Leo Forster 2213-018

Absorbance-Concentration Relationships

Research Question:

How will changing % SDS concentration affect the rate of diffusion of beetroot pigments from beetroots cubes placed in water, measured by means of a visible spectrometer

Hypothesis:

Being a common and important component in DNA extraction protocols, Sodium Dodecyl Sulphate (SDS) functions in that it creates electrostatic repulsion in proteins, and subsequently negates all protein activity within the cell membrane. If applied in the case of beetroot cubes placed in water, the SDS will split up the cell membranes; thus allowing the color pigments (betanine) to exit the cytoplasm and enter its surroundings. Once in the water, the betanine will dissolve, and color the water red.

In this experiment, as the concentration of SDS is increased, there will be more SDS molecules available for interaction with the cell membrane. Hence, the cell membrane will fall apart faster, and more pigments will be able to enter the water outside of the cell. Then, after measuring the absorbance of the pigments at their respective λmax, the rate of diffusion can be found with the following formula, where absorbance has no unit and time is in hours:

Consequently, the expected graph from this experiment is as follows:

Graph 1: Expected quantity of betanin pigments as SDS concentration is increased.

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Leo Forster 2213-018

Background/Introduction:

Diffusion is the movement of molecules from a higher concentration gradient to a lower concentration gradient. So, then, until the two gradients have reached equilibrium - the state in which the molecules are evenly distributed - movement from an area with one concentration to an area with another will exist. Diffusion can also occur between two separate parts of fluid across a membrane – where in this case, the two fluids are the SDS solution, and the betanin-containing dyes; and the membrane being crossed are the remnants of the cell walls previously destroyed by the SDS.

There are a few factors which can negatively affect the cell membrane – either rupturing it in places, or destroying it completely. These factors include, but are not limited to: temperature, pH, enzymes, lysis (by means of agitation or otherwise), or with the addition of chemicals which will weaken the intermolecular bonds present within the membrane. This experiment will investigate the effects that the addition of the compound, Sodium dodecyl sulfate (SDS) has on the rate of diffusion of the pigment, Betanin, from beetroot samples placed in water.

In this case, a visible spectrometer will be used to quantify the data - as it will be able to record the change in absorbance of light caused by the diffusion of the red pigments from the cells of the beetroot into the SDS-solution. As there is a direct relationship between concentration of solute and absorbance at its respective λmax, and in this case, the concentration of the solvent being used is known, it is easy to investigate into the cell-destructive abilities of SDS.

In this experiment, the diffusion of betanin - a red glycosidic food dye found in beetroots - from the mother cell into distilled water will be measured using a visible spectrometer.

Materials:

- Beetroot

- Visible Spectrometer

- 0.100M SDS solution

- Logger Pro 3.7

- Cork borer

- Cuvettes

- Micropipettes

- Microplate

- Knife

- Ruler

- Distilled water

- 100 ml Beaker

- Forceps

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Leo Forster 2213-018

Variables:

Variable Description Method of Measuring / Controlling

Independent SDS Concentration Using a stock solution of 0.100M SDS, a serial dilution will be

performed to achieve solutions of 20%, 40%, 60%, 80%, and

100% SDS concentration using a micropipette

Dependant Rate of diffusion of pigments from beetroot host

Measure the amount of pigments with the spectrometer

at λmax, 535.4 nm

Controlled Cuvette used Same cuvette will be used for all trials and will be washed after

every trial

Beetroot part The “meaty” middle part of the beetroot will be obtained with a

cork borer

Beetroot shape/size The beetroot will be cut using a cork borer, and then diced with

a knife and ruler

Volume of SDS-solution used 5 ml of varying concentrations of 0.100M SDS-solution will be

added to the beetroot

Temperature Experiment will be conducted in one sitting, so that room

temperature does not change and stays constant at 25°C

λmax Absorbance will be measured at same λmax every time

Table 1: Variables and their methods of controlling/measuring

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Leo Forster 2213-018

Procedures:

1. Using the cork borer, extract a sample of beetroot from the vegetable.

2. Cut the beetroot sample into 0.5 cm long pieces, taking care to discard the pieces which were close to the surface of the vegetable. Rinse beetroot pieces in distilled water for several minutes.

