تدريب Shadowing: Introduction to HPLC - Lecture 1: HPLC Basics - تعلم التحدث بالإنجليزية عبر الفيديو

Hello, everybody.

Welcome to another ChemComplete lecture series.

And today's lecture series, we are going to be introducing an introduction course to HPLC chromatography.

So HPLC stands for High Performance Liquid Chromatography.

And in this first lecture, we're going to take an overview of HPLC

and learn some of the basics and then we will get into more detailed parts as the lecture series continues,

into different types of columns and how to manipulate mobile phases and things of that nature.

So let's go ahead and get started with a general overview.

The first thing that we want to have an understanding of is what chromatography is.

So when we use the term chromatography, we're talking about a separation of compounds.

Now traditionally when chromatography was first discovered,

it was through basically just a separation on silica or alumina type gel.

And you could literally see the colors separating out from these different organic compounds.

And that's where the name chromatography comes from.

The chroma part with the color is referring to that separation where you could visually see colors.

Now in today's more modern labs, we usually have two options when it comes to instrumentation.

The first one is gas chromatographs, which is GC, gas chromatography.

And then the second one, which we will be talking about, is obviously HPLC.

Now, the difference between these is when you want to use GC, certainly you're going to be dealing with things going into a gas phase.

But one of the distinctions is that you need whatever you are putting through the GC to be volatile.

So whatever compounds you're going to work with, they have to be volatile compounds.

And that means that GC tends to be better situated for compounds that have a lower molecular weight.

And that's simply because due to the volatility, compounds with lower molecular weight have fewer intermolecular forces, and therefore they tend to be more volatile.

And then obviously GC is going to be operating at higher temperatures, because whatever volatile compounds we put in there,

we are going to need to push them into the gas phase.

And that requires a higher temperature, both internally in the column and at the injection port where we are injecting our samples.

So on the other hand, if we take a look at HPLC,

HPLC is acceptable for both volatile and you can do non-volatile compounds in HPLC.

Because we're not trying to push things into the gas phase, it's liquid chromatography.

The only thing you really need to worry about is solubility.

Now, solubility is going to be important, especially when we start talking about mobile phases and picking out solvents,

how things adhere to columns and how they are washed off of the columns and how that affects retention time.

One of the other benefits of this is that it's less destructive than gas chromatography is.

You can recover your samples most of the time

if you only have a small amount and you're attempting to purify something by HPLC, that is possible.

And one of the reasons it's also less destructive is most of the time you can run these at room temperature, so around 25 degrees Celsius.

There are no high temperature requirements where you have to move these compounds into a gas phase.

So that's sort of an overview of chromatography, the different methods we have available, and which one might be useful in which case.

So the next thing that we want to ask is how does HPLC actually work?

So now that we know some of the parameters, we want to take a look at how HPLC actually separates compounds and works.

And the way that this occurs is there are two phases that we consider,

and this is usually true of any type of benchtop chromatography as well.

So when we're talking about column chromatography, the original chromatography we mentioned, this is true.

So you're going to have a mobile phase, and as the name implies,

this is going to be a phase involved in the movement of compounds through the column.

And your mobile phase in HPLC is going to be a liquid,

and usually it's some sort of solvent or a mixture of water and organic solvents.

And you can manipulate the polarity of the mobile phase by mixing and matching.

So certainly water, alcohols, those are going to lead to more polar mobile systems, and that will mobilize polar compounds, whereas you can scale it back.

You could go mid-range with something like ethyl acetate or methylene chloride

and then you could scale back even further and

if you really want to get more into the non-polar region you could start working with maybe some hexanes

or some of your ethers where you would have a better movement of your non-polar compounds in a situation like that.

All right so you have the mobile phase and then the other phase that you have is known as the stationary phase.

Now the stationary phase, again by the name, is going to be a phase that is not involved with movement.

It is going to be keeping compounds sort of adhered or stuck where they are.

