effect of on the activity of amylase using visible spectrophtometer

Seung Soo (Jason) Lee

002213-065

Internal Assessment – Investigating the Relationship between Concentration of

Sodium Chloride and the Rate of Reaction of Enzyme Amylase

Research Question:

How will changing the percentage of sodium chloride concentration affect the rate of reaction of

enzyme amylase, measured using the absorbance of starch and iodine with a spectrophotometer.

Introduction:

Amylase is an enzyme that is involved in the human digestive process. Found in both the human

pancreas and the human saliva, amylase breaks down starch into sugar so that large molecules can be

easily digested1. Like all enzymes, amylase must be kept in a certain condition in order to function

properly. In this experiment, the effect of sodium chloride concentration on the rate of reaction of

amylase will be investigated with the use of starch and iodine.

When starch is mixed with iodine, the coils of beta amylose molecules found in starch trap iodine,

causing the mixture to turn into a shade of blue-black. 2 When starch is broken down into glucose,

however, the monosaccharide does not react with iodine. Therefore, glucose does not change color

even when it’s mixed with iodine. Correspondingly, when drops of amylase are inputted into a blue-

black mixture of starch and iodine, the starch molecules will be broken down into glucose molecules,

causing the mixture to turn colorless. Thus, the rate of reaction of amylase correlates to the absolute

value of the rate of change in absorbance of the solution. A rapid decrease in the absorbance of the

blue-black color equates to a high rate of reaction of amylase, whereas a slow decrease in absorbance

signifies a low rate of reaction. In this experiment, an external variable of sodium chloride will be

manipulated into the amylase enzyme to determine the effect the concentration of sodium chloride on

the rate of reaction of amylase.

Rate of Reaction = │

│

1 " A m y l a s e . " W i k i p e d i a . N . p . , n . d . W e b . 1 2 J a n 2 0 1 1 .

< h t t p : / / e n . w i k i p e d i a . o r g / w i k i / A m y l a s e > .

2 S e n e s e , F r e d . " H o w D o e s S t a r c h I n d i c a t e I o d i n e ? . " N . p . , 1 5 F e b 2 0 1 0 . W e b . 6 J a n 2 0 1 1 .

< h t t p : / / a n t o i n e . f r o s t b u r g . e d u / c h e m / s e n e s e / 1 0 1 / r e d o x / f a q / s t a r c h - a s - r e d o x - i n d i c a t o r . s h t m l > .


002213-065

Hypothesis:

As aforementioned, amylase, like all enzymes, must be kept under a certain set of conditions in order to

function properly. Factors such as pH level, temperature, and salt concentration could all denature the

enzyme and decrease its activity. . When a substrate can no longer bind to the active site of an enzyme

due to its conformational change, the enzyme activity and the rate of reaction of the enzyme drops

significantly. For instance, a high concentration of sodium chloride would alter the electrostatic

interactions between charged amino acids, causing conformational change in the enzyme and

destroying its active site.3 Furthermore, the presence of sodium chloride will only have little impact on

the enzyme structure unless the sodium chloride concentration is very high, when it could completely

denature the enzyme. Therefore, an enzyme should experience an exponential decrease in its rate of

reaction as the concentration of sodium chloride is increased

Figure 1: Prediction of the Effect of Sodium Chloride Concentration on Rate of Reaction of Amylase Enzyme

Thus, the hypothesis for this experiment is that if the sodium chloride concentration is increased, then

the rate of reaction of amylase will decrease. A high concentration of sodium chloride will denature the

enzyme amylase and, as a result, it will no longer be able to break down starch into glucose. The figure

above demonstrates that the average rate of change in absorbance will undergo an exponential

decrease as the concentration of sodium chloride is increased.

3 " R u l e o f P r o t e i n S t r u c t u r e . " N . p . , n . d . W e b . 6 J a n 2 0 1 1 .

< h t t p : / / u s e r s . r c n . c o m / j k i m b a l l . m a . u l t r a n e t / B i o l o g y P a g e s / D / D e n a t u r i n g P r o t e i n . h t m l > .

