Tank Chromatography.

Tank Chromatography.
Chromatography is a method used to separate both organic and inorganic compounds so that they can be analysed. Scientists often use chromatography to figure out which basic constituents makes up a specific mixture. Chromatography relies on the principle of selective absorption. A botanist named M.S. Tswett in 1906 first discovered chromatography. Tswett used a method called ?column chromatography? to separate chlorophylls by pouring petroleum-ether extract of green leaves into a column of powdered calcium carbonate. The compounds slowly move downwards through the glass column and each compound of the chlorophyll is absorbed into beds at different heights depending on their individual absorption affinity. Forensic scientists heavily rely on chromatography currently when solving crimes. It is also used for various purposes such as determining the presence of cocaine in urine, alcohol in blood and how much lead there is in water. Each of these are very important uses and chromatography is the method chosen as it is very reliable and easy to perform. Aim: Our aim was to use paper chromatography to separate various inks (both permanent and water based) into their core pigments. We could use the formula Rf = the distance moved by each solute (measured to the centre of each solute stain) / the distance moved by the solvent. This would give us the Rf values for each pigment in the ink. These Rf values should be constant for each individual pigment. Apparatus: Ø Chromatography Paper Ø Three permanent pens (Red, Green, Blue) Ø Three water based pens (Red, Green, Blue) Ø Chromatography tank pre-filled with acetone Ø Drying Rack Ø Pencil (for various markings) Ø Scissors (used for serration) Ø Ruler (used for all measurements) Ø Bag Ties Method: The base of the chromatography paper was marked and then cut to form a serrated edge. This edge was made to ensure that there was a controlled flow of acetone up the chromatography paper. If a full edge was placed in the acetone, the chromatography paper would absorb the acetone too quickly and we would run the risk of the different pigments blending and smudging. After the edge was serrated, a line one cm. up was marked across the paper and twelve ink spots were placed across the line at equal intervals. These spots were placed above the ?points? of serration (see Dia. 1) and were in the order of: Permanent Red, Water Based Red, Permanent Green, Water Based Green, Permanent Blue, Water Based Blue and then a repeat of these colours in the same order. The repeat was made due to the width of the paper and therefore gave us a repetition of results, enabling us to make averages of the two to give more reliable end results. Once the initial ink spots were placed, these were allowed to dry for 10 seconds and then another spot of the same colour was placed on top and was allowed to dry once more. This was repeated several times to build up the concentration of the ink at the specific point and saturate the paper, which would inevitably improve the visibility of the results. The spots were continually allowed to dry between dabbings so that the paper would not absorb the ink and cause the size of the ink spot to increase greatly which would make the results anomalous. Once the line of ink spots was complete, the chromatography paper was tied to another piece of identically set up chromatography paper and the glass lid of the chromatography tank was removed. The two sheets of paper were then hung over the frame inside the chromatography tank (see Dia. 2). The tips of the serrated edges were allowed to hang inside the acetone in the bottom of the tank and left to absorb the acetone. A great deal of care was used to make sure the chromatography paper wasn?t touching the inside of the chromatography tank, as this would spoil the experiment. The edges of the lid were covered in petroleum jelly to form an airtight seal with the ground glass edge of the tank, the lid was then replaced to ensure a constant environment and to enable the vapours of the acetone to saturate the air within the tank. The tank is then left for twenty-five minutes/so that the acetone has reached roughly ¾ of the way up the paper and the pigments have been separated. At this point we had to extract the paper from the tank and leave it until it had completely dried. Once the paper was dry, a series of measurements (see Dia. 3) was then taken. Using a ruler to measure from the starting line, the distance of the solvent front and then various distances of the centre of each pigment stain were then noted. As the solvent front was not a straight line, I measured the solvent front distance at the nearest point to the starting line and then at the furthest point to the starting line and then took the average of the two readings. All the data collected was then placed into a table and made into a bar graph (see later section). Throughout the experiment, the chromatography paper should be handled as little as possible and any necessary handling should be contained within the top ¼ of the page. This is because there are natural oils on ones hand that can disrupt the experiment and thus handling the page in the top ¼ (where the chromatography doesn?t take place) should reduce the risk of experimental error. It must also be remembered to do all markings on the paper with a pencil rather than a pen or the ink from the pen would start to take part in the process. This would completely jeopardise the experiment and give very unreliable results. Safety Issues: When working with acetone we must not: ü Get any on our hands as acetone dissolves fatty substances and would therefore irritate the hand. If this occurs we must wash the acetone off immediately under a running tap. ü Expose the acetone to sparks, open flames, heat and other ignition sources; prolonged period to direct sunlight as it is highly flammable. When working with acetone we must: ü Use