Scenario

You are head of the purification group of a small company selling enzymes for research. Most of your products have sales that are insufficient to justify research projects to optimize the purification schemes. Instead, general adsorbents are employed that usually purify enzymes quite well. There are many adsorbents from which to choose, and the capacities and resolving powers differ for various enzymes. Note that these adsorbents are quite expensive, but there is no relation between price and performance.

One of your products has been manufactured and sold in very small quantities for years. Suddenly you have a rush order for 40 grams, an amount that is far greater than you can make quickly with your old method. The implications for future sales and for developing an optimum process will not concern you yet; you are asked to fill this order fast. Any enzyme from test runs can be combined and sold with the main batches.

Six adsorbents are likely prospects for achieving purification of this enzyme. If you are lucky and can select a good one, there is no point in testing them all. There are no useful specifications on them because no one has ever evaluated properties with this enzyme.

You select from batches of partly purified enzyme. You may also rework chromatography fractions that do not meet the purity specification. A more pure feed stream means better operation of thechromatographic step, but with increased cost because of the previous losses.

The chromatograms show two other major proteinaceous impurities from which your enzyme must be resolved. Fractions of column effluent of over 95 per cent purity (the minimum specification for salable enzyme) are pooled and saved. Increased loading smears the chromatograms. In order to save time and money, you want to use larger column diameters and moderate loadings.

Solubilities of various proteins as functions of ammonium sulfate concentration were formulated in simple expressions and used to construct a plot to help you decide how to operate. Note that the yield of desired enzyme is zero until a certain concentration of ammonium sulfate is reached, and there is a peak followed by a decline in yield if too much is added.

Batches may be combined, and additional batches are created automatically from side fractions in the next purification step of column chromatography. You will discover that the peaks can spread and overlap with poor matches of column diameter and loading.

In response to the prompt for kilograms of liver, a small number doesn't provide much crude enzyme to work with. Several hundred kilos is too much. Ten or twenty kilos is reasonable as you are gaining experience, but you will need more later.

You are asked for the column diameter (in centimeters). A small column is okay for analytical work but too tiny for a big production run. Although today's columns can be quite large, this program has only a smaller range of column sizes available. The computer tells you the loading divided by the capacity of that resin. If this ratio is more than one, the column is overloaded and can give no separation. Initial choices of resin are strictly luck. You should experiment but with columns that are not very big because smeared chromatograms will tell you little. Study your results and continue making more batches or performing chromatography to accumulate the desired amount of pure enzyme.

Tips for Playing the Chromatography Game

1.  Go to the web page for the game.
2. Choose the amount of kilograms of liver that is adequate for your batch specifications.
3. Choose the desired concentration of Ammonium Sulfate (NH₄)₂SO₄ that will maximize the initial purity.
4. Make a batch by clicking on the button that reads Click to make batch with.
5. The cost of the liver, the cost for the specific (NH₄)₂SO₄ concentration, and the cost of the labor and equipment will automatically appear in boxes appropriately labeled. These three factors will all be figured into the calculation for the total cost of the run. The total cost is also automatically calculated. It can be found in the window that reads Cost of this run. The object of this game is to make the desired amount of product with the highest purity in as few batches as possible, in order to cut down on costs, so pay close attention to how much each individual batch costs.
6. Make a number of batches in the range of one to six and then move on to chromatography, by clicking on the link which reads Move on to chromatography.
7. The next part of this game allows for further purification by chromatography. Select which crude batch to purify. Anything that you do adds to the total cost. Fractions above 95% pure become salable product. Side fractions above 50% pure become batches to purify again. First highlight which batch you want to purify and enter in how many milligrams of that existing batch that you want to initially purify. Once those parameters have been entered click the button that reads Get material.
8. Choose a resin type from one to six. All of the resins behave differently so it is trial and error to find the best resin for your batches. You can change resin type for each batch if you so desire.
9. Next choose a column diameter. For this game a larger diameter works better for purification.
10. Verify that the loading/capacity ratio is between 0 and 1, if the number is not in the specified range then change the column diameter, respectively.
11. You are now ready to run the chromatography. Click on the button that reads Run Chromatography.
12. You can get material and purify as many times as there are open batch slots. Note you do not have to make all six batches.
13. The next step is to make more batches or pool. When the batch weight and purity both read 0 for a particular batch, that batch is empty. If you do not have any free batches then you must pool, because there is simply no room for any new batches to be made. If you have a choice then you must make the decision whether you want to make a brand new batch or pool existing batches based on cost and purity.
14. To make a new batch you simply follow the above instructions, paying close attention that when you get material you assign it to the new batch.
15. Pooling involves two existing batches. Simply choose which two batches you want to pool and enter the numbers into the appropriate boxes and then click the button that reads Click to pool. You can pool as many batches as you want, but remember pooling is expensive so you want to make intelligent decisions about how you mix your batches.
16. Follow the steps for making new batches and pooling existing batches until you reach the desired amount of product without sacrificing cost requirements too much. Remember a good engineer wants the most yield for a minimum amount of batches (time) and minimum process costs.
17. Note the program will tell you when you have made a mistake, by writing in the box that the error has occurred in. For example if you try to get material for a batch with no product then No such batch will appear in the box titled Enter how many mg. Likewise if you try to pool an empty batch then the same message will appear in the appropriate batch # input box.

The following is a simple lab to introduce the student to the elements of chromotography.

This link is also a game, and allows the student to run Virtual Gas Chromotographs.

To learn more about how a GC is set up, click here.

To learn more how to run a GC, complete with videos, click here.