Concentration of Sodium Chloride, %

Rate o

f Reactio

n o

f Am

ylase, Ab

ss-1


002213-065

Variables:

Variable Description Units / range Method of Measuring / Manipulating

Independent Concentration of

sodium chloride

% The independent variable will be

manipulated by a process of serial

dilution, from 20% concentration of

sodium chloride to 10%, 10% to 5%, 5% to

1%, and 1% to 0.1%.

Dependent Rate of reaction

of amylase │

│

(ΔAbss-1)

This will be measured with a

spectrophotometer and Logger Pro.

Because amylase breaks down starch into

glucose, and glucose does not react with

iodine, the enzyme activity of amylase

will lower the blue-black absorbance of

starch+iodine. Therefore, the rate of

decrease in absorbance over time

correlates to the absolute value of the

rate of reaction of amylase. The change in

absorbance will be measured from 0-20

seconds, and the rate of reaction can be

calculated by finding the slope of the

absorbance vs. time graph. The

uncertainty can be considered negligible.

Controlled Concentration of

starch & iodine

% This will be kept constant by using the

same mixture created through steps 1-3

of procedures for every trial. (0.05%

starch + 300μl of iodine)

Amount of

solutions inside

the cuvette

μl For every trial, 2.5ml of starch & iodine

solution and 500μl of sodium chloride &

amylase solution is put inside the cuvette.

Temperature °C Temperature is kept constant by

conducting the experiment at room

temperature (about 25 °C) for every trial.

Table 1: List of Variables


002213-065

Apparatus and Materials:

Electronic balance (±0.001g) 100 cm3 & 10 cm3 volumetric flasks 10 cm3 pipette (±0.02 cm3) 1000 μl & 50 μl micropipettes 3 cm3 cuvettes 2 cm3 micro tubes Microcentrifuge Five small beakers for serial dilution

1 medium sized beaker Sodium Chloride Iodine Starch Hot plate Vernier Spectrophotometer Logger Pro

Procedures:

Preparation of 0.05% starch mixed with iodine

1. 0.05g of starch and 100cm3 of distilled water are poured into a medium sized beaker.

2. The beaker is placed on a hot plate, and then stirred several times using a plastic stirrer until a homogenous solution is made.

3. 300μl of iodine is put into the starch solution. It is stirred several times using a plastic stirrer until a blue-black solution is made.

Preparation of sodium chloride of various concentrations (serial dilution)

Figure 2: Serial Dilution of Sodium Chloride Solution

5 cm3 distilled water

20% 5% 0.1% 1% 10%

5 cm3 5 cm3 2 cm3 1 cm3




002213-065

4. 2g of sodium chloride and 10cm3 of distilled water is poured into a small beaker.

5. The beaker is stirred several times using a plastic stirrer until a homogenous solution is made, creating a 20% sodium chloride solution.

6. 5 cm3 of the obtained solution is transferred into another small beaker using a 10 cm3 pipette, and another 5 cm3 of distilled water is added into the beaker. The beaker is then stirred using a stirrer until a homogenous solution is made, creating a 10% sodium chloride solution.

7. The serial dilution of sodium chloride is continued, according to the layout in figure 2, to obtain 5%, 1%, and 0.1% concentrations of sodium chloride solutions.

Conducting the experiment

8. The Vernier spectrophotometer is calibrated using distilled water. Then, the wavelength at which to measure the absorbance is determined using the maximum wavelength of the blue-black mixture of starch and iodine.

9. 450μl of 20% sodium chloride solution is put inside a micro tube using a 1000μl micropipette. 50μl of amylase solution is added into the micro tube using a 50μl micropipette. The micro tube is then placed inside a microcentrifuge so that the solution will mix together.

10. Step 9 is repeated three times for all concentrations of sodium chloride, creating three mixtures of sodium chloride and amylase for each of the five variables.

11. 2.5cm3 of the starch & iodine mixture is put into a 3cm3 cuvette using a micropipette. 500μl of the sodium chloride & amylase mixture is added into the cuvette using a micropipette.

12. The solution is squeezed in and out three times using the micropipette to ensure that amylase spreads throughout the starch solution. After mixing three times, the “start” button on Logger Pro is clicked. Steps 11 and 12 are performed with the cuvette placed inside the spectrophotometer to minimize error.