So this is usually going to be a solid chemical phase,

and this is the material that you're going to find in your column.

So it's solid chemicals that are going to be bound into a column.

Now you may have heard if you've ever experienced HPLC or GC because we use columns in GC as well.

You may have heard of the term column.

The column itself is just a very long tube

and then the columns properties depend on what has been coded inside of the column.

So maybe you've heard of things, and we'll show an example later, something like a C18 column, a C8 column, a C4 column, an ion exchange column.

All of those types of columns are going to have a different interior makeup or a different interior stationary phase,

and that will be responsible for binding the compounds as they're moving through

and you really have sort of a give

and take between the mobile phase and the stationary phase so

when you're doing this you will take your sample it will

be loaded with the auto injector into the column along with some mobile phase

and then your sample will adhere to the stationary phase

so it almost like glue

or some sort of a sticky pad the compounds will adhere to the stationary phase

and then they will slowly be washed along depending on their

preference or their binding to the stationary phase so the mobile phase is pumped through at a steady rate.

And then depending on the polarity of that mobile phase and the polarity of the stationary phase, that will determine how quickly your materials move along the column.

And the goal with HPLC is you want to separate these materials out as best you can.

You don't want them clustering together.

Again, going back to something like a column

that you would be running on a benchtop in an organic synthesis type of manner.

When you have a column like that, the goal is to separate the compounds out into different sort of aliquots of solvent.

You don't want to just bleed all of them down together, otherwise you haven't really done any type of chromatography and separation.

All right, so to do a very simple picture, and we will have a larger visual as we wrap this first lecture portion up in a little bit.

But to give a very simple diagram here, the way that this would work is you have your mobile phase.

Now I'm just going to draw a single container of solvent.

Many times in HPLC, you're going to end up having several different solvents

and the HPLC pumps can mix and match them at different rates.

So let's say it's called a solvent gradient.

I want to start with 50% methanol,

50% water, and then I want to move to 75% methanol five minutes into the run for better separation at that point.

You can tell or program your HPLC to pick

and choose different amounts that it puts through the pump at a given time, all right?

But in general, you're going to have your bottles filled with mobile phase, and the mobile phase is going to be connected, okay, into the HPLC.

It'll go through the pumps.

It also goes through something called a degasser, which we will talk about in a couple of minutes.

But the idea is that eventually, okay, along with your compound that is injected,

it's going to find its way onto the stationary phase, which is your column.

Okay, and in the stationary phase, it's going to, as we described travel along

and separate out at different times

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and once it comes off of the column it's going to hit the detector

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so the detector is going to be using usually we use

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a lot of uv vis you can use a fluorometry type of detector where you're doing fluorescence

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if you have the proper derivatives and materials

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but you have to have some way of detecting what's coming off of that stationary phase.

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And then that is what will be output into a chromatogram.

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So that's where we see the actual graph.

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And you've got the peaks as they go along.

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And those peaks have different tension times.

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Okay.

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And again, we'll talk about all of this in a little bit more detail.

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But the key here is that the compounds are going to travel at different rates.

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And therefore, they hit the detector at different time periods.

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And those different times, referred to as retention times is how we can really identify what is coming off of the stationary phase or the column.

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All right, so a mobile phase is oftentimes going to be a mixture of water

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and organic solvents when we take a look at it.

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And the polarity of the mixture or the lack thereof, because you could certainly be working with non-polar ones as well,

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that will determine how long the compounds are going to stay on the column or adhere to the column, and that's known as retention time.

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And then the stationary phase, the columns are packed with different materials, usually some sort of organic materials often applied to a silica bed.

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And those will bind to the compounds that are being separated

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and sort of spread them out as the mobile phase is going through doing its thing.

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All right.

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So one of the things I mentioned here was there are different types of material that get packed in your columns.

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And as the chemist, you have to decide what type of column you want to pick out in order to get the best separation.