in smallest possible amounts in a well ventilated area separate from the storage area. Avoid generating vapours or mists. Prevent the release of vapours and mists into the workplace air. ü Never return contaminated material to its original container. (Some of the above safety precautions written by Canadian Centre for Occupational Health and Safety) Diagrams: [image] [image] [image] Table of Results: please see attached sheet #1 for the table Interpretation of Results: To help interpret my results, I immediately made a colour-coded bar graph showing the mean Rf values for each pigment per ink. The mean Rf values of the two repeats is worked out by using the formula Mean = sum of results/n, where n is the number of repeats (in our case 2). To view this graph please see attached sheet #2. Each bar on the graph is colour coded and represents a different pigment. I decided to make the colours of the bars the same colour as the pigment for viewing ease. After making the graph I decided to work out the standard deviation to show the range of results in comparison to the mean results. ?The definition for the standard deviation is the square root of the variance. That is, the standard deviation is the average squared deviation (variance) returned to standardized format.?(Statistics the Easy Way (Easy Way Series) by Douglas Downing) The more varied the results are from the mean, the higher the standard deviation is from zero. The standard deviation formula is: s = [image] Where x stands for the list of numbers of which the standard is being worked out, x-bar (the x with the bar above it) stands for the average of x, n stands for the amount of numbers in the list, S (uppercase ?sigma?) stands for the sum of the list of numbers and s (lower case ?sigma?) is the symbol for standard deviation. Instead of calculating all the above, as I was using a spreadsheet on my PC to help organise all the collected data, all I needed to do was highlight the information I wanted to standardise and then just use the stdev function. This saved me a lot of time and effort and made sure my standardised results were accurate (i.e. no chance of calculation error on my part). Once the standard deviation was worked out, I could then work out the standard error for my results. ?The standard error of a statistic is the standard deviation of the sampling distribution of that statistic. Standard errors are important because they reflect how much sampling fluctuation a statistic will show.? (http://davidmlane.com/hyperstat/A103397.html). To work out the standard error, the standard deviation must be worked out then divided by the square root of n. The formula is (σ / [image]n), n still standing for the amount of numbers in the list. When looking at the graph and table of results, the first thing I noticed was that all the permanent inks separate into two different pigments and all the water-based inks separate into only one pigment. This observation isn?t consistent with the blue inks where the permanent blue only separates into one pigment and the water based separates into two. Thus for the red and green permanent inks there are two Rf values but for the blue permanent ink there is only one Rf value and the opposite way round for the water-based inks. In the case of the permanent inks, the coloured pigment closest to the original colour travels the furthest with the secondary pigment not travelling as far. Once again this doesn?t apply to the blue ink as only one pigment was recorded. The pigments closer to the solvent front/further away from the original ink spot will always have a higher value than the pigments further away from the solvent front/closer to the original ink spot. This is because the solvent used was acetone, which is a non-polar solvent, if water (a polar solvent) was used the water-based pigments would have travelled further and the permanent pigments wouldn?t have travelled as far. ?The polarity of the solute and solvent molecules will affect the solubility. Generally polar solute molecules will dissolve in polar solvents and non-polar solute molecules will dissolve in non-polar solvents. The polar solute molecules have a positive and a negative end to the molecule. If the solvent molecule is also polar, then positive ends of solvent molecules will attract negative ends of solute molecules. This is a type of intermolecular force known as dipole-dipole interaction.?(Seth Martin ? Louisiana State University, Department of Chemistry) The pigment with the higher Rf value in the permanent red and the pigment with the higher Rf value in the permanent green (when looking at the average results of the two repeats) have very similar Rf values with readings of 0.96 and 0.98.The only Rf value calculated for the blue (once again looking at the average of the two repeats) is very similar to the previous two with a reading of 0.96. The pigment with the lower Rf value in the permanent red and the pigment with the lower Rf value in the permanent green (when looking at the average results of the two repeats) have identical Rf values. This is because all three permanent inks were taken from pens made by the same company and different companies use different dyes to make their ink. Some are doing it to produce special physical or visual effects; some are doing it so they cannot be accused of breaking copyright laws. This means if we had taken a selection of pens made by different companies, and therefore different inks, the results would have been much more varied. The Rf value itself, is the retention/retardation value of the substance. This means how much the substance moves when it is dissolved in the solvent, which depends on the substance?s affinity to the solvent. The different pigments have different affinities for gripping the paper, and those that grip hardest to the cellulose in the paper will stop first, and those that grip the weakest will travel further up the chromatography paper before