13. The rate of change in absorbance of the mixture is measured using Logger Pro for 20 seconds.

14. Steps 11-13 are repeated for triplicate trials for all five concentrations of sodium chloride.


002213-065

Data Collection:

Qualitative Data:

Even with the naked eye, one could observe the disappearance of color inside the cuvette,

from a dark, blue-black coloration to a clear, colorless state.

Quantitative Data:

*** Refer to the Appendix for a complete table of raw data from Logger Pro.


002213-065

Data Processing:

Sodium Chloride Concentration

/ %

Rate of Decrease in Absorbance / ΔAbss-1

Trial 1 Trial 2 Trial 3

20.0 -0.001240 -0.001280 -0.001223

10.0 -0.001517 -0.001299 -0.001402

5.0 -0.001812 -0.001523 -0.0012304

1.0 -0.001836 -0.001703 -0.001714

0.1 -0.001845 -0.001985 -0.001931

Control (no sodium chloride): -0.002830

Table 2: Rate of Decrease in Absorbance for All Trials5

Table 3: Calculation of Average Rates of Reaction

**Because the rate of reaction must be a positive value, the average rate of reaction was taken as an absolute value.

4 This value was neglected in data processing because it was considered as an outlier. 5 The rate of decrease in absorbance was determined by finding the slope of absorbance vs. time graph

using linear regression on Logger Pro software. 6 The processing of standard deviation is shown in table 4

Sodium Chloride

Concentration / %

Calculation Average Rate of Reaction

(±Standard Deviation)6 / ΔAbss-1

20.0

0.001248 ± 0.000029

10.0

0.001406 ± 0.000109

5.0

0.001668 ± 0.000204

1.0

0.001751 ± 0.000074

0.1

0.001920 ± 0.000071

Control (no sodium chloride): 0.002830 ΔAbss-1


002213-065

Data Presentation:

Figure 4: Graph of Raw Data from Logger Pro7

7 Slopes of lines that have values closest to the average slope value for each concentration of sodium chloride is shown in boxes.

LEGEND

Run 4: Control

(no sodium

chloride)

Run 5: 20%

sodium chloride

Run 10: 10%

sodium chloride

Run 11: 5%

sodium chloride

Run 14: 1%

sodium chloride

Run 19: 0.1%

sodium chloride


002213-065

Figure 5: Graph of Average Rates of Reaction against Concentration of Sodium Chloride8 9

8 Vertical error bars represent standard deviation for triplicate trials. 9 Though they are difficult to discern, horizontal error bars represent the absolute uncertainty of sodium chloride concentration.

y = -3E-05x + 0.0018

R² = 0.914

0.0011

0.00135

0.0016

0.00185

0 5 10 15 20

Ave

rage

Rat

e o

f R

eac

tio

n /

ΔA

bss

-1

Sodium Chloride Concentration / %

Effect of Sodium Chloride Concentration on the Rate of Reaction of Amylase


002213-065

Uncertainties:

Standard Deviation:


/ %

Rate of Reaction of Amylase / ΔAbss-1 Average / ΔAbss-1 (±Standard Deviation)

Trial 1 Trial 2 Trial 3

20.0 -0.001240 -0.001280 -0.001223 0.001248 ±

0.000029

10.0 -0.001517 -0.001299 -0.001402 0.001406 ±

0.000109

5.0 -0.001812 -0.001523 -0.00123010 0.001668 ±

0.000204

1.0 -0.001836 -0.001703 -0.001714 0.001751 ±

0.000074

0.1 -0.001845 -0.001985 -0.001931 0.001920 ±

0.000071

Table 4: Standard Deviation at Different Concentrations of Sodium Chloride

Example of Standard Deviation Calculation:

[Sodium Chloride Concentration] = 20%

≒ 0.000029

Same calculations were done for 10%, 5%, 1%, and 0.1% sodium chloride concentrations.

