So you have a new polymer, unknown protein, or material combination but don’t know its molecular weight? An easy way to measure molecular weight of your sample is gel permeation chromatography (GPC). GPC is an analytical technique that separates molecules in polymers by size and provides the molecular weight distribution of a material. GPC is also sometimes known as size exclusion chromatography (SEC). So how exactly does GPC work?
Figure 2. Gel permeation chromatography layout.
Briefly, How GPC Works
Before you can use GPC to determine the molecular weight of your sample you must dissolve it. In what solvent depends a bit on your material to be studied. Once dissolved in appropriate solvent you can inject your sample into continuously flowing mobile phase of the same solvent. Your dissolved sample and the mobile phase is then pumped through a column. This finally leads to a detector and the data system. But component of GPC is equally important to your final result, the molecular weight of your sample components, so let’s take a closer look at them.
More Detailed Look at GPC
Choosing an appropriate solvent for your sample is very important. There are a wide range of solvents used in GPC, from non-polar to aqueous. The critical part is to select a solvent that fully dissolves your studied material. The solvent must also be compatible with the column being used. A common non-polar solvent is tetrahydrofuran (THF), while even water can be used as an aqueous solvent.
Most GPCs have an autosampler with an injector. Using an autosampler, you can set up a very large run and let it run overnight, while you are working on other tests, or (and I would never do this!) while you check Facebook. Obviously the important part of using an autosampler is to keep track of the order of your samples. Trust me, you don’t want that confusion after you’ve run a test!
Figure 3. GPC column. Image by ANTEC GmbH Analysen.
A GPC column (Figure 3) is made up of the mobile phase and a stationary phase of porous particles all packed together into a column. You can select your needed pore size and a wide range exists for GPC. In order to select an appropriate pore size, you must have some idea of a molecular weight range of your sample. Otherwise, you are stuck with finding the correct pore size via trial and error. And you might ruin a few perfectly good columns in the process.
GPC columns separate material by size. And how they do so is a little counter-intuitive. In other techniques like gel electrophoresis we are used to the smaller material moving faster than larger material. But in GPC, higher molecular weight material passes through the column first. While lower molecular weight material enters the pores and takes longer to elute out of the column. You can imagine that these smaller particles “get stuck” in the small pores, while large molecules simply pass by.
Many different types of GPC columns exist and column selection should be based on your sample. A good column would have the absence of ionizing groups and a low affinity for the samples being studied. You don’t want your sample sticking and clogging the column (they are expensive- often around $2,000 or $3,000).
The pump pushes the mobile phase and your samples through the column. Pump pressure and flow rate are two very important variables in GPC. Too much pressure and you can damage your sample and the column, but too little pressure and your sample may never elute out of the column. Flow rate is also critical. A flow rate that is too slow makes the test unnecessarily slow, while a flow rate that is too fast can provide inaccurate measurements.
Numerous types of detectors also exist for GPC, including ultraviolet, refractive index, infrared, density, etc. Normally two detectors are used in conjunction with each other, so that molecular weight can be directly calculated. The actual chromatogram from GPC shows the amount of material that exited the column at a certain time. Using a calibration curve of polymers with known molecular weights, the molecular weight distribution of your chromatogram can be calculated.
Figure 4. Molecule size distribution of a material in millivolts versus time in minutes. Image by Chem538grp2w09.
Figure 4 shows the chromatogram output of GPC, before molecular weight is calculated. In this case, the two peaks would translate to different molecular weights. The peak on the left would have a higher molecular weight, while the peak on the right would have a lower molecular weight.
So what exactly can GPC tell you about molecular weight? GPC can provide number average molecular weight (Mn), weight average molecular weight (Mw), z average molecular weight (Mz), molecular weight distribution (MWD), and the polydispersity index (PDI). A molecular weight distribution typically looks like a bell curve, with one end indicating high molecular weight and the other end indicating low molecular weight. A broad molecular weight distribution peak indicates that there are many different molecules with different molecular weights – that the sample is very polydisperse. A sharp molecular weight distribution peak indicates a sample that has mostly one narrow molecular range – also known as a monodisperse sample.
Besides obtaining an overall molecular weight for a sample, GPC also provides insight into the different components of a sample. Because the process separates different sized molecules from each other, you can learn a lot about a sample with multiple parts. For instance, even if you already know an overall molecular weight, you can determine the size distribution of the molecules.
One thing to keep in mind is that, while GPC can provide much needed insight into sample molecular weight, samples often have overlapping molecular weights. In these cases, you might not be able to get an accurate molecular weight of a certain component of your sample. Also, some studied samples are more finicky with GPC than others when it comes to solvent and column selection.
There can be a small learning curve with GPC, but once you get the hang of it, it’s actually a very easy test to run and very hands off after setup. Multitasking at its finest!
Have you had good experience with GPC? Let me know!