This document summarizes an experiment on ultrafiltration and gel electrophoresis. For ultrafiltration, the goals were to understand membrane separation principles and parameters like osmotic pressure and fouling. Experiments tested filtration rate with different protein concentrations. Results showed osmotic pressure had a greater effect on mass transfer than fouling. For gel electrophoresis, the goals were to separate and measure molecular weights of pepsin and rennet using SDS-PAGE. Results showed crude pepsin, pure pepsin, and rennet-pepsin mixture were close to literature values, while rennet alone had some unexpected bands possibly from breakdown.

1. Ultrafiltration and Gel
Electrophoresis
Rashid Alsuwaidi
Ahmed Alhazeem
Matt Liu
Wanying Chia

2. Ultrafiltration

3. Goals
• Understanding the principles and parameters involved in membrane
separations
• Asses the roles of osmotic pressure and membrane fouling on
membrane flux decline
• Determine relevant solute mass transfer coefficients in membrane
filtration process

4. Ultrafiltration
• Purifying solvents
• Concentrating solutes
• Utilizes pressure gradients
• 𝑄 𝑚 ∝
𝑛 𝑝∗𝑑 𝑝
4
𝑙∗𝜂
• 𝑣 = 𝑄 𝑚 ∆𝑃 − ∆𝜋
• 𝐶𝑠 =
∆𝜋
𝑅𝑇
• 𝑘 𝑐 = 𝑣/ ln
𝑣 𝑃−𝑣 𝐵
𝑅𝑇
𝐶 𝐵

5. Experiment
• 20 mM Acetate Buffer
• 1 mg/mL porcine pepsin
• 2 mg/mL porcine pepsin

6. Filtration Rate
2.00E-03
2.50E-03
3.00E-03
3.50E-03
4.00E-03
4.50E-03
5.00E-03
5.50E-03
6.00E-03
30 32 34 36 38 40 42 44 46 48 50
AvgFlux(mL/cm^2*sec)
Applied Pressure (Psi)
Avg. Flux Due to Applied Pressure
20 mM Acetate Buffer
1 mg/mL Protein Solution
20 mM Acetate Buffer After
2 mg/ml
𝝂 = 𝑸 𝒎(∆𝑷 − ∆𝝅)

7. Osmotic Pressure
𝝅 = 𝒄 𝒔 𝑹𝑻
-2.0
0.0
2.0
4.0
6.0
8.0
10.0
12.0
30 32 34 36 38 40 42 44 46 48 50
OsmoticPressureΔπ(psi)
Pressure ΔP (psi)
Osmotic Pressure Over Pressure
1 mg/mL
2 mg/mL

8. Membrane Clogging?
• Qm (before) = 1.01 x 10-4 mL/cm2*sec*Psi
• Qm (after) =9.74 x 10-5 mL/cm2*sec*Psi
• t-test results in p= 0.54
• Therefore Osmotic Pressure is greater
at affecting mass transfer
y = 1.08E-04x - 3.21E-04
R² = 9.88E-01
y = 8.78E-05x + 3.98E-04
R² = 9.98E-01
2.00E-03
2.50E-03
3.00E-03
3.50E-03
4.00E-03
4.50E-03
5.00E-03
5.50E-03
30 35 40 45 50
AvgFlux(mL/cm^2*sec
Pressure (Psi)
Comparison of Permeability Coefficient
Flux Before
Flux After

9. 4.00E-04
6.00E-04
8.00E-04
1.00E-03
1.20E-03
1.40E-03
1.60E-03
30 32 34 36 38 40 42 44 46 48 50
Kc(cm/s)
Pressure (Psi)
Comparison of Kc Values
Ideal 1 mg/mL
Ideal 2 mg/mL Solution
Jonnson 1 mg/ml Solution
Jonnson 2 mg/mL
0.0
100.0
200.0
300.0
400.0
500.0
600.0
700.0
800.0
900.0
1000.0
30 32 34 36 38 40 42 44 46 48 50
Cs(mg/mL)
Pressure (Psi)
Comparison of Solute at Membrane Surface
Ideal 1 mg/mL
Ideal 2 mg/ml
Jonnson 1 mg/ml
Jonnson 2 mg/ml