10 This value was neglected in data processing because it was considered as an outlier.


002213-065

Uncertainty due to dilution of glucose solution:

*Uncertainty due to 10cm3 pipette = ±0.02 cm3

Concentration of Glucose / %

Uncertainties

Volume of sodium chloride solution added /

cm3

Volume of distilled water added / cm3

Total percentage error for

concentration of glucose / %

Absolute uncertainty for concentration of

glucose / %

20.000 – – – –

10.000 5.00 ± 0.02cm3 = 5.00 ± 0.4%

5.00 ± 0.02cm3 =

5.00 ± 0.4%

±0.80 0.008

5.000 5.00 ± 0.02cm3 =

5.00 ± 0.4%

5.00 ± 0.02cm3 =

5.00 ± 0.4%

±0.80 0.004

1.000 2.00 ± 0.02cm3 =

2.00 ± 1%

8.00 ± 0.02cm3 =

8.00 ± 0.25%

±1.25 0.013

0.100 1.00 ± 0.02cm3 =

1.00 ± 2%

9.00 ± 0.02cm3 =

9.00 ± 0.22%

±2.22 0.011

Table 5: Uncertainty for Concentration of Glucose Solution


(±Uncertainty) / %

Average Rate of Reaction (±Standard

Deviation) / ΔAbss-1

20.000 0.001248 ± 0.000029

10.000 ± 0.008 0.001406 ± 0.000109

5.000 ± 0.004 0.001668 ± 0.000204

1.000 ± 0.013 0.001751 ± 0.000074

0.100 ± 0.011 0.001920 ± 0.000071

Table 6: Combined Uncertainties for Independent & Dependent Variables


002213-065

Conclusions:

The hypothesis was supported by the results to the extent that an increase in sodium chloride

concentration decreased the rate of reaction of enzyme amylase. However, the decrease in the rate of

reaction was not exponential; rather, the relationship between NaCl concentration and average rate of

reaction was pretty linear. As sodium chloride concentration increased, the average rate of reaction

decreased at a fairly constant rate. Furthermore, once extrapolated, the graph in figure 5 demonstrates

that the rate of reaction will be 0 when the sodium chloride concentration is at 60%. From this data, one

could conclude that the enzyme amylase will completely cease to catalyze reactions at NaCl

concentration of 60%.

In the hypothesis, it was stated that a slight presence of sodium chloride will not affect the rate of

reaction of amylase significantly, but as the concentration of sodium chloride increases, the enzyme will

undergo a rapid decrease in its rate of reaction. This is due to the fact that, as more sodium chloride ions

are present in amylase, the ions associate with oppositely charged groups in the enzyme protein,

increasing protein hydration and denaturing the enzyme.11 Contrary to the hypothesis, where the rate of

reaction was predicted to undergo a slight decrease up until a certain concentration of sodium chloride,

then a rapid decrease as the concentration is at a level high enough to denature the enzyme, the graph

below displays the fact that even 0.1% of sodium chloride was enough to largely decrease the rate of

reaction of amylase. Although the 0.1% sodium chloride did not completely denature amylase, it was

still enough to cause the greatest decrease in the rate of reaction of amylase.

Figure 6: Graph Demonstrating the Relationship between Sodium Chloride Concentration and Rate of Reaction, Including the Control

11 " P r o t e i n D e n a t u r a t i o n . " N . p . , n . d . W e b . 7 J a n 2 0 1 1 . < h t t p : / / c l a s s . f s t . o h i o -

s t a t e . e d u / F S T 8 2 2 / l e c t u r e s / D e n a t . h t m > .

0.001

0.0015

0.002

0.0025

0.003

0 5 10 15 20Ave

rage

Rat

e o

f R

eac

tio

n /

ΔA

bss

-1

Sodium Chloride Concentration / %

Effect of Sodium Chloride Concentration on the Rate of Reaction of Amylase


002213-065

Evaluation:

Overall, the results of this experiment seem fairly accurate and reliable. There are no striking outliers –

except for the one value shown in table 2 – and although the standard deviations are bit sizeable for

some values, they are not critical enough to negate the conclusions drawn. As the model in figure 5

represents, the relationship between sodium chloride concentration and the rate of reaction of amylase

is clearly a negative correlation. On a separate note, while it is true that the best-fit line in figure 5 is a

linear one, the best-fit line for the graph in figure 6 would more likely be an exponential one. Going back

to one of the conclusions drawn, the relationship shown in figure 6 represents an exponential decrease

because of the fact that the control is also included in the graph. The jump from 0% sodium chloride to

0.1% sodium chloride is largely significant – more significant than any of the other increases in sodium

chloride concentration. Thus, such results encourage the next experiment to, perhaps, incorporate an

even smaller concentration of sodium chloride. The results of this experiment support the idea that a

miniscule NaCl concentration such as 0.1% was still significant enough to disrupt the electrostatic bonds

within the enzyme. In order to observe the effect of NaCl concentration on the activity of enzyme more

efficiently, it would be apt to utilize even more miniscule concentrations of sodium chloride.