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And each of those can go into their own lecture.

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So we can talk about, and we will make a list,

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but we can talk about things such as ion exchange or reverse phase or normal phase HPLC.

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Those all have different meanings.

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They have different type of mobile phase and stationary phase setups.

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all right but for example

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if we were to talk about using a c18 column a

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c18 column I'm drawing the inside of the column here okay

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so imagine that this is the inside of the column what you would find is a silica binding portion

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that sort of lines the bed of the column okay now a lot of times

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when we do wet bench sort of a desktop chromatography we use silica gel right

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or alumina so you have this as the base

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and then once you have your silica gel you can adhere other types of organic material to the side

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so what you end up with is in a c18 column, you get very long 18 membered carbon chains.

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So what I'm going to write here is CH2.

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We have 17 of those, and then the 18th is going to be a CH3, right?

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And then you would see that at every single silica site, all right?

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So it's going to get a little crowded here, but I'll draw another one, all right?

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So you'd go like this.

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You have the silica with the two methyl groups, and then you would have a CH2, right?

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And you have 17 of those and a methyl.

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So you have these very, very long hydrophobic, and that's important in a case like this, okay, hydrophobic chains.

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And I mentioned that that's important because you could also have a hydrophilic or a polar type of chain.

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This one in particular is going to have better adherence to other nonpolar compounds.

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All right.

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And it would sort of go on where throughout the column you would have this type of a binding site.

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And as the materials come through, they interact through intermolecular forces with these other compounds here,

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right, these long chains that are hydrophobic.

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And that sort of adheres your organic materials to the column.

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And the mobile phase will sort of push them along

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and depending on how strongly they adhere to it

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that is going to be what determines how quickly they will come off of the column

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which is relating to retention time there so

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for something like a c18 you're dealing with a highly non-polar column

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so non-polar analytes are going to be retained at a longer

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rate right they're going to stay on the column longer before they get pushed off onto the detector.

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Now this is very important the choice of column

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and solvent phases will always play a role in your separations

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and you want to basically maximize the efficiency of those separations

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which again could be its own lecture in terms of how

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do we maximize retention time how do we get good resolution

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so the peaks aren't too clustered together and we'll address that as we go along in this course.

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So what are some of the modes of HPLC

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that we can talk about because if you remember I mentioned

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that there are several modes

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and each one of them could have their own lecture in terms of what's involved

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so the first mode that we would talk about is just normal okay

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and for a normal phase you would usually be talking about

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a situation where the column is more polar than the mobile phase

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so the mobile phase is going to be non-polar in that case

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and the column is going to be more polar okay one of the other very common ones is reverse phase

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okay c18s tend to be used in reverse phase a lot

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and with a reverse phase HPLC it's just the opposite of what we said

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so something like a c18 you have a very non-polar column

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but then your mobile phase tends to be more polar

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so you might have methanol acetonitrile water things of

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that nature in different ratios okay then you've got some other ones when you start dealing with ion pair.

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Okay, so you can have an ion pair and you can also have an ion exchange phase.

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And then one of the last ones that sometimes gets mentioned is you can also have a size exclusion chromatography.

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Okay, so each of these has their own unique way of dealing with stuff.

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So sometimes when you're dealing with the ion exchange columns, you might be using some sort of a buffer, like a sodium buffer at different concentrations.

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Whereas when you're doing just the regular phase or the reverse phase, you're using water and organic solvents instead of a salt or a buffer system.

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All right, so each of these is its own lecture, as I mentioned, but I did want to list these here so that we could sort of talk about them briefly

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and give you an idea of some of the upcoming content.

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We will cover each of these in its own right, but it'll be its own probably 20-minute,

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30-minute lecture dealing with everything that goes into each one of these types of phases.

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So with that being said, what I want to do, I want to bring up an image here

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as we get ready to start wrapping up this first lecture

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and really go through each of the parts of an HPLC

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because it's one thing to talk about it in theory with

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this sort of like simple drawing I have up here in blue.

