do you let this go overnight? Or just come back every hour to see the progress? Click on the Channels tab, and then click on the Update button. At this point you should see your sample in position 1 under SampleName. Right click on your sample name and select Review. Once you click on Review you will be shown your chromatogram. Click on the integrate icon, followed by the quantitate icon. This is will give you your MWD. Comment [BR10]: You don t have to pick the peak like you do with NMR? 7

8 Click on File > Save > Result You should now go back to Find Data, and click on the Results Tab. Once you click the Update button your saved result will appear. Right click on your result and select Preview. This will put your data into a template that you can save onto a USB drive. Once you are done taking samples make sure that you turn off the lamp, put the flow rate back to 0.1 ml/min, and move the waste line back to the clean THF bottle. Comment [BR11]: Looks good so far!! As mentioned previously, let s build on this by having some words on some more sophisticated issues like the ones you brought up during group meeting, and anything else along those lines (how to interpret your data, signs of aggregation in the GPC, etc.). While the manuals are not intended to teach everything there is to learn about GPC, it is an opportunity for more experienced users to pass on those tips and tricks that everybody wishes they knew about when they first started using the instrument. 8