3. See Table 2 for instructions on preparation of SDS solution for each well.

Concentration of SDS solution used, C / %

Respective Volumes

Volume of 0.1 M SDS solution added / cm3

Volume of Distilled Water

added / cm3

Total Volume of Solution /

cm3

0 0 2.5 2.5

20 0.5 2 2.5

40 1 1.5 2.5

60 1.5 1 2.5

80 2 0.5 2.5

100 2.5 0 2.5

Table 2: Preparation of SDS-solution for destruction of beetroot cells

4. Noting the time, place one 0.5 cm long beetroot piece into each well.

5. After approximately one hour, give each well a short stir with a toothpick or appropriate tool – take care not to cross-contaminate samples.

6. Remove 2.5 ml (the entire contents) of the fifth well – the solution should be red in color – and place it in a cuvette

7. Place the cuvette in the visible spectrometer, having previously calibrated said machine with a cuvette full of distilled water.

8. At λmax, measure the absorbance of the pigments, and plot it against the concentration of SDS solution.

9. Repeat steps 6-9 with the fourth through first cuvettes, collecting triplicate samples of each SDS concentration in order to aid in the calculation of the mean and standard deviation

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Leo Forster 2213-018

Data Collection:

Qualitative Data:

As the SDS concentration in each solution increased, the solutions became increasingly red as time passed. Those cuvettes with a higher concentration of SDS became a deeper shade of red than those with less SDS.

Quantitative Data:

Raw Data:

Table 3: Absorbance of betanin at λmax, 535.4 nm

a Standard Deviation and average have been computed using the available triplicate samples

b, c, d, e Data point was not included in computation of average and standard deviation, as it did not fit with the other two data points of said trial, and so was probably influenced by some sort of experimental error.

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Absorbance at λmax, 535.4 nm

Concentration of SDS solution used, C / % Trial 1 Trial 2 Trial 3 Average ± St. Dev.a

0 0.024 0.000 0.004 0.009 ± 0.002

20 0.595b 0.296 0.321 0.321 ± 0.018

40 0.405c 0.679 0.613 0.646 ± 0.047

60 0.501d 0.729 0.704 0.717 ± 0.017

80 0.639 0.491 0.557 0.562 ± 0.074

100 1.397e 0.698 0.708 0.703 ± 0.007

Leo Forster 2213-018

Data Processing:

The rate of diffusion was measured by the diffusion of the red betanin pigments into the distilled water, and was quantified by the visible spectrometer. The absorbance that was taken is shown in Table 3, and the calculations of the Rate of Diffusion can then be made with the following formula:

Absorbance has no unit, and time is in hours. The calculations can be seen in Table 4.

Calculation of Rate of Diffusion

Concentration of SDS solution used, C / %

Initial Absorbance, A Average Absorbance after 1 hr

Rate of Diffusion / hr-1

0 0.000 0.009 0.009

20 0.000 0.321 0.321

40 0.000 0.646 0.646

60 0.000 0.717 0.717

80 0.000 0.562 0.562

100 0.000 0.703 0.703

Table 4: Finding the Rate of Diffusion

Sample Calculation:

Note that Absorbance has no unit, while Time is in hours.

This calculation was repeated for the remaining data values, and the results noted in Table 4.

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Error Analysis / Uncertainties:

Tables 5 and 6 show the calculations for the uncertainties involved in the amount of solution used and the % SDS concentration

For Amount of Solution used:

Concentration of SDS solution used,

C / %

Amount of 0.100M SDS used ± pipette

uncertainty / ml

Percent Uncertaintyf /

%

Amount of H2O used ± pipette

uncertainty / ml

Percent Uncertaintyg

/ %

Total Percent Uncertainty /

%

Absolute Uncertaintyh

/ ml

Amount of Solution Used ± Uncertainty / ml

0 0 0 2.5 ± 0.005 0.2 0.2 0.005 2.5 ± 0.005

20 0.5 0.2 2 ± 0.004 0.2 0.4 0.01 2.5 ± 0.01

40 1 0.2 1.5 ± 0.003 0.2 0.4 0.01 2.5 ± 0.01

60 1.5 0.2 1 ± 0.002 0.2 0.4 0.01 2.5 ± 0.01

80 2 0.2 0.5 ± 0.001 0.2 0.4 0.01 2.5 ± 0.01

100 2.5 0.2 0 0 0.2 0.005 2.5 ± 0.005

Table 5: Calculating uncertainty in Amount of Solution Used

f Sample calculation for Percent uncertainty in amount of 0.100M SDS used:

g Sample calculation for Percent uncertainty in amount of H2O used:

h Sample calculation for total Absolute Uncertainty:

Effect of different % SDS Concentrations on the Absorbance

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

-10 10 30 50 70 90 110

SDS Concentration / %

Abs

orba

nce

Data Presentation:

Graph 2: % SDS Concentration against Absorbance

i The calculated Standard Deviation was used to create the Y error-bars

j The error in amount of solution used was used to create the X error-bar

i

j

Effect of different % SDS Concentrations on the Rate of Diffusion

y = 0.0122x + 0.0563

R2 = 0.943

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

-10 10 30 50 70

SDS Concentration / %

Rate

of D

iffus

ion

/ hr

Graph 3: % SDS Concentration against Rate of Diffusion

k The calculated Standard Deviation was used to create the Y error-bars

l The error in amount of solution used was used to create the X error-bar

k l

Conclusion:

From the graphs on the previous page, it is possible to discern that as SDS concentration increases, the ability for the SDS solution to rupture cell membranes and allow the betanin pigments to escape also increases. Therefore, as SDS concentration increases, both the rate of destruction of cell membranes, as well as the rate of diffusion of betanin pigments from said cells (the two are intimately related) into another medium will also increase. Hence, there exists a positive relationship between the two variables, and as such, the data does support the hypothesis posed above.

The second graph in particular, is valuable when attempting to answer the research question. This is due to the fact that there are multiple points at which the data does not agree with what was expected or projected – or with the other data points, for that matter. For example, after a steady, linear, increase of absorbance from an SDS concentration of 20%, 40%, and 60% – from 0.321 to 0.717 – there is a sudden drop in absorbance when SDS concentration reaches 80%: back below the 60% level, to an absorbance of 0.562. This discrepancy can be, to some extent, explained by Beer-Lambert’s Lawm; which makes the suggestion that at low concentrations, the relationship between concentration and absorbance will be linear – but that this relationship will become void once the concentration becomes relatively high. Applied in this case, then, it seems logical to simply omit the last two points, and apply Beer-Lambert’s Law to the first four points.

In addition to this, it was difficult to both remove the entire contents of each well of the microplate and to subsequently remove the entire contents of the cuvette and dry it properly. As the visible spectrometer is extremely accurate, and gives readings to eight decimal places, it is probable that any number of water molecules left in the cuvette after rinsing will induce unnecessary error. Despite this though, the standard deviation and experimental error present in this investigation were rather consistent and quite small. This aids in the justification of the results discussed above, and establishes the validity of the hypothesis as well.

Finally, there were a number of sources of smaller possible errors (which have already been addressed above, but not elaborated upon) which could have, and probably did, the outcome of this experiment. It may be interesting to note that these errors themselves are not variables that were simply not controlled properly, but were, for the majority, rather small issues or problems with the experiment which were emphasized due to their not being constrained properly due, in great part, to time constraints present while conducting the experiment.

__________________________

m For further information, see: http://www.chemguide.co.uk/analysis/uvvisible/beerlambert.html

Evaluation:

Procedural Weakness Hypothesized Improvement

Inconsistent or contaminated cuvette face Thoroughly clean cuvette face with a wet paper towel after every use; and make sure that the cuvette is consistently placed into the colorimeter with the same face toward the spectrometer’s sensor.

Measurement of absorbance at incorrect λmax Correct calibration and choosing of sample with which to find λmax is important in this case. If a sample that is only vaguely red is used to find λmax, there is a rather large chance of it not exactly correlating to the actual λmax of the pigments inside of the solution. It is advised that the λmax always be found with the darkest, deepest colored sample.

Inconsistencies or mistakes in spectrometer calibration

Always make sure that spectrometer is calibrated with the solution in which the pigments will be dissolved – in this case SDS-solution – so as to remove any possibility of different absorbance based on the makeup of the solution in which the pigments are dissolved.

Table 7: Procedural errors and their negation.