Wednesday, January 23, 2013

Lets talk about Lab Techniques - Chromatography

ChromatographyHello gorgeous people...

In case you weren't aware there are several different types of chromatography that are commonly used that separate (aka extract aka resolution) compounds based off subtle differences in properties including polarity, size charge and/or affinity for other compounds.

Examples include....
  • Column (Flash) Chromatography
  • Gas Chromatography
  • Gel Filtration
  • Ion Exchange Chromatography
  • Thin-Layer Chromatography (TLC)
  • Affinity Chromatography can be done but these beads are uber modified ie antibodies for a virus or a bead with a particular DNA fragment and in reality I can't explain the general concepts behind these because it would take too long. Just know its anything you want anyway you want and your beads fish out whatever you seek.
I'll spend some time delving into each.....

You may ask why bother when I can wiki this?

This is true but I'm going to try to make this user friendly so everybody can understand. Plus now you don't have to switch tabs every 5 seconds. I'm just trying to help....By the way and as always PLEASE click an ad if you like what you see. It helps me out so I can continue to keep helping you all out!

Column Chromatography

I'm lazy and colum chromatography will be explored in further detail in the description of Gel Filtration Chromatography and  Ion-Exchange Chromatography...

I'm just gonna say that it would probably be a good idea to know that its a pretty decent way to separating bulk compounds. It involved filling a column up with silica gel (just like the silica of the TLC plate yes, SiO2). In this case you would have a polar stationary phase because those silica beads aren't going anywhere and a non polar mobile phase gets poured through this column that has been packed with the polar silica beads.

Main concept behind how this works: Like associates with like. The polar stuff will stick the polar beads. The non polar stuff will stick with the non polar mobile phase and be repelled by the polar beads it will go through the column without interacting with the polar silica beads....As I said the like will associate with the like. Polar with polar non polar with non polar...This all occurs in a column....hooray!

Gas Chromatography 

Lets be honest Gas Chromatography is kind of rare and in reality you/me/we probably won't be using it all that much in our scientific careers. In Gas Chromatography the liquid is the stationary phase and the gas is the mobile (moving) phase.  The ultimate goal is still to separate components and the main characteristic you'll use is their differences in volatility. Remember highly volatile means its boils easier or at a lower temperature. 

Fun Fact: Gas Chromatography has proven to be particularly useful for stuff that's super volatile

The procedure consist of loading a sample into a syringe and injecting it into sample port of the chromatography machinery/device, it then passes into the column which is under 24/7 temperature controlThe sample is vaporized and carried by an inert gas (usually Helium). The more volatile the component the less it will interact with the stationary phase so it can travel faster through the column (ie it finishes traveling first). As the components come out of the mixture a detector detects them (the identity of the component and the amount are recorded) and reports to a computer that usually puts it into some sort of visual form. This visual form can be integrated kind of like NMR to determine the percent of the compound the component was. Luckily enough we live in an age that the computers can do all the math and present the rentention times.

Note: The detector usually has a flame portion that helps in the process of detecting. The components pass through the flame become ions that affect the electronegativity. Detectors can also aid in the process of mass spectroscopy to aid in the process of Mass Spectrometry, this is called Gas Chromatography Mass Spectrometer (GCMS).

Gel Filtration Chromatography

Gel Filtrationis a type of Column Chromatography. The main physical attribute it uses to separate components is that of size.  It can be thought of as a molecular sieve and is sometimes/also called size exclusion chromatography.

You may ask, what is used to control the sizes of compounds that will be selected for?
The answer is actually pretty simple "beads" made of cross linked dextran. Dextran is a polysaccharide that is insoluble in water. The amount of cross linking determines what can pass through based on how porous it is. More links = more tangled = smaller pore sizes. Like the story of goldielocks and the bears a compound can either be too big, too small or jusssssttttt right.

The degree of porousness is commonly referred to as the fractionation range.

 For example Sephadex G-25 beads have a fractionation range of 1000–5000.

