Question 4 please! I posted 3 for help on question 4

Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
Chapter1: The Human Body: An Orientation
Section: Chapter Questions
Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...
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Question 4 please! I posted 3 for help on question 4
3. A mixture of proteins shown in the table below are dissolved in water and injected into a size exclusion
chromatography system. Given the calibration curve for the SEC column, draw a qualitative curve to show the
expected results (chromatogram) of running the SEC by showing detection units (RI or UV absorbance) on the y-
axis and time on the y-axis. Relative peak heights (areas under curves) and times should be as quantitative as
possible.
ID
1
2
3
4
5
6
Protein
Collagen
Lactoperoxidase
Insulin
Fibronectin
Lysozyme
Trypsin
Molecular Weight
68,000
92,600
5,800
55,000
16,000
47,000
Note: as shown in the calibration curve, the beads in the
column can separate molecules less than ~120,000
molecular weight, but any proteins larger than that will
elute at ~5 minutes. The minimum molecular weight that
can be separated in the column is is 20,500; smaller
proteins elute at 25 minutes. The equation of the
calibration curve is shown on the graph.
Conc
0.5 g/L
1 g/L
12 g/L
5 g/L
2 g/L
10 g/L
log(Molecular Weight)
8765432
pl
7.8
6.5
0
7.3
4.6
4.6
2.5
Type
fibrous a-helices; structural
antibacterial enzyme
globular protein
fibrous protein
cell-lysing enzyme
digestive enzyme
Calibration Curve for SEC Column
3log (MW) = 5.262 -0.0378t
R² = 0.9991
10
20
Elution Time, min
30
Transcribed Image Text:3. A mixture of proteins shown in the table below are dissolved in water and injected into a size exclusion chromatography system. Given the calibration curve for the SEC column, draw a qualitative curve to show the expected results (chromatogram) of running the SEC by showing detection units (RI or UV absorbance) on the y- axis and time on the y-axis. Relative peak heights (areas under curves) and times should be as quantitative as possible. ID 1 2 3 4 5 6 Protein Collagen Lactoperoxidase Insulin Fibronectin Lysozyme Trypsin Molecular Weight 68,000 92,600 5,800 55,000 16,000 47,000 Note: as shown in the calibration curve, the beads in the column can separate molecules less than ~120,000 molecular weight, but any proteins larger than that will elute at ~5 minutes. The minimum molecular weight that can be separated in the column is is 20,500; smaller proteins elute at 25 minutes. The equation of the calibration curve is shown on the graph. Conc 0.5 g/L 1 g/L 12 g/L 5 g/L 2 g/L 10 g/L log(Molecular Weight) 8765432 pl 7.8 6.5 0 7.3 4.6 4.6 2.5 Type fibrous a-helices; structural antibacterial enzyme globular protein fibrous protein cell-lysing enzyme digestive enzyme Calibration Curve for SEC Column 3log (MW) = 5.262 -0.0378t R² = 0.9991 10 20 Elution Time, min 30
4. A. Using ultrafiltration as the only separation method (with as many different MWCO membranes as needed from
those listed in the class notes on filtration, reproduced below), develop a process to isolate/purify two
products from the same mixture in question 3: insulin and lactoperoxidase. (that is come up with two
different products that contain the highest concentration of (1) insulin and (2) lactoperoxidase, with
minimal other components in these two product streams.
Note: % recovery in the table refers to the % of each biochemical recovered in the retentate; the rest passes
through the membrane into the permeate, and can be recovered in the stream that crosses the membrane.
Dashes in the table indicate that that the particular biochemical was not evaluated in that membrane.
Solute
B. Without using filtration or size exclusion chromatography, discuss one other technique covered in class that
could work if the desired product from this mixture were pure trypsin.
Solute MW (Kd)
Vitamin B12
Aprotinin
Cytochrome C
Chymotrypsinogen A
Ovalbumin
BSA
Phosphorylase B
IgG
Thyroglobulin (1 mg/mL)
1,335
6,200
12,400
25,000
45,000
67,000
97,400
156,000
677,000
MWCO
Spin Time (min.)
% Recovery
% Recovery
% Recovery
% Recovery
% Recovery
% Recovery
% Recovery
% Recovery
% Recovery
3K
15
7
99
100
-
.
10K
10
-
51
89
97
97
-
-
30K
8
-
11
77
94
92
100
95
-
-
100K
5
-
-
1.8
2.1
3
26
91
97
100
300K
3
-
2.
1.5
1
1.5
91
Transcribed Image Text:4. A. Using ultrafiltration as the only separation method (with as many different MWCO membranes as needed from those listed in the class notes on filtration, reproduced below), develop a process to isolate/purify two products from the same mixture in question 3: insulin and lactoperoxidase. (that is come up with two different products that contain the highest concentration of (1) insulin and (2) lactoperoxidase, with minimal other components in these two product streams. Note: % recovery in the table refers to the % of each biochemical recovered in the retentate; the rest passes through the membrane into the permeate, and can be recovered in the stream that crosses the membrane. Dashes in the table indicate that that the particular biochemical was not evaluated in that membrane. Solute B. Without using filtration or size exclusion chromatography, discuss one other technique covered in class that could work if the desired product from this mixture were pure trypsin. Solute MW (Kd) Vitamin B12 Aprotinin Cytochrome C Chymotrypsinogen A Ovalbumin BSA Phosphorylase B IgG Thyroglobulin (1 mg/mL) 1,335 6,200 12,400 25,000 45,000 67,000 97,400 156,000 677,000 MWCO Spin Time (min.) % Recovery % Recovery % Recovery % Recovery % Recovery % Recovery % Recovery % Recovery % Recovery 3K 15 7 99 100 - . 10K 10 - 51 89 97 97 - - 30K 8 - 11 77 94 92 100 95 - - 100K 5 - - 1.8 2.1 3 26 91 97 100 300K 3 - 2. 1.5 1 1.5 91
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