10. Cs and Mass transfer coefficient

11. Conclusions
• Osmotic Pressure has a greater effect on mass transfer coefficient
• Pressure increases mass transfer coefficients as well asProtein
Concentration on the membrane surface

12. Gel Electrophoresis

13. Experiment Objectives
• To learn how to assemble and perform gel electrophoresis.
• How to use gel electrophoresis to measure the molecular weight of
pepsin and mucor rennet and to compare them to the literature
values.

14. Gel Electrophoresis• Used for separating and detecting molecules based on size.
• By applying an electric field to the gel matrix the particles
migrate and separate based on size towards the positive
pole.
http://creationwiki.org/Gel_electrophoresis

15. SDS-PAGE
SDS
(sodium dodecyl sulfate)
• Detergent
• Denatures protein to their
primary structure
• Covers molecules with a
negative charge to be able to
migrate to the positive pole
PAGE
(polyacrylamide gel electrophoresis)
• A polymer of acrylamide
monomers
• Separates based on size of
molecules.
• Allows different sized proteins to
move at different rates.
http://www.bio.davidson.edu/genomics/method/SDSPAGE/SDSPAGE.html#SDS

16. Experiment Procedure
• 5 mg/ml crude porcine pepsin
• 5 mg/ml mucor rennet
• 5 mg/ml mix of crude pepsin and rennet
• 5 mg/ml of pure porcine pepsin
http://en.wikipedia.org/wiki/Polyacrylamide_gel_electrophoresis

17. Results - Gel
Total migration distance:
6.3 cm
Lanes 2,3,6,9: Protein
standard marker
Lane 4: Crude pepsin
Lane 9: Pure pepsin
Lane 5: Mucor Rennet
Lane 8: Crude
pepsin/rennet mixture

18. Results – Calibration Graph
y = -0.0131x + 2.4800
R² = 0.9824
0.5
0.7
0.9
1.1
1.3
1.5
1.7
1.9
2.1
2.3
2.5
0 10 20 30 40 50 60 70 80 90 100
Log(MW)
% migration distance
Log(MW) vs. % migration distance

19. Results – Values
Sample Band migration distance (cm) % migration distance MW (kDa) min MW (kDA) max MW (kDa) Literature Value
Crude Pepsin 1 4.6 73.0 33.4 18.9 59.0 34.6
Mucor Rennet 1 4.5 71.4 35.0 19.8 61.9 38
2 5 79.4 27.6 15.6 48.7 38
3 5.5 87.3 21.7 12.3 38.4 38
4 5.7 90.5 19.7 11.2 34.8 38
Mixture 1 4.4 69.8 36.7 20.8 64.9 38
2 4.6 73.0 33.4 18.9 59.0 34.6
Pure Pespsin 1 4.8 76.2 30.3 17.2 53.6 34.6
2 5.6 88.9 20.7 11.7 36.6 34.6

20. Possible Causes of Error
Cross-contamination between the sample mixtures:
• From sample preparation (e.g. contacted other solutions).
• During loading onto gel.
Break-down of mucor rennet

21. Conclusion
• Overall, three out of the four protein samples had results that clearly
showed the MW of each sample as being close to the literature value.
• Main error in the experiment was with the mucor rennet lane as
more unexpected bands showed up from unknown sources.

22. Reference
• Ming Cai, Wei Li, Hanhua Liang, Effects of ultrasound parameters on ultrasound-assisted ultrafiltration using
cross-flow hollow fiber membrane for Radix astragalus extracts, Chemical Engineering and Processing:
Process Intensification, Volume 86, December 2014, Pages 30-35, ISSN 0255-2701,
http://dx.doi.org/10.1016/j.cep.2014.10.008.

23. Q & A