Ultrafiltration and Gel Electrophoresis

Rashid AlsuwaidiAhmed Alhazeem

Matt LiuWanying Chia

Ultrafiltration

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

Ultrafiltration• Purifying solvents• Concentrating solutes• Utilizes pressure gradients

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

Filtration Rate

30 32 34 36 38 40 42 44 46 48 502.00E-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

Avg. Flux Due to Applied Pressure

20 mM Acetate BufferLinear (20 mM Acetate Buffer)1 mg/mL Protein Solution

Applied Pressure (Psi)

Avg

Flux

(mL/

cm^2

*sec

)

Osmotic Pressure

30 32 34 36 38 40 42 44 46 48 50

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

12.0

Osmotic Pressure Over Pressure

1 mg/mL

2 mg/mL

Pressure ΔP (psi)

Osm

otic P

ress

ure

Δπ (p

si)

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

30 32 34 36 38 40 42 44 46 48 502.00E-03

2.50E-03

3.00E-03

3.50E-03

4.00E-03

4.50E-03

5.00E-03

5.50E-03

f(x) = 8.78290268225185E-05 x + 0.000398009512442898R² = 0.997636414417587

f(x) = 0.000108401663283696 x − 0.000321281391542577R² = 0.988014651958772

Comparison of Permeability Coefficient

Flux BeforeLinear (Flux Before)Flux AfterLinear (Flux After)

Pressure (Psi)

Avg

Flux

(mL/

cm^2

*sec

30 32 34 36 38 40 42 44 46 48 504.00E-04

6.00E-04

8.00E-04

1.00E-03

1.20E-03

1.40E-03

1.60E-03

Comparison of Kc Values

Ideal 1 mg/mL

Ideal 2 mg/mL Solution

Jonnson 1 mg/ml Solution

Jonnson 2 mg/mL

Pressure (Psi)

Kc (c

m/s

)

30 32 34 36 38 40 42 44 46 48 500.0

100.0

200.0

300.0

400.0

500.0

600.0

700.0

800.0

900.0

1000.0

Comparison of Solute at Membrane Surface

Ideal 1 mg/mL

Ideal 2 mg/ml

Jonnson 1 mg/ml

Jonnson 2 mg/ml

Pressure (Psi)

Cs (m

g/m

L)

Cs and Mass transfer coefficient

Conclusions• Osmotic Pressure has a greater effect on mass transfer coefficient• Pressure increases mass transfer coefficients as well asProtein

Concentration on the membrane surface

Gel Electrophoresis

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.

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

SDS-PAGESDS

(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

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

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

Results – Calibration Graph

10 20 30 40 50 60 70 80 90 1000.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

2.1

2.3

2.5

f(x) = − 0.013126531196024 x + 2.47995708466254R² = 0.98240464241078

Log(MW) vs. % migration distance

% migration distance

Log(

MW

)

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

Possible Causes of ErrorCross-contamination between the sample mixtures:

• From sample preparation (e.g. contacted other solutions).• During loading onto gel.

Break-down of mucor rennet

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.

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.

Q & A