Paper, Thin Layer, and Liquid ChromatographyExperiment 17

Sally Li (S.L)2/27/14

Chem 113-102TA: Stéphanie Le Clair

Group Members:Alexis Lingenfelter (A.L)

Joseph Mattei (J.M)Kelly Menges (K.M)

Introduction:

Chromatography is essentially based off development, or the movement of a

solvent along filter paper (1). There are two phases that allow for component

separation: the mobile and stationary phase. The stationary phase in paper

chromatography is the solvent, and the mobile phase is a result of the interactions

between the solvent and the paper(2). As the solvent moves up the paper through

capillary action, dye components of varying molecular structures are drawn up as

well(3). Since the molecular structures vary, the polarity of the dye components

also varies, which results in the components being deposited along different

segments of the filter paper. The separated components can then be analyzed in

order to form a complete picture of the analyzed material.

Chromatography was first used to separate chlorophylls by a Russian

botanist, Mikhail Tswett, and was later rediscovered when the need to analyze

wool structure arose. Martin and Synge found that partition chromatography

separated the complex products that came with reacting the wool with hot acid in

order to glean the wool's components(3). Since then, chromatography has proven

to be a useful tool in multiple fields. One such field, is the forensics field where

small samples are typical and time is of the utmost importance(4). Paper

chromatography allows forensics chemist to compare to a given standard

chromatograms and identify substances rapidly(5).

Another method, aside from paper chromatography, that can be used in the

identification of ink is electrophoresis, which is useful with water-soluble dyes,

most of which are positively charged(6). Electrophoresis involves running a current

of a certain voltage through an electrolytic solution. The positively charged dyes

are then drawn to the anode, causing the dye components to separate.

The method of ink component separation and identification that is the focus

of the lab is paper chromatography. Upon completing Section A, which involves a

sample group of an assortment of food dyes and pen inks, the results showed that

the dye components for the pen travelled with the 2:1 1-propanol:water solvent

mixture, thus no dye separation was observed because of the strong bonds

between the mobile phase and the components. From the food coloring molecular

structures seen in the Chemtrek, the food dyes are polar due to polar side groups

(3). Since the food dyes do separate along the length of the chromatography paper,

there are strong bonds between the food dye and the stationary phase, making the

stationary phase polar and the mobile phase, less polar, and the pen inks nonpolar

as well.

If the polarity gap between the mobile and stationary phase decreases, in

such a way that the mobile phase is more polar, yet has a low polarity than that of

the stationary phase, there will be a distinct separation of the pen dye components.

The pen components would still travel with the mobile phase. However, the pen

components will not travel to the solvent front line because the attraction between

the components and the mobile phase is weaker due to the polarity change.

In order to test this hypothesis, solvent mixtures of PIs ranging around 5.9,

the PI of the 2:1 1-propanol:water solvent mixture used in Section A, will be tested.

Should the polarity be lower, dye separation will be poor and the dye components

indiscernible on the solvent front line. On the other hand, should the polarity be

too high, the dye components will still be indiscernible on the origin line and the

separation will not occur because the components will move in and out of both the

stationary and mobile phase, due to the small polarity gap between the two.

Procedure (3):

1. Dye samples are dotted along a graphite-drawn line half a centimeter away

from the base of the chromatography paper with a stretched pipet tip. After

applying each sample, the pipet tip is to be cut to avoid contamination of the

samples and to ensure that the sample sizes applied are of equal sizes.

2. The dotted chromatography paper ends are stapled together such that there

is a visible gap between the ends. The resulting upright paper column is

then placed into a Petri dish, which is filled with a mobile solvent phase.

3. The mobile solvent phase to be used is a 2:1 1-propanol: water solution. The

origin line with the sample dots should be well above the solution level in

the dish.

4. A plastic cup or tank should be used to cover the paper column over the

Petri dish in order to prevent rapid evaporation of the solvent.

5. The mobile solvent will gradually travel up the chromatography paper. Once

the mobile solvent front is approximately half a centimeter away from the

top of the paper column, the paper should be removed to keep the dyes from

running off the paper.

6. Upon removal, the solvent front line should be marked and the paper set out

to dry for later analysis, in which spot diffusion, separation, and location

must be noted.

7. In analysis of the paper chromatography results, spot diffusion, separation,

and location must be noted in order to obtain the retention factor or Rf

value.

8. Upon evaluation, fault is found with the polarity of the mobile solvent used

especially with the pen inks used, as shown in Figure 1 and Table 1. New

solvents of varying polarities are tested. The polarities can be calculating,

accounting for the ratio of solutions as well as the respective polarities,

which can be found on the Snyder Polarity Index.

9. A new chromatography sheet is then spotted with 15 different pens of an

assortment of brands and colors. From there, steps 1 to 5 are repeated with

mobile solvents of different polarities, though the order of the spots will

remain unchanged. The key is shown in Table 2: Paper Chromatography Pen

Order.

10.Once the most suitable

mobile solvent is found, set up a paper chromatography test for unknown

inks using the solvent found. Using the original paper chromatography that

was run using the best solvent with known pen inks, compare results to

match the unknown inks with those that are known.