The sizeable nature of the standard deviation could be caused by the discrepancy created by human

error. Although a standard was set at the beginning of the experiment, to mix the amylase and starch in

the cuvette – in & out using the micropipette three times – then pressing “start” on Logger Pro, this

process posed the biggest error throughout the experiment. The time taken between the moment when

enzyme amylase was put into the cuvette – thus starting to interact and break down starch – and the

moment when the “start” button was clicked varied, though only by little, for every trial. Furthermore,

mixing the solutions in the cuvette three times – and taking up time in the process – may have been a

bad idea, for that time could have been sufficient for the enzyme to do all of its work. Moreover,

another problem during the procedures could have occurred with the mixing of amylase with sodium

chloride. Because 15 separate micro tubes had to be filled one by one, and then mixed through the

microcentrifuge one by one, some of the amylase solutions in the micro tubes had longer time to

interact with sodium chloride. This could have meant longer time for the sodium chloride to denature

the enzyme, thus lowering its rate of reaction. Though it’s not certain, this could have been another

source of error in the experiment.

Overall, however, this investigation was successful in terms of the accuracy of its results. The increased

presence of sodium chloride did lower the enzyme activity of amylase, as predicted in the hypothesis,

and as accepted as a scientific fact. Although improving on minor errors could strengthen the

investigation, the experiment successfully produced consistent and reliable data, leading up to a solid

conclusion.


002213-065

Improving the Investigation:

Error Impact Improvement

Time discrepancy

between the moment

amylase is inserted

into the cuvette and

Logger Pro reading is

started

It could have allowed more time for

amylase to break down starch in

some trials than in others, causing

differences in rate of reaction from

trial to trail and increasing standard

deviation

There are a few ways to improve this

error. One way would be to get a help

of another person, allowing him to

press the “start” button on Logger Pro

as soon as the amylase is mixed three

times. Another method would be to

simply take out the mixing process, and

start the Logger Pro reading as soon as

amylase is inserted into the cuvette.

Time discrepancy in

the amount of time

sodium chloride was

allowed to interact

with amylase

between each trial

It allowed more time for the sodium

chloride in some micro tubes to

denature amylase than in other

micro tubes, allowing the possibility

for further decrease in the enzyme

activity for amylase used in some

trials compared to other trials.

All 15 micro tubes could be incubated

for an allotted amount of time – around

30-40 minutes – to equalize the

amount of time that sodium chloride is

allowed to interact with amylase.

450μl of sodium

chloride solution was

inputted into the

micro tube, while only

50μl of amylase

solution was inputted,

causing imbalance

As mentioned in the conclusion, the

results demonstrate a huge decrease

from no sodium chloride to 0.1%

sodium chloride. This suggests that

too much sodium chloride was

incorporated throughout the

experiment, compared to the

amount of amylase. The abundance

of sodium chloride could have

disrupted the enzyme activity of

amylase too much.

The amount of sodium chloride

solution and the amount of amylase

solution could be balanced, to about

250μl each used for every trial. This

change could perhaps produce results

that are closer to those that were

hypothesized.

10 cm3 pipette used

during serial dilution

of sodium chloride

Decreased precision & increased

range of uncertainty

Since only about 1.5ml of each sodium

chloride concentration was necessary

for the experiment, a micropipette

could have been used to perform the

serial dilution, which would have

lowered the range of uncertainty.

