Water | Turkish Chemistry
May 12

Chromatography

Column chromatography is one of the most common methods of purification. Like many of the techniques on this site, it is as much an art form as a science. Proteins vary hugely in their properties, and the different types of column chromatography allow you to exploit those differences. Most of these methods do not require the denaturing of proteins.

To be very general, a is passed through a column that is designed to trap or slow up the passing of proteins based on a particular property (such as size, charge, or composition).

There are three main steps to purification:

    1. Capture. You need to get your into a concentrated form. If, for example, you are trying to isolate a you have synthesized in an E. coli , you could be looking at a to junk ratio of 1:1,000,000. For capture purification you need a high capacity method that is also fast. You need a speedy method because your crude solution is very likely to contain proteases in addition to your of interest that can quickly chew up your .

    2. Intermediate. Intermediate purification requires both speed and good resolution.

    3. Polishing. For the final step of purification you need a system that has both good resolution and speed. Capacity is usually irrelevant at this stage.

Some of the more common columns include:

  • IEX: Ion exchange chromatography. Good for capture, intermediate, and polish.
  • HIC: Hydrophobic interaction column. Good for intermediate purification.
  • AC: Affinity chromatography. Good for capture and intermediate purification.
  • GF: Gel filtration (size exclusion) chromatography. Good polishing step.

Let’s look at these types of columns in more detail.

Ion exchange chromatography

Ion exchange chromatography is based on the charge of the you are trying to isolate. If your has a high positive charge, you’ll want to pass it through a column with a negative charge. The negative charge on the column will bind the positively charged , and other proteins will pass through the column. You then use a procedure called “salting out” to release your positively charged from the negatively charged column. The column that does this is called a cation exchange column and often uses sulfonated residues. Likewise, you can bind a negatively charged to a positively charge column. The column that does this is called an anion exchange column and often uses quaternary ammonium residues.

Salting out will release, or elute, your from the column. This technique uses a high salt solution. The salt solution will out compete the in binding to the column. In other words, the column has a higher attraction for the charge of salts than for the charged , and it will release the in favor of binding the salts instead. Proteins with weaker ionic interactions will elute at a lower salt, so you will often want to elute with a salt gradient. Different proteins elute at different salt concentrations, so you will want to be sure you know the properties your well for best results.

Also be aware that changes in pH alter the charges in proteins. Be sure you know the isoelectric point of your (the isoelectric point is the pH at which the charge of a is zero) and make sure the pH of your system is adjusted and buffered accordingly.

The basic steps in using an ion exchange column are:

    1. Prep the column. Pour your buffer over the column to make sure it has equilibrated to the required pH.

    2. Load your solution. Some proteins in the solution don’t bind and will elute during this loading phase.

    3. Salt out. Increase the salt to elute the bound proteins. It is best to use a salt gradient to gradually elute proteins with different ionic strengths. At the end bump the system with a very high salt (2-3M) to make sure all proteins are off the column.

    4. Remove salts. Use dialysis to remove the salts from your solution.

Temperature doesn’t have a huge effect on column . However, it is better to work cold since proteins are more stable cold.

Hydrophobic interaction chromatography

Where ion exchange chromatography relies on the charges of proteins to isolate them, hydrophobic interaction chromatography uses the hydrophobic properties of some proteins. Hydrophobic groups on the bind to hydrophillic groups on the column. The more hydrophobic a is, the stronger it will bind to the column.

Load the proteins in the presence of a high of ammonium sulfate (not ammonium persulfate). Ammonium sulfate is a chaotropic agent. It increases the chaos (entropy) in water, and thereby increases hydrophobic interactions (the more disordered the water, the stronger the hydrophobic interactions). Ammonium sulfate also stabilizes proteins. So as a result of using an HIC column you can expect your to be in its most stable form.

The hydrophobic column is packed with a phenyl agarose matrix. In the presence of high salt concentrations the phenyl groups on this matrix binds hydrophobic portions of proteins. You can control elution of different column-bound proteins by reducing the salt or by adding solvents.

Affinity chromatography.