stopping. The Rf value itself can never be above 1 as this would mean the substance has travelled past the solvent front which would be impossible. Limitations, Sources of Error and Modifications: Paper chromatography has a few limitations but the most major one is the fact that it can (usually) only be used when looking at compounds made up of various pigments. This can be overcome by adding certain chemicals to the compound that could be seen under a UV light. Another limitation is the amount of time we had to do the experiment and the amount of care we had to take over it. This isn?t a general limitation to paper chromatography but was merely a limitation to us at the time of experimentation and therefore isn?t such a problem. There are several noteworthy sources of error. These consist of: handling the page when serrating the edge and handling the page when marking it, different amounts of the page being submerged in the acetone in different places, the airtight seal on the tank and the purity of the acetone. All of these would affect the experiment and would likely give a small margin of error. By handling the page, grease from your hands can saturate it and affect the way the solvent acts. By not having a consistent amount of acetone submerging the page, the solvent would travel slower/quicker in some areas and would therefore change the Rf values. If the airtight seal (in our case Vaseline) sealing the lid on the tank was not perfectly spread, very unlikely that it would be, air would still get in and therefore disturb the constant environment within. This would also happen when taking off the lid to put the chromatography paper inside, air would get in and it would have a delay for the environment to reach equilibrium. To modify this experiment firstly I would use gloves or tongs to handle the paper (both of which would have to be chemical free) and I would also make the serrated edge on the bottom of the paper longer so that only a fine point is submerged within the acetone. This would make the acetone travel even slower and really let it travel at an equal pace across the page. If possible, given the choice, I would have used purer acetone which would help to get more accurate results. The last modification I would make to this experiment would be to use a selection of pens and therefore a selection of inks. This would give more varied results and therefore would enable the chromatography process to be shown more clearly. Conclusion: In all chromatography there is a mobile phase and a stationary phase. The mobile phase moves through the stationary phase picking up the compounds to be tested. As the mobile phase continues to travel through the stationary phase it takes the compounds with it. At different points in the stationary phase the different components of the compound are going to be absorbed and are going to stop moving with the mobile phase. This is how the results of any chromatography are obtained, from the point at which the different components of the compound stop moving and separate from the other components. (Ann VanBlaricum ? High school student) Another significant fact of chromatography is that the Rf value of a substance should remain constant if the experimental conditions don?t change. Forensic scientists in crime investigations often use paper chromatography. The first case to be solved by paper chromatography of inks was when a man in Miami falsified travel and expense vouchers. However, the ink pen he used had ink that wasn?t available commercially until 3 years after the trips had taken place. Thousands of dollars of taxable income were involved. Many other cases have been solved by this simple yet affected method of substance separation; two high status cases include the Bill Clinton and Monica Lewinsky scandal and the O.J. Simpson murder case. Apart from forensic science, paper chromatography doesn?t play such a large role in science as other forms of chromatography do. This though, doesn?t mean that paper chromatography has a less significant role than the others. Since chromatography was first used, many new and improved methods have been thought up to give clearer and more accurate readings. These other types of chromatography are: Absorption Chromatography (which includes paper chromatography) - Absorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibriation between the mobile and stationary phase accounts for the separation of different solutes. Partition Chromatography ? This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibriates between the mobile phase and the stationary liquid. Ion Exchange Chromatography ? In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. Molecular Exclusion Chromatography ? Also known as gel permeation or gel filtration, this type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. The pores are normally small and exclude the larger solute molecules, but allows smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones. Affinity Chromatography ? This is the most selective type of chromatography employed. It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. For example, the immobilized molecule may be an antibody to some specific protein. When solute containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later extracted by changing the ionic strength or pH. (Introduction to Biochemical Engineering) Chromatography in general has many uses, which include: Ø Test water samples to look for pollution Ø Detecting Bombs in Airports Ø Identify and quantify such drugs as alcohol Ø Used in forensics to compare fibres found on a victim Ø Detecting pesticide or insecticide residues in food Ø Separating amino acids and anions Ø rna fingerprinting Ø Separating and testing histamines This shows the wide variety of uses that chromatography offers and each and everyone of them benefits us even if we don?t think it directly effects us

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