Table 7: Ways to Improve the Investigation


002213-065

Appendix:

20% Trial 1 20% Trial 2 20% Trial 3 10% Trial 1 10% Trial 2 Time (s) Abs -

614.6nm Time (s) Abs -




614.6nm

0 0.325344 0 0.332057 0 0.313663 0 0.324849 0 0.328899

1 0.323938 1 0.329859 1 0.312331 1 0.324127 1 0.322839

2 0.322158 2 0.328055 2 0.310414 2 0.327022 2 0.323331

3 0.320197 3 0.326182 3 0.308982 3 0.32371 3 0.323634

4 0.319295 4 0.325192 4 0.306824 4 0.317347 4 0.318245

5 0.317086 5 0.321327 5 0.30595 5 0.316302 5 0.317459

6 0.316004 6 0.322082 6 0.303372 6 0.311371 6 0.315669

7 0.31459 7 0.32031 7 0.301999 7 0.312996 7 0.315408

8 0.31396 8 0.319333 8 0.301567 8 0.310745 8 0.314665

9 0.312072 9 0.317908 9 0.299804 9 0.309092 9 0.313663

10 0.310708 10 0.315929 10 0.298299 10 0.309422 10 0.311851

11 0.309642 11 0.314813 11 0.297477 11 0.309973 11 0.310488

12 0.308286 12 0.313737 12 0.296372 12 0.305913 12 0.309019

13 0.306788 13 0.312848 13 0.294631 13 0.303481 13 0.307299

14 0.305986 14 0.311556 14 0.29378 14 0.302649 14 0.305913

15 0.304786 15 0.31034 15 0.29332 15 0.301747 15 0.305404

16 0.303916 16 0.309459 16 0.292472 16 0.300774 16 0.304278

17 0.302866 17 0.308469 17 0.291767 17 0.298908 17 0.303553

18 0.302396 18 0.307774 18 0.290852 18 0.298586 18 0.302758

19 0.301495 19 0.306788 19 0.29029 19 0.297549 19 0.301639

20 0.301098 20 0.305658 20 0.289237 20 0.296479 20 0.300631

10% Trial 3 5% Trial 1 5% Trial 2 5% Trial 3 1% Trial 1

Time (s) Abs - 614.6nm





0 0.32863 0 0.339073 0 0.322687 0 0.346723 0 0.320573

1 0.327137 1 0.333645 1 0.320762 1 0.342346 1 0.320573

2 0.324811 2 0.329398 2 0.316787 2 0.34045 2 0.31675

3 0.322271 3 0.328362 3 0.314999 3 0.341792 3 0.312109

4 0.323028 4 0.328055 4 0.314108 4 0.339938 4 0.310892

5 0.320875 5 0.322536 5 0.31115 5 0.333917 5 0.30781

6 0.32178 6 0.320988 6 0.31012 6 0.332986 6 0.30635

7 0.31847 7 0.31832 7 0.31034 7 0.331864 7 0.302613

8 0.315743 8 0.316675 8 0.306642 8 0.331285 8 0.301711

9 0.314293 9 0.316302 9 0.305113 9 0.330668 9 0.299194

10 0.311814 10 0.313515 10 0.303843 10 0.329744 10 0.299266


002213-065

11 0.311556 11 0.312183 11 0.3033 11 0.328515 11 0.296978

12 0.31012 12 0.310561 12 0.301567 12 0.327787 12 0.294701

13 0.308506 13 0.308359 13 0.299445 13 0.326144 13 0.292014

14 0.306605 14 0.307189 14 0.298514 14 0.325039 14 0.290817

15 0.306532 15 0.306059 15 0.299338 15 0.3239 15 0.289623

16 0.304641 16 0.304097 16 0.298192 16 0.323255 16 0.288467

17 0.303952 17 0.303807 17 0.296408 17 0.322498 17 0.287106

18 0.303264 18 0.302469 18 0.295625 18 0.321667 18 0.286131

19 0.302721 19 0.30135 19 0.294276 19 0.321365 19 0.285609

20 0.301387 20 0.300092 20 0.294063 20 0.320235 20 0.286235

1% Trial 2 1% Trial 3 0.1% Trial 1 0.1% Trial 2 0.1% Trial 3






0 0.337622 0 0.337035 0 0.342465 0 0.341121 0 0.326678

1 0.334461 1 0.33485 1 0.340529 1 0.340253 1 0.323066

2 0.332212 2 0.33256 2 0.337152 2 0.335317 2 0.321252

3 0.33009 3 0.329898 3 0.333762 3 0.331246 3 0.318695

