Mr. Paul Johnson: Hi. I'm Paul Johnson, part of the Global Marketing Team here at Bio-Rad. I'm responsible for training our worldwide sales team, along with developing traditional marketing collateral as well. I have over 10 years of practical lab experience, including working at Pfizer in antibody process development
I'm here today to tell you a little bit about the basics of chromatography and also tell you about our new NGC product and how it may help your research. Welcome to Chromatography 101. This is part one, an introduction to medium pressure protein chromatography
Chromatography actually comes from a term coined by a Russian botanist who was doing some work to separate chlorophylls and carotenoids from plant extracts that had different colors. And from that, he came up with the word chromatography that came from the Greek chroma and graphein. And what we're talking about when we're talking about chromatography is it is a separation process where you have a mixture of components in a solution and that solution is passed by a stationary phase. We call that, a lot of times, the matrix or the media. And in the mobile phase this is where we would have our mixture of components. These compounds are passed by the stationary phase and separated based on their traction or impulsion from that stationary phase
At the very end of all this separating we often have a detector that will provide some type of quantitative estimate of what we have in solution. So, let's look in-depth at what's actually happening within this column process in chromatography
So, as I mentioned, has a mixture of components are passed by a solid phase. And, again, as I mentioned, this is usually called the media or the resin sometimes. They're used synonymously. But, what happens is you will have some type of targeting molecule, in this case we call it the ligand, that will be there for the expressed purpose of attracting a protein. So, we call that target molecule the ligand. And so, the media it is several support structures with ligands attached meant to attract a protein. In this illustration you see that many of the proteins and solutions do not interact with that ligand. And that’s actually the intended purpose here. We only want to capture the protein of interest. So, you might wonder how exactly are we attracting a protein to this ligand. Well, there's actually quite a few different ways you can do that. And that is the basis of the different type of chromatography media you may have seen in your lab or in other labs. So, the type of techniques that exist out there include those using charge, hydrophobicity, solubility, size or a combination thereof to attract a protein. Some of the most popular techniques include ion exchange. So, this takes advantage of protein and charge characteristics. There are size exclusion chromatography that takes advantage of size differences of proteins. There is hydrophobic interaction. And what this does is it takes advantage of those proteins that don't like to be in polar solution such as water. And the very popular technique that I'll tell you a little bit more about here in a just a few moments is affinity chromatography that combines many of these characteristics
So, let's go a little bit more in-depth with chromatography and what exactly is happening. So, I already told you that we would have a tube that would be filled with some type of solid chromatography media. And that tube is where we would allow flow of buffer that contains sample and the buffer that would allow traction to occur to pass through the media
Our goal here is to have that sample approaching of interest bind to that media in most cases. And then we would want to, in a lot of cases, use some kind of difference in the buffer properties to separate that protein from the media. And this is usually accomplished by either a gradient, which I'll tell you a little bit more about, but essentially that’s changing solution properties over time or if you know something about your protein you can actually use a sudden step to knock the protein off of that media
But there are techniques out there, such as size exclusion, that allow isocratic flow of buffer, meaning there's no change in the buffer composition during the protein purification. During all of this process, we would be collecting what is called eluent or elute. This is essentially what we are separating in terms of the mixture components, one from another. And when we separate these out we do what is called collecting fractions. And what you see here on the right is a chromatogram. And this is essentially an output of what you're doing in this whole protein purification process
Notice on the right you see a very small peak. This is actually our protein of interest. And this is not uncommon for a first step that you see a lot of components in a mixture that actually aren't the ones we're most interested in. And I'll go into great depth about this in just a moment. But this gives you an idea of what a typical protein purification procedure might look like. So, let's take a look at the chromatogram. I just showed you an example, but what exactly are we looking out with this type of trace? Right now it looks like a mountain range, but I'll explain it so it makes a little bit more sense to you. We'll start with the axis. So, first let's start with the Y axis. And you see on the Y axis we're saying that typically this is where you would measure the amount of protein you have in solution through a UV monitor
I would also make a note that sometimes systems will allow two axis and a lot of times you might see the concentration of salt in solution on the right axis. You might also see pH measured as well. But, the most typical chromatogram will measure protein concentration and that’s usually, again, 280 nanometers
On the X axis we see time or volume being measured. And this really is dependent on the researcher's choice of what they want to see. Sometimes you might see it in milliliters. Sometimes you might see it in column volumes or then you might see it in minutes in most cases. Now, let's look at the peaks a little bit closer. The peak on the far left you see noted as visa vero [sp] is the flow volume peak. Now, this means basically whatever passed through the column and did not interact with it in any way, that would be the first thing you would see, that is called the void volume peak