Affinity chromatography relies on the biological functions of a to bind it to a column. The most common type involves a ligand, a specific small biomolecule. This small is immobilized and attached to a column matrix, such as cellulose or polyacrylamide. Your target is then passed through the column and bound to it by its ligand, while other proteins elute out. Elution of your target is usually done by passing through the column a solution that has in it a high of free ligand.  This is a very efficient purification method since it relies on the biological specificity of your target , such as the affinity of an enzyme for a substrate.

Gel filtration, or size exclusion, chromatography separates proteins on the basis of their size. The column is packed with a matrix of fine porous beads.

It works somewhat like a sieve, but in reverse. The beads have in them very small holes. As the solution is poured on the column, small enter the pores in the beads. Larger are excluded from the holes, and pass quickly between the beads.

These larger are eluted first. The smaller have a longer path to travel, as they get stuck over and over again in the maze of pores running from bead to bead. These smaller , therefore, take longer to make their way through the column and are eluted last.

May 12

Fuel Chemistry
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Molecular Size

Alternative fuels tend to be made up of small, fairly simple molecules; for example, here are schematic chemical diagrams (C denotes a carbon atom, H is hydrogen, and O is oxygen) of
Methane, CH4methane (CH4), the primary constituent of liquefied or compressed natural gas, and
Propane, C3H8propane (C3H8), the primary constituent of liquified petroleum gas.

Petroleum fuels are blends of lots of different chemical species; in general, the molecules of a liquid petroleum fuel are pretty big and complex. Here is
Isooctane, C8H18isooctane (C8H18), typical of the molecules found in gasoline (I had to spread out the structure a bit to get all the hydrogen to fit in the picture–all of these molecules are, of course, three-dimensional, but some squish into a plane better than others!), and
Cetane, C16H34
this monster is cetane, or n-hexadecane (C16H34), typical of diesel fuel.

Incomplete Combustion

When a hydrocarbon fuel (that is, one that is made up of hydrogen and carbon) burns completely, the oxygen in the air combines with the hydrogen to form water (H2O) and with the carbon to form carbon dioxide (CO2). If the burning is not complete, then some of the carbon only combine with one oxygen atom rather than two, to form carbon monoxide (CO), a highly poisonous gas.

Some of the carbon may remain stuck together with each other and with some of the hydrogen as well, so that unburned hydrocarbon molecules (mostly smaller than the ones in the original fuel) can also come out the tailpipe. These unburned hydrocarbons (plus any fuel hydrocarbons that evaporate from the fuel system before getting into the engine to be burned at all) react with nitrogen oxides (another pollutant from combustion) in the presence of sunlight to form ozone, which is a lung irritant (the “ozone layer” in the stratosphere is a shield against the sun’s ultraviolet light, but at ground level ozone is the main component of “photochemical smog”). Carbon can also remain stuck to one another with few or no hydrogen attached, especially during incomplete combustion of diesel fuel, producing soot.

This is one of the reasons alternative fuels are less polluting than gasoline and diesel: their simpler molecules are easier to burn more completely in an engine, so that less carbon monoxide, soot, and unburned hydrocarbons come out the tailpipe. In addition, any unburned hydrocarbons that are produced are less reactive than those that come from incomplete burning of gasoline or diesel fuel, and so they produce less ground-level ozone; methane in particular is almost incapable of forming smog.

Oxygen Content

Some alternative fuels are not hydrocarbons; alcohols and biodiesel contain oxygen as well as carbon and hydrogen. Here are the chemical structures of the common alcohol fuels,
Methanol, CH3OHmethanol (CH3OH) and
Ethanol, C2H5OHethanol (C2H5OH).
(Biodiesel molecules are “monoalkyl esters”, but I haven’t been able to trace down anything more specific. The “ester” part of that name, however, indicates that the molecules include oxygen .)

In many parts of the USA, gasoline is “oxygenated” during at least part of the year; this means that oxygen-bearing compounds are added to the fuel mixture. The reason for doing this is that having some oxygen as part of the fuel molecules to start with promotes more complete combustion, so that less carbon monoxide, soot, and unburned hydrocarbons come out the tailpipe, as described above. Alcohol fuels and biodiesel carry this one step further, in that the oxygen-bearing compound is not an additive at the 5 to 10 percent level, but a major constituent of the fuel, which increases the benefits of oxygenation.