Size What will happen and why?
<1000 g/mol These molecules are small, and in this case they're too small. They can freely enter and exit the pores as the wish they will come out entirely in the total volume because of this freedom.
1000-5000 g/mol
Anything in this range will come out between the void volume (discussed below) and the total volume (also discussed below). These beads are in the right size to enter the matrix created by our lovely porous beads....aww how nice, no? In general it is said that...

k (log molar mass) = Elution volume divided by the Total Volume

You may ask what does this mean...It means that the volume in between the void and total is thought to be directly related (proportional) to the log of its molecular weight. The elements are separated by size and that equation shows the relationship between their size and the volume in which they eluted.
>5000 g/mol Anything 5,000 g/mole or greater in size will be toooooooo big to actually go through the pores of the beads. Being that it can't enter the matrix it bypasses it and it is through the be excluded from the beads. As a result the stuff that is too large elutes (leaves) first and is said to be found in the void volume of the column.

Terms and other stuff that you should consider knowing: 
  • Total Volume of column = Void Volume + Internal Volume
  • Void Volume = volume outside the beads
  • Internal Volume = Volume occupied by the beads
  • k (log molar mass) = Elution volume / Total Volume

Ion Exchange Chromatography 

Ion Exchange Chromatography involves the separation of molecules on the via their charge. In reality these is pretty much a modified version of gel filtration using modified/custom beads that have specific/chosen characteristics. There are two broad categories Anion and Cation Exchangers.

Anion Exchangers:

Often involves Diethylaminoehtyl dextran because of its overall positive charge.

Anion exchangers contrary to what you would guess actually have a positive charges. Due to their positive charge they bind to molecules with an overall negative charge. Meaning molecules that are positively charged and/or neutral can pass right on through well strictly speaking that is still based on their size and the pore size also known as the fractionation range.

Cation Exchangers :

Often involves CM Sephadex = carboxy methyl sephadex, has an overall negative charge.

Like anion exchangers the name can confuse you but yet again contrary to what you would expect they have an overall negative charge allowing them to bind with cations (molecules with positive charge). The Negative and Neutral stuff pass right on through at a rate determined by the good old fractionation range.

Both Anion and Cation Exchangers play with the idea of charge affinity. In order to separate what has bound to the beads additional mobile phases are added that vary characteristics like salinity or pH that will take the position on the beads kicking off the stuff that was previously bound to it. The get "exchanged" hence the name.

You can think of it like the increased anion or cation concentrations in the mobile phase compete with the previous compound that was bound to it. That compound gets kicked off, eluted and you collect it. This method is extremely sensitive and it must be done in the right order with proper timing or else you don't get the desired separation.

Thin-Layer Chromatography (TLC)

Thin-Layer Chromatography has already been mentioned in the experiment so for funsies feel free to go here to learn about the Purification of Caffeine.

But it can't hurt to talk about it a little bit more.

Thin Layer Chromatagraphy once again allows for separation based on polarity. You get have a polar stationary phase that is the silica plate which is coated with a thin layer of which is an absorbant silica (SiO2) and a non polar mobile phase.

 You carefully spot your sample and some sort of comparison or known compound at the origin of the plate. You should probably mark it with a pencil or something. You then place the plate in the stationary phase which is usually in a beaker which is covered with aluminum foil to prevent evaporation of the mobile phase. You let the sample run technically known as developing the plate till the solvent gets really close to the top of the plate.

Note that Capillary action is the "force" and/or "reason" that draws the nonpolar mobile phase up the plate.

When the plate has fully developed quickly mark where the solvent reached so you can calculate the ratio to front, also known as the beloved Rf value.

Before you do any calculations know that the more polar components have stronger interactions with the polar plate and are less likely to move with the solvent, well that is in comparison to nonpolar components that are more likely to travel with the nonpolar stationary phase.

Long story short the more like the stationary phase the component is the more it will travel.

To Calculate the Rf Value do as follows.

Divide the distance that the compound traveled from the origin by the distance the solvent traveled. Units don't really matter because they will cancel out, but I personally like centimeters.

Semi Important points...
  • Rf values should never be greater than 1. Why? Well, because its impossible and the compound couldn't actually travel further than the solvent. 
  • The closer to 1 the Rf value is the more the component was like the stationary phase.
  • The Rf value will always be positive. Why? Well, because there is no way to get a negative value, however you can get Rf values that are very close to 0 if the compound didn't move.

Paper Chromatography

Such thing exists and in reality it follows the same rules as long as the mobile phase is not polar and the stationary phase is polar.  usually is the opposite of TLC discussed above. Unless the polar and non polar components are switched.

Rf values are calculated in the same fashion with the same implications.

The paper is usually composed of some sort of polysaccharide with copious amounts of OH groups (making it polar)


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