You will also see some other terms over there that are more specific to size exclusion chromatography, such as visa B [sp] and visa T [sp]. Those mean you bind the dilution and the volume or the total volume. But, what I'm trying to point out here in this illustration is when you are looking at a chromatogram, what does a chromatographer typically want to see? They want to see the best case scenario, that is the difference between V subzero there and visa B. You want to see baseline separation in your peaks
And what you see on the right hand side is a less than ideal situation. This is where you have peaks that are not well resolved. Sometimes you might see an example of this being called a shoulder. So, a shoulder is where you see part of a peak, kind of in a shoulder fashion or shape attached to another peak. But, the point is we want to optimize our procedure such that we get as close to baseline separation as possible
So, I mentioned that there are several steps in a protein purification procedure. I made reference to the fact that we are going to bind the protein and then somehow we're going to get that protein back off the column. Well, what we're talking is elution. And you see there are two different forms of elution represented in the top and bottom of this illustration. So, we see isocratic elution and we see gradient elution. So, with isocratic elution, essentially what we're doing is we are keeping everything the same in terms of our binding conditions. And, at a certain point, we know something about our protein and we're going to hit it with a specific concentration of salts, in most cases, to get that protein back off of the column. So, in other words, everything is the same until a certain point we decide we're going to use a defiant concentration of salts or, perhaps, we change the pH, but it's going to be a very defined pH. So, everything happens at once to knock that protein off the column
Now let's contrast that with gradient elution. With gradient elution we're actually going to change conditions over time. And what that means is we'll start with a set concentration of salt, say, 0 percent. And over time we will increase to a total of, in this example you see 50 percent. So, we're applying that buffer bead because we have typically two pumps on a chromatography system and, in general, people will use the second pump, the pump B, to use their elution, to introduce the higher salt concentration. And I'll tell you a little bit more about that in a moment. But, I'm really trying to make the point that there are two typical methods that researchers will use to elute their protein off of the column
And you see one is a very sudden step, or isocratic elution. The other is gradient elution or changing the concentration over time. One thing I should also point out you see there are some other steps in this purification protocol listed. And maybe I'll just spend a moment to describe what's happening here
So, on the far left, so focus your attention on the top left illustration, you see something called equilibration. So, this means essentially making sure that the conditions in your sample and the conditions in the column are the same in terms of cell concentration, pH, et cetera. When we're sure that everything is the same, then we will actually fire sample to that column. We'll wash. So, what we mean when we're washing is we're washing away any unbound or non-specific finding. And when we're sure that our protein is the only thing bound to that column, we'll elude it off. And then our last step will be actually to try to knock anything else that may have bound accidently to the column off. So, we call that our wash step
And on the far right you see regenerating the column. This is essentially getting your column ready for the next procedure you want to perform, so, a little introduction to the steps in chromatography
But, the point of this slide is really to introduce you to what's happening at elution. Again, this is where we're knocking the protein that’s been bound to the column off so then we can go and study that protein, analyze it or whatever else is planned for it
I mentioned one very popular technique. It's called affinity. And essentially this is a lock and key mechanism whereas your protein of interests binds specifically only to one protein of interest targeting ligand or essentially the lock in the key. So, in this case the key would be the ligand on the matrix media and your lock would be, then, the protein of interest
This is a very popular technique because it doesn't really require, in many cases, a very fancy set up. You can usually achieve very high purity, most times greater than 80 percent. And people will use a combination of gravity, stem columns or syringes to perform this procedure. Essentially you get that lock and key mechanism in place and you might change your solution properties in salt concentration or pH or some combination thereof. And that will unlock both the ligand and the protein of interest, very simple, very straight forward. Now, you might be wondering, based on your academic experience, why I haven't mentioned some of the techniques you see here on the slide in the conversation of chromatography. Well, these are non-chromatography separation techniques, some of the most popular in the academic world include ammonia sulfate cut for salting out, which essentially takes advantage of your proteins elusive properties or solubility properties relative to all the other proteins in a solution. But you might also see people using centrifugation or filtration to take advantage of size differences in proteins. You might have also heard about gel electric resits as a way of isolating a protein. But, this is really an analytical technique that only provides a very small amount of protein and typically it's not very useful for activity type of experiments
