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Part I.

Analysis and Identification of Amino Acids

1) Table 8-1 (p. 72) Information


Table 1: Amino acid identification using paper chromatography.
Amino Acid Color of Distance Distance Rf Values
Spot(s) Moved by Moved by
Amino Acid, Solvent, cm
cm
Aspartic Acid (asp) Blue 0.9 7.6 0.1184
Histidine (his) Dark Purple 2.5 7.6 0.3289
Leucine (leu) Light Purple 5.8 7.6 0.7632
Lysine (lys) Purple 4.5 8.2 0.5488
Proline (pro) Yellow 3.3 8.2 0.4024
Valine (val) Purple 1.5 8.2 0.1829
Unknown X Yellow 4.0 7.6 0.5263
Blue 1.0 0.1316
Unknown Y Purple (2 spots) 4.5 8.2 0.5488
1.7 0.2073

Unknown X is suggested to be aspartic acid and proline as X demonstrated to have both


blue and yellow spots. The blue spot for the unknown sample traveled 1.0 cm while the yellow
spot traveled 4.0 cm. This is closely related to aspartic acid and proline as the former was shown
to travel 0.9 cm while the latter traveled 3.3 cm.

2) Photo of Developed Chromatogram

Figure 1: Photo of Developed Chromatogram. After preparing the amino acids aspartic acid (asp), histidine (his),
leucine (leu), and unknown X on chromatography paper, we took a picture of the developed chromatogram and
proceeded to determine the Rf value for each amino acid.
Part II. Absorption Spectra and Standard Curve

1) Page 47/48: Attached at the back


2) Methods
1. Four colored water solutions were tested in order to determine their absorption
spectra and maximal absorbance (λmax)
2. After allowing the spectrophotometer to warm up for five minutes and setting it to
400 nm, we pipetted 4 mL of distilled water into a test tube as it would be our
blank sample.
3. 4 mL of each of the colored solutions (blue, green, red, and yellow) were already
pipetted into test tubes and placed on our bench. We wiped each test tube with
Kimwipes.
4. In order to "blank" the instrument, we placed the test tube with distilled water into
the spectrophotometer, pressed 0 ABS and waited for it to display 0.000
absorbance.
5. We then put each of the colored water tubes one by one into the instrument and
recorded the absorbance displayed on the window for the wavelength set. The
first wavelength set was 400 nm.
6. We measured the absorbance of the colored dye samples for every increment of
25 nm after 400nm up to 700 nm (ex. 400nm, 425nm, 450nm…700nm). Before
we changed the wavelength at any time we repeated step four.

3) Results
1.6 1.4 y = 1.18x + 0.014
1.4 R² = 0.9985
1.2
1.2
Absorbance

Concentrations
Absorbance

1
1 of Red Sample
Blue 0.8
0.8 vs Absorbance
0.6 Green 0.6
0.4 Yellow 0.4 Linear
0.2 0.2 (Concentrations
Red
0 of Red Sample
0
vs Absorbance)
675
400
425
450
475
500
525
550
575
600
625
650

700

0 0.25 0.5 0.75 1


Wavelength (nm) Concentrations of Red Colored Water

Figure 2: Absorption Spectra. The absorbance, as Figure 3: Standard Curve. Absorbance was tested after
recorded by the spectrophotometer, of each colored dye four different concentrations of the red colored sample
samples (blue, green, red and yellow) along the range of were prepared and adjusted to four milliliters. Zero
400nm to 700 nm was organized into a graph form and contained 4mL of distilled water, 0.25x was 3mL distilled
λmax was documented for each. The λmax value for blue water and 1mL of the red sample, 0.5x was 2mL of
was 625nm; green was also 625nm; yellow was 425nm, distilled water and 2 mL of the red sample, and 1x was
and red was 525nm. 4mL of the red sample. Each sample's absorbance was
tested in the spectrophotometer and a standard curve was
created.

Equation: 1.18x + 0.014


R2 = 0.9985
Part III. Quantitative Analysis of a Protein

1) Pages 74/75: Attached at the back


2) Results: We tested unknown sample C. The unknown sample contained 0.395 mg of
BSA. The final concentration was determined to be 0.0789 mg/mL with an absorbance of
0.073 at 560 nm. We measured the final concentration of each BSA sample shown in
Figure 4 at 560 nm as it was confirmed to be the λmax for test tube 5 (final BSA
concentration was 1.00 mg/mL. Figure 5 demonstrates that tube 5 absorbed the most light
at 560 nm. Thus, we tested for our unknown's absorbance at 560 nm and confirmed it to
be 0.073. We then plotted a graph showing the relationship between the final
concentrations of BSA and the absorbance at 560 nm as indicated by Figure 6. Plugging
in the value of 0.073 for y in the trendline's equation y=0.294+0.0498, the final
concentration of BSA for unknown C was calculated to be 0.0789 mg/mL. After
multiplying this number by five mL, the total amount of BSA was found to be 0.395 mg
3) Figures
A. B.
0.33
0.3
Absorbance

0.27
0.24
Tube 5
0.21 Absorb
0.18 ance
0.15
490 500 510 520 530 540 550 560 570
Wavelength (nm)
Figure 4: BSA Standards and Unknown Samples. Each test Figure 5: Absorption Spectrum. Tube 5 contained the most
tube had varying amounts of BSA, biuret reagent and distilled BSA and was the most concentrated. Based on the purple
water. Test tube 1 had 0 mg of BSA for final BSA color that tube 5 transmitted, it was found that wavelengths
concentration of 0 mg/mL. Test tube 2 had 1.25 mg BSA and ranging from 490-570nm are absorbed. Tube 5's absorbance
0.25 mg/mL BSA concentration. Test tube 3 had 2.5 mg BSA was then tested in this range in increments of 10nm and the
and 0.50 mg/mL BSA concentration. Test tube 4 had 3.75 mg λmax was determined to be 560 as it is at this value the tube 5
BSA and 0.75 BSA concentration while test tube 5 had 5.00 absorbed the most light.
mg BSA and 1.00 mg/mL BSA concentration.
C.
0.35 y = 0.294x + 0.0498
Absorbance at 560nm

0.3 R² = 0.9962
0.25 BSA
0.2 Concentration
0.15 vs Absorbance
0.1 Linear (BSA
0.05 Concentration
vs
0
Absorbance)
0 0.25 0.5 0.75 1
Final Concentration of BSA (mg/mL)

Figure 6: Standard Curve for BSA. Each of the standards (tubes1-5) were tested for their absorbance at 560 nm compared to
their final concentrations.
Equation of trendline: y=0.294x + 0.0498 , R2= 0.9962