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But it's another thing when you actually walk into a lab and you see the HPLC,

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there's a lot of containers or boxes for this instrument that are sort of stacked on top of one another.

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So I made a rough sketch of an HPLC setup.

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It's very generalized, but it'll get the point across as we're talking about this.

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So let me bring that up and we'll take a look at

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that together okay

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so here we are we have the setup for what the HPLC would ideally look like

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if we had the system

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and we could kind of briefly look at the insides again this is still a simplified drawing

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but it's more detailed than what I showed a few minutes ago

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so the very top part here you can see the bottles

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that are hanging out at the top this is where you're going to find your mobile phase components okay

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so these are your solvents your water whatever it might be it could be your buffer solutions

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but your mobile phase is usually found at the top of the HPLC instrument

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and it will be in you know maybe four liter solvent

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bottles it might be in a one liter plastic container again

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most of the time you're going to see multiple containers

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because many HPLC techniques use what's called a gradient solvent system where you're changing it over time

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and either increasing or decreasing the polarity at certain time markings on the column.

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So that's what's being hosted at the top there where you see the different colored bottles.

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Those are your mobile phases.

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So the mobile phases send the liquids into the next component, and that's the component we see right here,

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and that is known as a degasser.

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Now, a degasser has one simple job, and that is literally to degas the solution.

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So the degasser, as it accepts the mobile phase, these solvents, it's attempting to remove any air bubbles

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or any sort of air pockets that might be found in your mobile phase, because you cannot have,

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or you certainly don't want to have air pockets as you're going along in your column

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because that can disrupt the flow and the rate at which these components come out.

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It can screw up your baseline particularly.

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So having a good degassing system is crucial in making sure

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that you establish a steady baseline and you have nice clean separation of peaks

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when they finally hit the detector and you get your chromatogram.

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So the mobile phase is pumped into the degasser and after any type of air is removed,

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this liquid comes down and is sent to the pump system.

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So these are the pumps.

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Now, when you have your HPLC pumps, they use a back pressure in order to continuously,

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and it is very important that it's continuous and at a steady rate,

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move the mobile phase down through the rest of the HPLC system across the column and into the detector.

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And it's important that your pumps are in good shape in order to maintain this regular flow, okay, which you could call the flow rate of solvent.

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You can adjust how quickly these pumps are working and what pressure in order to change the flow rate.

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When you change the flow rate, you obviously change how quickly or slowly things will come off of your column and hit the detector.

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So changing any of your pump settings will be able to change the retention times for the compounds.

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So sometimes that can be a good thing, sometimes that can be a bad thing.

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But you've got your pumps, they are going to help mix these mobile phases

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and then pump them down and get them ready to go into the stationary phase.

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All right, so after the pumps have thoroughly mixed it, it's going to come in to the next part of the HPLC in

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which we have the mixing for the auto sampler and the injector.

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All right, so what you see on the left over here is your auto sampler, or at least my best representation of one.

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and the auto sampler is going to take your samples.

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So these little blue vials here are your samples.

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You've processed them in a lab.

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You've been given these samples.

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You put them into a rack.

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You load that into the auto sampler.

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And then through the software, the auto sampler will come, pick up, give an amount.

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You can choose the injection amount.

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It's usually a tiny amount like five uh not 550 to 100 microliters excuse me

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and that will send this material

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and mix it right into the injection port where it will also meet up with the mobile phase

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and that will be sent down into the column.

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So that's the next part right here, right?

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After everything is thoroughly mixed, it is sent down and your material from the autoloader is going to adhere to the column

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and then your pump is doing its thing.

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It's continuously pushing the mobile phase along.