You might have also heard of amino precipitation. This is specific to antibodies in most cases and it's really most useful for batch purification. So, let's look a little bit at the different approaches you might have in using column chromatography. So, in the first illustration what we see is the most basic setup. And you might have seen this in a biochemistry 101 type of lab. So, this is your typical glass column that has glass wall and, perhaps, some cotton at the very end. But, essentially what we're looking at is at the very top of the column we would have a frit or some type of net to keep your solid phase in that container. The top is usually open, which allows you to apply your sample once you're sure that the column in your sample conditions are the same and ready for application. Essentially what you would do is use a pipette a lot of times or just dump it from a beaker straight on to the column. It would pass through and then you would collect, a lot of times by hands, which is the painful way to do it. You would collect your fractions there at the bottom
This type of methodology is allowed and enhanced actually by columns produced by Bio-Rad, such as the Econo-Column. So, these are meant for very simple applications as I just described. What's nice about these is they actually have attachments that allow you to slowly flow buffer, if you so choose, if you don't want to go through a gravity type of setup. But, these are really meant for very low pressure, low-tech type of situations. But, for those researchers choosing a low pressure type of application these are great columns for that type of use
Let's contrast that with what we're calling the improved column situation. So, what you see up top is one of our partridge columns at Bio-Rad. But, you will also see on the right kind of a more traditional chromatography column. This is a representation of what one of our new enriched high resolution columns. And when we're talking about columns like this, essentially what they do--what gives them an advantage over what we were just looking at is they have plungers or adapters that allow the bed to stay in place such that you can flow at a higher flow rate and, therefore, allow higher pressures and faster finding in elution
So, basically you would want a column like this any time you're attached to, in most cases, a low to medium pressure system or a high pressure system. But, essentially, this is most useful for a medium and higher pressure situations. But they allow the bed volume to stay the same and allow you to flow at a faster rate
So, I was just making reference the different type of chromatography systems that exist out there. Why might you choose a low pressure or a medium pressure system? Well, this is really based on the complexity of your purification strategy and how much automation you want. So, in other words, do you want to have to stand by your system and collect the fractions yourself or would you rather have the flow rates and the elutions and the collection done by the machine for you? And that’s what we're talking about with automation. Also, the column you use will dictate the type of system you're using. So, most columns have a pressure limit. So, that’s why we would use a low pressure column for something like an Econo-Column and a medium pressure system for something like the high resolution column I was just telling you about. So, you see a wide range of systems available through Bio-Rad, including our new NGC there on the bottom right
One point of confusion when I talk to people who are new to chromatography is what exactly is happening on the chromatography system. So, what I'd like to do is just kind of describe each component of the system, so whether it's the NGC there that you see or whether it's any other system you might have in your lab. They all work the same. So, what I'm gonna do is give you a brief introduction to the system through illustration. So, let's take it component by component and kind of describe what's happening here. I think this will eliminate some of the confusion you may have and make it easier for you to look at your own system and understand what's happening
So, first let's start with the system pumps. What's happening with the system pumps? Well, as you see here, we're going to flow buffer through the tubing we have attached to the buffer into the system through the system pumps. Okay, that seems very intuitive. Remember I told you that there was an A and a B pump? Well, you can see here that on the left, the bottom left, is our system A pump and on the bottom right is our system B pump. We'll tell you a little bit why--a little bit more why you'd want two pumps here in just a second, but this is essentially how we're going to get the buffer into the system and on to the column
Next is the mixer. So, the mixer is where we're going to proportion the amount of fluid from both pumps A and B into the system and on to the column. So, remember we were talking about elution. And elution there is a set amount of salt that’s used to both bind and elude the protein. So, this is where you will use a strategy of how much of your wetting buffer or equilibration buffer in pump A and how much of your high salt concentration buffer or any other type of component that might elute your protein of interest in pump B. So, the mixer is going to dictate, based on your chromatography software, how much of A and B are introduced into the column. So, next we'll go to the injection valve. And what the injection valve will do in your system is allow the sample to be applied at only the time that it's appropriate. So, this is sort of a traffic cop. The injection valve will essentially say buffer can go to the column, away from the column or sample can go with buffer to the column. I have an illustration that'll make that clear in just a moment. So, please bear with me. But, that’s essentially what an injection valve will do for you. In this case we've drawn in a little sample loop, but I'm sure many of you are familiar with sample loops. So, that’s a great example of what you might use in your lab