Table 17.1: Paper Chromatography Pen OrderSpot

NumberPen

Color Pen Brand

1

Black

Papermate2 Pilot Easy-Touch3 Bic4 Staples5 Pilot B-ball6

Red

Papermate7 Pilot Easy-Touch8 Bic9 Staples

10 Pilot B-ball11

Blue

Papermate12 Pilot Easy-Touch13 Bic14 Staples15 Pilot B-ball

Table 17.2: Experimental Solvent Mixture Constituents, Ratio, and PolarityExperimental

Solvent Mixture Person Mobile Phase Solvent Components RatioPolarity

Index (PI)A A.L (7) 1-propanol:water 1:1 6.7B S.L ethanol: 1-propanol 2:1 4.9C J.M (8) methanol: water 2:1 7.4D K.M (9) 1-propanol:water 3:1 5.5E S.L ethanol: 1-propanol: water 1:1:1 6.2F K.M (9) ethanol: water 2:1 6.5G A.L (7) ethanol: water 1:1 7.1H J.M (8) ethanol: methanol 1:1 5.9I J.M (8) water: methanol 2:1 8.2

Results:

Figure 17.1: Section A Paper Chromatography with 2:1 1-propanol:water mobile

phase

Polarity of 2:1 1-propanol:water mobile phase

Solution Polarities

1-Propanol: 4.3

Water: 9

Mobile Solvent Polarity

= [2(4.3) + 9] / 3

=5.9

Solvent Mixtures

Solvent Mixture Set 1

Figure 17.2

A) 1:1 1-propanol: water solvent mixture tested by A.L (7) , PI 6.7

Compared to the paper chromatography results for the pens in Section A, there is better distinction in the separation between the dye components. The improvement is especially notable in the red inked pens that, progressing up the paper, display yellow and then pink dye spots. However, the Papermate, Pilot Easy-Touch, and Bic red pens are difficult to differentiate. The same applies to the blue and black-inked pens, the results of the Papermate and Bic pens also appear to be similar. Progressing up the paper for the black-inked pens, the ink separated from purple to yellow. Dye separation was not present for the blue-inked pens, as the components traveled up to the solvent front line. For the most part, the dye components did travel too close to the solvent front line, as the centers of the spots were found near the solvent front line, which indicates that the solvent used was of too low of a polarity, as the nonpolar components were strongly bonded to the mobile solvent mixture.

Figure 17.3

B) 2:1 ethanol: 1-propanol solvent mixture tested by S.L, PI 4.9

The solvent mixture mobile phase used was of an even lower polarity than before. The results reflect the polarity change in the lack of spots and the streaky results. The results also make identification of pens immensely difficult as the spectrums shown appear to be replicated through pens of a color. The colors observed for all of the pens remained unchanged. Red-inked pens, starting from the bottom, were yellow and then pink. The only exceptions were the red Staples and Pilot V-Ball pens which did not separate at all. The separation for the Black Pilot V-ball pen was exceptional in this test, as a clear spectrum could be seen, starting from black at the bottom, to purple, blue, green, and then yellow. Aside from the pens mentioned, all others failed to separate well due to the low polarity of the mobile phase solvent mixture used.

Figure 17.4

C) 2:1 methanol: water solvent mixture tested by J.M (8), PI 7.4

Compared to the original results from Section A, as well as the other tests performed, the dye separation exceptionally clear. Aside from the Black Pilot V-ball, distinct spots can be seen as the dyes separate along the paths. The same colors are seen for the black and red pens, however, dye components of the blue pens can be seen for the first time. Progressing up the paper, there are two shades of blue, the lighter shade separating first, followed by the darker shade. From the results seen, it is difficult to differentiate between the blue Papermate, Pilot Easy-touch, and Staples pens as the gap between the dye components remains constant for those pens. In addition, the similarities shared between the black Papermate and Bic pens, and between the red Papermate and Pilot Easy-touch pens, make identification difficult. However, judging from how the spots were well below the solvent line and that the separation was clear, the polarity of the solvent mixture used in this test is more suitable.

Figure 17.5

D) 3:1 1-propanol: water solvent mixture tested by K.M (9), PI 5.5

The results of this chromatography test were on par with those of the original in Section A. The black Papermate, Bic, and Staples pens are difficult to differentiate. All but the red Pilot V-ball pen are similar, as are the blue inked-pens, which do not show a separation, merely shading differences. The same color spots that were seen in the previous results are still present, though streaks are prevalent were there would, ideally, be spot. The streaks resulted from a polarity that was far too low, as the dyes were strongly bonded to the nonpolar solvent mixture and thus travelled up to the solvent front line.

Solvent Mixture Set 2

Figure 17.6

E) 1:1:1 ethanol: 1-propanol: water solvent mixture tested by S.L, PI 6.2

The results of this test, were almost the same as those of the 2:1 ethanol: 1-propanol solvent mixture. The unique spectrum that was seen for the black Pilot V-ball is present yet again, showing a spectrum of black, purple, blue, green, to yellow at the top. Separation is lacking in the blue pens and in the black Pilot Easy-touch pen. The colors observed in the red pens are similar and are difficult to differentiate, save for the red Pilot V-ball pen which appears to have darker pigmentation than the rest. Since the dye components were on the solvent front line, the polarity of this solvent mixture is not high enough, as the ink travelled far with the mobile phase solvent mixture.