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so your materials will start traveling on the inside of your column at a given rate right

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and we've talked about the inside of the column and how

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that works the adherence

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and then at some point they will come off of the column they will be sent down into the detector

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so again your detector could be fluorescence it could be uv those are probably the two most common

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but when you hit the detector

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that is where the signals are going to be received

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and then you can output into a chromatogram and

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when you get to the chromatogram that's the actual printout

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that you would be looking at where you have the peaks

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and their retention times and that is the final step for the HPLC at

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that point you would be using software in order to analyze your actual chromatogram okay

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so a general chromatogram is going to look something like this you're going to have

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so along the y-axis you're going to usually some see some

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sort of a response okay usually it's just called abundance

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or detector response or something of that nature and then on the x-axis you're going to see the time.

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This is typically in minutes.

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It could be in a different unit depending on how you set it up, but it's almost always seen in minutes.

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And so you have your baseline.

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Again, to have a steady baseline, you need to make sure that you've got a good degasser for your mobile phase.

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And then at some point, you're going to see, if you have compound, peaks that start coming off.

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Okay, now you might just have one peak if it's a pure substance, but most of the time

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when you're doing HPLC it's to separate out several components from one another right

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so you can see an example like this now there's two important parts here

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and that is you have what's called T naught or T zero

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which is the initial amount of time that it takes for anything

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that has not adhered to the column to just come out

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and directly be flushed okay so there is a compound called uracil

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that is typically utilized in order to detect when this hits however again t-naught is going to be the elution time

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for anything that was not retained so for unretained material

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and when you have that sometimes you'll hear it called

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or referred to as dead time again this is simply time

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that elapses when you have essentially the first dump of mobile phase into the detector

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that did not have any type of adherence to the column.

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All right, so the other important thing that we've been talking about over and over again is the retention time.

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Now, this goes certainly beyond the dead time that we have there out to whatever peak you're currently looking at.

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So let's say that we're looking at this last peak right here,

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the amount of time that it takes to go from the dead time out here is TR,

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and that is considered to be the time of retention.

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Now, when we start talking about things like resolution and the distance between peaks, it's going to be important to understand both of these,

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because both of these components are used in calculating how to improve things like resolution okay

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but retention time okay and what's important about retention time

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and i know i mentioned this once earlier is

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that this is really we can identify compounds by retention time

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so the compound identity is associated by retention time and that's really how we look at compounds when we do HPLC.

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Now one of the other things that we discuss with HPLC is we do discuss the area under the peak.

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So the retention time kind of identifies what compound we're working with.

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The area under the peak is going to tell us how much of that compound there is.

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Okay so there's essentially the area under the peak is going to be proportional to the analyte amount.

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Now the way that you determine this

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because you have to have something to compare it to is that you would need to use a calibration curve.

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So let's say that I'm working with amino acids and I want to analyze the amount of lysine.

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So if I'm going to analyze the amount of lysine, I need to have a calibration curve of lysine at several different concentrations.

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And then ideally, I want

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that range to cover from min to max any type of sample of lysine that I might be expecting.

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So it can be a little bit of trial and error

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when you're first setting up your calibration curve if you don't know a whole lot about your samples.

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But once you have a reliable one, you'll know the area under the peak for the various concentrations of your lysine,

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and therefore you would be able to determine the amount based on any sort of unknown sample going through.

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And you always know that it's lysine based on the retention time.

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So that would just be a example there.

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All right, so we're closing in on half an hour.

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I think I'm going to wrap it up there in terms of the introduction to HPLC,

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and we will continue forward with these lessons and start to outline some more detailed parts to the HPLC,

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particularly the different types of phases that we can undergo, as well as some troubleshooting stuff.

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So what happens if all my peaks are too close together?

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How can I improve that?

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Things of that nature.

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So hopefully everybody found this introduction course to be useful.

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So I will see everybody for the next lecture.

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Thanks a lot, guys.

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تدريب Shadowing: Introduction to HPLC - Lecture 1: HPLC Basics - تعلم التحدث بالإنجليزية عبر الفيديو

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