So, the next step after all this is really to get the fluid and your sample onto the column. And you see our illustration of a column there. But, how do we know what's happening on the column? Well, that’s why we have detectors on a system. And what you see here is a representation of a UV detector. And that’s the first place our fluid is going after reaching the bottom of the column. And the most typical applications will use either 260 or 280 in [unintelligible] absorbance. So, 280 is used to basically measure protein absorbance. And 260 is for monitoring DNA. You might also see on a chromatography system next in this flow path, a connectivity monitor. And what this does is it measures the amount of electrolytes in solution and typically you measure that in millisiemens per centimeter. Finally, after everything that I've just described you will go to some type of fraction collector. And what we're doing there is we're collecting discreet amounts of buffering sample and we're separating those from each other so that we can later analyze those and use those in our research. So, now that we've described the flow path a little bit, let's talk a little bit more about the individual components. What we're showing you here are different versions of Bio-Rad system pumps. And essentially what these do are speeding up the process of binding and eluding. So, as I mentioned, gravity is one way that you can bind and elude your protein. But, it's not the most efficient nor is it the fastest way. So, this is why you would have humps in the first place
Now, let's look at the injection valve. As I mentioned, the injection valve is sort of the traffic cop in all of this buffer flow. And if you'll look down at the bottom left, you see, in this case, there is a sample loop that’s not in line with the system. Okay, so, essentially what the injection valve is saying is I'm going to allow buffer to go from the pump to the column, but I'm not going to apply my sample. When we're ready for the sample to be applied then the injection valve will turn. And when it turns, now the loop is in line and you will go from your pump, through your loop and to the column. There's also a waste position if, perhaps, you want to purge your system where neither the sample loop or the column are in line. Finally, let's talk a little bit more about detectors. So, basically, what's happening on my column? I mentioned that 280 is a very common application for detection using a UV detector and that’s because proteins have certain amino acids that will absorb at that wavelength. So, that’s a great way to quantitatively estimate how much of each protein you have in a solution. But, you can also tune to a frequency like 260 to look at the composition of nucleic acids in solution. This shows a chromatogram of a 280 absorbance that also shows the fractions that are being collected and why that is important is when you have the fractions collected you would often run them on some type of electrophoresis gel as you see here on the right. And you can match those fractions to what you're seeing on the gel and get an idea of the identity of each of those--the fraction components
So, besides a simple UV detector, what else is available to give you an idea of what's in that solution and what's being separated? As I mentioned, a very simple type of detector, like a 280 or 260 is available. Often on systems you will see a multi-wave detector. What that allows is looking at a wider range, such as 400 to 500-range where proteins that have color can absorb. You might also have a connectivity detector, as I mentioned before. This essentially is measuring the ionic strength or the salt concentration in solution. And this is most useful when you're seting up a gradient you want to know exactly what salt concentration your protein came off at
So, for example, you might have started your protocol with a gradient and you determined that your protein of interest eluded at a certain concentration then, perhaps, the next time you might want to use the isocratic method we told you about to really isolate just that protein in your elution protocol
If you're using pH in your experiments as some type of differentiator, you might want a pH meter. And that’s something that you will often see in line most often after a UV detector. And some people are interested in other type of activities such as fluorescents and they might have an external detector attached to their chromatography system as well
Finally, let's talk about fractioning. So, fractioning, as I mentioned before, you're going to collect very discrete amounts of solution or your opulent and basically you're going to separate all those components in the mixture from each other so you can use those in your studies. The very old fashion way of collecting fractions, often by hand, is using a timer and your own hand and a beaker and essentially taking little amounts of protein off at a time. Now, this is the least efficient way to collect fractions. Most people use an automated fraction collector, as you see below. And the automated fraction collector, in combination with your chromatography system, can use parameters such as self-concentration and pH et cetera to move from fraction to fraction and basically from that then, you can separate out your protein of interest from all the others in solution
So in summary, chromatography is a science of separating components by taking advantage of how they interact with a stationary phase. And I mentioned that the chromatography column is the most effective way to isolate proteins. But simple set ups such as gravity or syringes can be used. What a chromatography instrument provides is speed and reproducibility in getting to that answer and getting to that isolated protein. Such things as detectors and fraction collectors allow that type of automation. But there are other components that add to speeding up to your end goal
I showed you a wide range of Bio-Rad columns and systems. And these are all available for the type of applications, whether that be the more simple setups like an Econo-Column and gravity or whether that be using a pre-packed column, such as the BioScale mini, or a high resolution column such as our enrich series. And we do have systems available such as the econo system or the more versatile system the LP Jewel Flow [sp] or our brand new NGC
If you found this webinar useful you can stay tuned to other introductory webinars. We have one on ion exchange that'll be presented by Shawn Anderson [sp]. We also have size exclusion chromatography, the basic principles, presented by Jim Marr [sp]. That concludes this webinar.