Carbon Content

Even if, with the aid of electronic engine controls and efficient catalytic converters, a hydrocarbon fuel is burned completely to water and carbon dioxide, there is now growing concern about carbon dioxide as a greenhouse gas. Measures to cut back on production of carbon dioxide by automobiles without sacrificing performance can focus on efficiency, i.e., getting as much useful propulsive power out of a given amount of fuel as possible, which typically involves replacing the traditional drivetrain of a piston engine driving the wheels through a gearbox with a more efficient design.

However, some fuels inherently produce less carbon dioxide when burned completely than gasoline or diesel fuel. For example, counting the numbers of oxygen it takes to burn up an isooctane and a methane (typical of gasoline and natural gas respectively), one can calculate that 100 oxygen will combine with four isooctane molecules to produce 32 carbon dioxide molecules and 36 water molecules, while the same number of oxygen will combine with 25 methane molecules to produce 25 carbon dioxide molecules and 50 water molecules. That is, a given amount of air (oxygen) will produce about 25% less carbon dioxide if used to burn natural gas than if used to burn gasoline. (Of course, this advantage will be reduced if you have to open the throttle wider and burn an additional amount of air with natural gas to get the same amount of power, but in the real world the 25% figure turns out to be about right.)

Avoiding Carbon Dioxide Emissions Entirely

The other thing to consider is the source of the carbon in the fuel; if it came from the carbon dioxide in today’s air to begin with, like an alcohol fuel produced by fermenting biomass (as opposed to a fossil fuel, whose carbon came out of the air when the dinosaurs were around!), then returning it to the air now adds nothing to the net flow of carbon dioxide into the atmosphere. Alcohol fuels or biodiesel produced from plants, when burned, just return to the air the carbon dioxide that those plants took out of the air while growing.

Finally, there’s one fuel that, in itself, produces no carbon dioxide at all when burned, namely
Hydrogen, H2hydrogen; there’s no carbon there to produce carbon dioxide!
Of course, since free hydrogen molecules don’t occur in nature, it is typically produced by “reforming” a hydrocarbon or alcohol fuel or by using electricity to split water into hydrogen and oxygen. Then the size of the contribution of hydrogen fuel to carbon dioxide emissions depends on the source of the hydrocarbon fuel that was reformed or the source of the electricity used to split the water.

If a fossil fuel was the ultimate source of the energy that is, in effect, stored in the hydrogen, then you can still gain a large improvement in carbon-dioxide production if the hydrogen is used in an efficient drivetrain, as noted above; the same is true for the electrical energy stored in a battery-powered electric vehicle. In order to obtain the full benefits of reduction of carbon dioxide (or of ordinary air pollutants like carbon monoxide), of course, the energy used to split the hydrogen or charge the battery can be obtained from a renewable source like wind power or photovoltaics.

The nice thing about hydrogen- or battery-powered vehicles is that they can run on whatever is available–efficient natural-gas-burning powerplants today, with an increasing contribution from renewable energy as time goes on and the price of photovoltaic cells (solar cells) and other renewable energy sources continues to decline. As renewable energy becomes an ever larger part of the power generation mix over the next few decades, hydrogen- and battery-powered vehicles can switch over to the new power sources without a hiccup–it’s all electricity to them!

May 11

Vitamin C Determination by Iodine Titration
C (ascorbic ) is an antioxidant that is essential for human nutrition. C deficiency can lead to a disease called scurvy, which is characterized by abnormalities in the bones and teeth. Many fruits and vegetables contain C, but cooking destroys the , so raw citrus fruits and their juices are the main source of ascorbic for most people.

One way to determine the amount of C in food is to use a redox . The redox reaction is better than an -base since there are additional acids in a juice, but few of them interfere with the oxidation of ascorbic by iodine.

Iodine is relatively insoluble, but this can be improved by complexing the iodine with iodide to form triiodide:

I2 + I- <–> I3-

Triiodide oxidizes C to form dehydroascorbic :

C6H8O6 + I3- + H2O –> C6H6O6 + 3I- + 2H+

As long as C is present in the solution, the triiodide is converted to the iodide ion very quickly. Howevever, when the all the C is oxidized, iodine and triiodide will be present, which react with starch to form a blue-black complex. The blue-black color is the of the .

