Bio Spectroscopy Lab

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Introduction:
This investigation used spectroscopy to evaluate light absorption in different solutions. A spectrophotometer was used in the lab to determine these values. A spectrophotometer is an apparatus used to “measure the absorption of radiation in the visible and UV regions of the spectrum and allows precise at a particular wave length” (Jones et al., 2007). The amount of light absorbed by a substance is directly in relation to the concentration of the solute and also the wavelength moving through the solute (Jones et al., 2007). This is commonly referred to as Beer’s Law and can be expressed as A= εl [C]. Beer’s Law equation measures the absorbency of light, making it an effective measure as spectrophotometers give exact values for absorbency (Jones et al., 2007). The purpose of this lab is to identify the concentration of unknown 276 using absorption values obtained from the spectrophotometer. When using these absorbance values in relation to similar fast green dilution solutions, the concentration of unknown 276 was achieved by drawing a line of best fit on a scatter plot (refer to figure 2 within this lab). By using absorption values of other solutions such as fast green stock solution and chloroplast pigments, information about high absorbency wavelengths was gained. By plotting this information in an “absorption spectrum” graph, the maximum absorption was easily identified for fast green, chlorophyll A and chlorophyll B (refer to figures 1 and 4 within this lab). The graphs in general can be closely related and compared to Beer’s Law stated above. For example, when referring to figure 2 (within this lab) concentration curve for fast green, absorption is in relation to concentration, where as when the concentration of a substance is increased, higher values of light was absorbed (Jones et al., 2007).

Materials and Methods:
Please refer to Lab 4 in manual

Results:
Dilution concentration calculation:
(Using CiVi= CfVf formula)

Test tube1: (stock fast green solution only)
0.015mg/mL
Test tube2:
(0.015mg/mL)(10.0mL) = Cf (20.0mL)
Cf = (0.015mg/mL)(10.0mL) / 20.0mL
Cf = 0.0075mg/mL
Test tube3:
(0.0075mg/mL)(10.0mL) = Cf (20.0mL)
Cf = (0.0075mg/mL)(10.0mL) / 20.0mL
Cf = 0.00375mg/mL
Test tube4:
(0.00375mg/mL)(10.0mL) = Cf (20.0mL)
Cf = (0.00375mg/mL)(10.0mL) / 20.0mL
Cf = 0.001875mg/mL
Test tube5:
(0.001875mg/mL)(10.0mL) = Cf (20.0mL)
Cf = (0.001875mg/mL)(10.0mL) / 20.0mL
Cf = 0.0009375mg/mL
Table 1: Absorption spectrum values when measuring fast green test tube #1 WAVE LENGTHFast Green
460nm0.035A
480nm0.015A
500nm0.016A
520nm0.040A
540nm0.07A
560nm0.165A
580nm0.320A
600nm0.533A
620nm0.978A
640nm0.608A
660nm0.128A
680nm0.017A
700nm0.00A
Table 1 shows the absorption values fast green solution, concentration of 0.015mg/mL for every 20nm. Highlighted in red is when the fast green reached its maximum absorption. Distilled water was used to recalibrate the spectrophotometer after every wavelength. Note: the fast green peaked at 625nm with 0.996A

Table 2: Absorption values for each concentration
Test tube (concentrations)Absorption values
1 (0.015mg/mL)0.996A
2 (0.0075mg/mL)0.505A
3 (0.00375mg/mL)0.226A
4 (0.001875mg/mL)0.116A
5 (0.0009375mg/mL)0.069A
6 (water)N/A
7 (unknown)0.783A
Table 2 outlines the absorption values for various concentrations used throughout the lab. These concentrations all peaked at wave-length 625nm

Table 3: Absorption spectrum values when measuring Chlorophyll A and B WAVE LENGTH Chlorophyll AChlorophyll B
4001.969A0.588A
4202.437A0.927A
4401.964A1.294A
4601.077A1.670A
4800.607A0.457A
5000.159A0.093A
5200.121A0.090A
5400.139A0.099A
5600.172A0.111A
580 0.271A0.138A
6000.297A0.179A
6200.427A0.170A
6400.473A0.453A
6601.872A0.445A
6800.313A0.084A
7000.200A0.023A
Table 3 shows the absorption values for Chlorophyll A and Chlorophyll B for every 20nm. Highlighted...
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