4 0.327749 4 0.327864 4 0.331169 4 0.328208 4 0.316712

5 0.325192 5 0.325915 5 0.329475 5 0.326831 5 0.31333

6 0.323824 6 0.324241 6 0.326946 6 0.324925 6 0.311224

7 0.321365 7 0.322271 7 0.325687 7 0.322536 7 0.308872

8 0.319859 8 0.320423 8 0.323407 8 0.320047 8 0.307189

9 0.317721 9 0.317908 9 0.321554 9 0.317983 9 0.305258

10 0.315743 10 0.316302 10 0.319145 10 0.316339 10 0.302938

11 0.314331 11 0.314776 11 0.317684 11 0.314145 11 0.301423

12 0.31333 12 0.313293 12 0.316228 12 0.312737 12 0.300056

13 0.311888 13 0.311851 13 0.314702 13 0.310819 13 0.29812

14 0.310782 14 0.310488 14 0.313218 14 0.309679 14 0.296194

15 0.309422 15 0.308579 15 0.311298 15 0.308469 15 0.294595

16 0.308213 16 0.307372 16 0.31012 16 0.306642 16 0.29279

17 0.30635 17 0.306532 17 0.308909 17 0.304822 17 0.291696

18 0.304931 18 0.304822 18 0.30708 18 0.303409 18 0.290044

19 0.303011 19 0.303988 19 0.306205 19 0.302252 19 0.288992

20 0.302541 20 0.302685 20 0.30504 20 0.300056 20 0.287315


002213-065

Maximum Absorbance Control (No NaCl)

Wavelength (nm)

Abs Wavelength (nm)

Abs Wavelength

(nm) Abs Time (s)

Abs - 614.4nm

400 0.140983 523.68 0.269512 629.44 0.506694 0 0.356633

403.5 0.143298 526.6 0.276394 632.4 0.505749 1 0.351468

407 0.144298 529.52 0.284868 635.36 0.502435 2 0.352439

410.5 0.143858 532.44 0.293012 638.32 0.499854 3 0.345206

414 0.144867 535.36 0.303899 641.28 0.497412 4 0.341121

417.5 0.145449 538.28 0.313062 644.24 0.493168 5 0.34116

421 0.144269 541.2 0.322568 647.2 0.492372 6 0.337113

424.5 0.15575 544.12 0.332483 650.16 0.488485 7 0.337543

428 0.16144 547.04 0.34552 653.12 0.482216 8 0.329129

431.5 0.168923 549.96 0.356925 656.08 0.480191 9 0.327175

435 0.171384 552.88 0.367604 659.04 0.474983 10 0.323634

438.5 0.177023 555.8 0.378477 662 0.47441 11 0.321214

442 0.18106 558.72 0.389967 664.96 0.469595 12 0.318058

445.5 0.185164 561.64 0.402134 668 0.464303 13 0.314479

449 0.184681 564.56 0.413991 671 0.461757 14 0.312368

452.5 0.164762 567.48 0.424809 674 0.455878 15 0.311999

456 0.184657 570.4 0.435809 677 0.451873 16 0.309202

459.5 0.184207 573.32 0.446248 680 0.448952 17 0.306168

463 0.183719 576.24 0.456774 683 0.445418 18 0.304133

466.5 0.19358 579.16 0.46569 686 0.438446 19 0.303011

470 0.198735 582.08 0.47381 689 0.43383 20 0.301639

473.5 0.205575 585 0.48009 692 0.428318

477 0.209701 588 0.487579 695 0.425969

480.5 0.213411 590.96 0.490925 698 0.42161

484 0.216026 593.92 0.492981 701 0.416259

487.5 0.218792 596.88 0.501117 704 0.411412

491 0.221762 599.84 0.504044

494.5 0.224311 602.8 0.507599

498 0.227361 605.76 0.511082

501.5 0.232694 608.72 0.510439

505 0.235745 611.68 0.51301

508.5 0.240517 614.64 0.514248

512 0.245664 617.6 0.512766

514.92 0.253401 620.56 0.510628

517.84 0.258245 623.52 0.509945

520.76 0.264801 626.48 0.508682

effect of on the activity of amylase using visible spectrophtometer

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