This procedure is appropriate for testing the amount of C in C tablets, juices, and fresh, frozen, or packaged fruits and vegetables. The can be performed using just iodine solution and not iodate, but the iodate solution is more stable and gives a more accurate result.

Purpose

The goal of this laboratory exercise is to determine the amount of C in samples, such as fruit juice.

Procedure

The first step is to prepare the solutions. I’ve listed examples of quantities, but they aren’t important. What matters is that you know the concentration of the solutions and the volumes that you use.

Preparing Solutions

1% Starch Indicator Solution

 

  1. Add 0.50 g soluble starch to 50 near-boiling distilled water.
  2. Mix well and allow to cool before use. (doesn’t have to be 1%; 0.5% is fine)

Iodine Solution

 

  1. Dissolve 5.00 g potassium iodide (KI) and 0.268 g potassium iodate (KIO3) in 200 ml of distilled water.
  2. Add 30 ml of 3 M sulfuric .
  3. Pour this solution into a 500 ml graduted cylinder and dilute it to a final volume of 500 ml with distilled water.
  4. Mix the solution.
  5. Transfer the solution to a 600 ml beaker. Label the beaker as your iodine solution.

C Standard Solution

 

  1. Dissolve 0.250 g C (ascorbic ) in 100 ml distilled water.
  2. Dilute to 250 ml with distilled water in a volumetric flask. Label the flask as your C standard solution.

Standardizing Solutions

 

  1. Add 25.00 ml of C standard solution to a 125 ml Erlenmeyer flask.
  2. Add 10 drops of 1% starch solution.
  3. Rinse your buret with a small volume of the iodine solution and then fill it. Record the initial volume.
  4. Titrate the solution until the is reached. This will be when you see the first sign of blue color that persists after 20 seconds of swirling the solution.
  5. Record the final volume of iodine solution. The volume that was required is the starting volume minus the final volume.
  6. Repeat the at least twice more. The results should agree within 0.1 ml.
You titrate samples exactly the same as you did your standard. Record the initial and final volume of iodine solution required to produce the color change at the .

Titrating Juice Samples

 

  1. Add 25.00 ml of juice sample to a 125 ml Erlenmeyer flask.
  2. Titrate until the is reached. (Add iodine solution until you get a color that persists longer than 20 seconds.)
  3. Repeat the until you have at least three measurement that agree to within 0.1 ml.

Titrating Real Lemon

Real Lemon is nice to use because the maker lists C, so you can compare your value with the packaged value.

  1. Add 10.00 ml of Real Lemon into a 125 ml Erlenmeyer flask.
  2. Titrate until you have at least three measurements that agree within 0.1 ml of iodine solution.

Other Samples

 

  • C Tablet – Dissolve the tablet in ~100 ml distilled water. Add distilled water to make 200 ml of solution in a volumetric flask. 
  • Fresh Fruit Juice – Strain the juice through a coffee filter or cheese cloth to remove pulp and seeds, since they could get stuck in the glassware. 
  • Packaged Fruit Juice – This also may require straining. 
  • Fruits & Vegetables – Blend a 100 g sample with ~50 ml of distilled water. Strain the mixture. Wash the filter with a few milliliters of distilled water. Add distilled water to make a final solution of 100 ml in a volumetric flask.

Titrate these samples in the same way as the juice sample described above.

Calculations

 

  1. Calculate the ml of titrant used for each flask. Take the measurements you obtained and average them.average volume = volume / number of trials

     

  2. Determine how much titrant was required for your standard.If you needed an average of 10.00 ml of iodine solution to react 0.250 grams of C, then you can determine how much C was in a sample. For example, if you needed 6.00 ml to react your juice (a made-up value – don’t worry if you get something totally different):

    10.00 ml iodine solution / 0.250 g Vit C = 6.00 ml iodine solution / X ml Vit C

    40.00 X = 6.00

    X = 0.15 g Vit C in that sample

     

  3. Keep in mind the volume of your sample, so you can make other calculations, such as grams per liter. For a 25 ml juice sample, for example:0.15 g / 25 ml = 0.15 g / 0.025 L = 6.00 g/L of C in that sample

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