Figure 17.7

F) 2:1 ethanol: water solvent mixture tested by K.M (9), PI 6.5

Dye separation is marginally better compared to that in Section A. The red and blue-inked pens in particular do show separation. The dye components from the bottom up for red and blue are, respectively, yellow to pink, and sky blue to dark blue. Despite clear separation, the spots are streaky and extend beyond the solvent line, which indicates that the polarity of the solvent mixture is still too low at 6.5

Figure 17.8

G) 1:1 ethanol: water solvent mixture tested by A.L (7), PI 7.1

Dye separation is present although it's blurry and undefined. The colors components have separated for most of the inks, though the black Pilot V-ball is a black smear. The black pens now show purple, then yellow, and then a darker purple. There is a greater spectrum for the red pens as well, as they start off yellow, pink, orange, and then pink again. The blue pens also have a similar alternating pattern as the black pens. The colors seen are a dark blue, followed by a light blue, and then again, a dark blue. Watery appearances aside, pen inks could be differentiated through differences in pigmentation.

Figure 17.9

H) 1:1 ethanol: methanol solvent mixture tested by J.M (8), PI 5.9

Considering that the PI of this solvent mixture is the same as that of the solvent mixture used in Section A, the results are similar and the polarity is too low. Dye components are on the solvent front line, and as such, separation is unclear. Aside from the black and red Papermate, as well as the red Pilot V-ball pen, separation is not observed. The black Papermate components are yellow and then purple at the top. The red Papermate is yellow and then pink, while the red Pilot V-ball is bright oranges and then magenta at the top.

Figure 17.10

I) 2:1 water: methanol solvent mixture tested by J.M (8), PI 8.2

Compared to the original results from Section A, the results for this test contrast greatly as most of the ink components do not travel far from the origin line, indicating too high of a solvent mixture polarity. While the black, red, and blue Pilot V-ball pens do travel considerably beyond the origin line, dye separation is only distinct with the red Pilot V-ball pen, which shows magenta and then bright red. The black and blue Pilot V-ball pens show only a monochromatic streak.

Discussion:

From the results of the paper chromatography test performed in Section A using a solvent mixture of a polar index 5.9, consisting of 2:1 1-propanol:water, it was discovered that the solvent mixture polarity was too low to separate the pen inks. A higher solvent mixture polarity would bridge the polarity gap between the stationary and mobile phase, thus forcing the ink components to separate earlier on, as the attraction between the mobile phase and the components is weaker.

The hypothesis formulated stated that a solvent mixture with a low polarity would result in dye components on the solvent front line, and a solvent mixture of a

high polarity would have dye components on the origin line. In order to test that hypothesis, solvent mixtures of varying polarities both below and above 5.9 were used. The PI range chosen for the first set of experimental solvent mixtures was 4.9 to 7.4. The logic behind such a range was that both ends of the polarity spectrum could be analyzed and incorporated into the hypothesis. In retrospect, such a range was unnecessary since the suitable PI value was still unknown. After the first set of chromatograms, it was determined that the PI range should be shifted up to include solvent mixture that were too polar.

The lower range of the first set of chromatogram results succeeded in showing that the low PI values results in dye components travelling with the solvent mixtures up to the solvent front line. However, confusion regarding the better solvent polarity arose between solvent mixtures: 1:1 1-propanol:water and 2:! methanol: water. The result of the latter showed clear separation and dye components well below the solvent front line. However, the result of the former showed very distinct separation and dye components close to the solvent front line, but still below.

The PI range for the second set was decided to be 5.9 to 8.2. The results from the second set showed that PIs of 5.9, 6.2, 6.5, and 7.1 were still too low. While separation was present in some of the results, the separation wasn't as distinct as those of the best PIs tested in the first set. In addition, the dye component location continued to encroach upon the solvent front line. The results of the 8.2 PI showed a considerably lower dye component location near, and sometimes on the origin line. From this, it was determined that the best solvent mixture tested was the 2:1 methanol: water as the separation was consistently distinct and the dye components were located further down, allowing observation of dual spot centers in most of the pens.

In short, the chromatography results agreed with the original hypothesis, which led to the discovery of a suitable solvent mixture. Upon using the 2:1 methanol: water solvent mixture on the unknown inks, identification was fairly straightforward. There was minor confusion in identifying the pen on the fourth lane, but that was remedied upon the second try since there were only two likely inks.

Conclusion:

The ideal solvent mixture of PI 7.4, was suitable for chromatography of the pen inks as the unknown inks were identified in time with relative success. In the porcess of discovering said suitable ideal solvent mixtures, the hypothesis formulated at the beginning was proven to be correct by the results. Solvent mixtures of low and high polarities resulted in poor separation and dye components at the solvent front line and origin line, respectively. Future areas of study that could prove to be more challenging, regarding paper chromatography

could include finding a suitable solvent that could be used on both food dyes and pen inks on a single filter paper.

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