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BIOCHEMISTRY LAB REPORT 4 2025-2026| PORTAGE LEARNING|WITH PROCEDURES, EXPLANATION&RESULTS, Exams of Biochemistry

Pennsylvania State University - AbingtonBiochemistry

BIOCHEMISTRY LAB REPORT 4 2025-2026| PORTAGE LEARNING|WITH PROCEDURES, EXPLANATION AND FINAL RESULTS.

Typology: Exams

2024/2025

Available from 04/29/2025

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Biochemistry Lab Report
BIOCHEMISTRY LAB REPORT 4 2025-2026| PORTAGE
LEARNING|WITH PROCEDURES, EXPLANATION
AND FINAL RESULTS.
Experiment #: Lab 4
Title: Chromatography of molecules.
Purpose: The goals for the student performing the experiment are 1. Explain the
fundamentals of chromatography. 2. Demonstrate ion exchange and size- exclusion
chromatography. 3. Separate a Real-World sample. The student should be able to
use the techniques learned and be able to apply them to further use in the lab or in a
chemistry class again. Students should be able to fully recall and demonstrate how
to do these techniques when completed the lab.
Procedure:
Part 1:
•
Setting up the columns.
o Notes: The pH of a solution can impact the stationary phase and the
molecules to be separated.
o 1. The chromatographic media is poured in as a slurry.
o 2. The media is permitted to settle.
o 3. Buffer (mobile phase) is permitted to flow through the column.
•
Procedure:
o Step 1: Put on appropriate lab equipment based on what solutions
and chemicals will be used in the lab that they are performing. Ex:
Gloves, Lab coat, Closed toe shoes, Protective glasses, etc. Also
have the appropriate materials needed for completion of the lab.
o Step 2: Place glass wool in column so that the media has a barrier to
keep it from eluting.
o Step 3: Place column on stand. Be sure to label columns.
o Step 4: With prepared media place in column and let it settle for a
couple of minutes.
o Step 5: Let some of the buffer for the columns run through so that
they are in the gel. Never let the gel go dry, always keep the buffer
above the gel.
o Step 6: Your columns are not set up for separating test mixture
compounds.
Part 2:
•
Separation of two test mixtures.
o Notes:
▪
There will be two test mixtures applied to each column.
o Step 1: Put on appropriate lab equipment based on what solutions
and chemicals will be used in the lab that they are performing. Ex:
Gloves, Lab coat, Closed toe shoes, Protective glasses, etc. Also
have the appropriate materials needed for completion of the lab.
o Step 2: With columns already set up and have had buffer ran through
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Partial preview of the text

Download BIOCHEMISTRY LAB REPORT 4 2025-2026| PORTAGE LEARNING|WITH PROCEDURES, EXPLANATION&RESULTS and more Exams Biochemistry in PDF only on Docsity!

BIOCHEMISTRY LAB REPORT 4 202 5 - 2026 | PORTAGE

LEARNING|WITH PROCEDURES, EXPLANATION

AND FINAL RESULTS.

Experiment #: Lab 4 Title: Chromatography of molecules. Purpose: The goals for the student performing the experiment are 1. Explain the fundamentals of chromatography. 2. Demonstrate ion exchange and size- exclusion chromatography. 3. Separate a Real-World sample. The student shouldbe able to use the techniques learned and be able to apply them to further use in the lab or in a chemistry class again. Students should be able to fully recall and demonstrate how to do these techniques when completed the lab. Procedure: Part 1:

- Setting up the columns. o Notes: The pH of a solution can impact the stationary phase and the molecules to be separated. o 1. The chromatographic media is poured in as a slurry. o 2. The media is permitted to settle. o 3. Buffer (mobile phase) is permitted to flow through the column. - Procedure: o Step 1: Put on appropriate lab equipment based on what solutions and chemicals will be used in the lab that they are performing. Ex: Gloves, Lab coat, Closed toe shoes, Protective glasses, etc. Also have the appropriate materials needed for completion of the lab. o Step 2: Place glass wool in column so that the media has a barrier to keep it from eluting. o Step 3: Place column on stand. Be sure to label columns. o Step 4: With prepared media place in column and let it settle for a couple of minutes. o Step 5: Let some of the buffer for the columns run through so that they are in the gel. Never let the gel go dry, always keep the buffer above the gel. o Step 6: Your columns are not set up for separating test mixture compounds. Part 2: - Separation of two test mixtures. o Notes: ▪ There will be two test mixtures applied to each column. o Step 1: Put on appropriate lab equipment based on what solutions and chemicals will be used in the lab that they are performing. Ex: Gloves, Lab coat, Closed toe shoes, Protective glasses, etc. Also have the appropriate materials needed for completion of the lab. o Step 2: With columns already set up and have had buffer ran through

them know you are ready to begin separating test mixtures. o Step 3: Apply .3 mL of test mixture one to each column using an automatic pipette. o Step 4: Open stopper on the column for a couple of drops to let the mixture seep into the gel. o Step 5: Then place buffer on top of mixture trying to layer and not dilute the dye. o Step 6: Open the stopper again and let it drop a couple of drops and close and add more buffer to the top. o Step 7: Open the stopper again and let it drip till color comes out and replace beaker with a test tube. Remember to keep adding buffer so that the gel does not go dry. o Step 8: Collect every different color that comes out of the column with a new test tube. Do not let it drop into the same test tube theentire time. o Step 9: If a color is not eluting drop a couple drops with a higher concentration buffer. o Step 10: Repeat Steps 2 – 9 for each column. Suggest doing one column at a time so that student performing lab can maintain the adding of the buffer. o Step 11: On the DEAE column, if no color is moving use a few drops of HCl to influence the color movement. o Step 12: Write down and record results for further use in the lab.

- Separation of “real world” sample. o Notes: ▪ A ‘real world” sample will be separated using at least two columns. - Procedure: o Step 1: Put on appropriate lab equipment based on what solutions and chemicals will be used in the lab that they are performing. Ex: Gloves, Lab coat, Closed toe shoes, Protective glasses, etc. Also have the appropriate materials needed for completion of the lab. o Step 2: Prepare the three different columns which would be the DEAE, CM. o Step 3: Apply .7 mL of test mixture to the CM column using an automatic pipette. Would be easier to finish one column at a time so students can focus on results. o Step 4: Open stopper on the column for a couple of drops to let the mixture seep into the gel. o Step 5: Then place buffer on top of mixture trying to layer and not dilute the dye. o Step 6: Open the stopper again and let it drop a couple of drops and close and add more buffer to the top. o Step 7: Open the stopper again and let it drip till color comes out and replace beaker with a test tube. Remember to keep adding buffer so that the gel does not go dry.

o DEAE Column: ▪ First Color: Yellow Dextran ▪ Second Color: Orange/Pink Vitamin B ▪ Third Color: Blue Dextran Discussion Questions:

- Discussion Question 1: The elution from the columns is different because in the IEC it is picking out the opposite charged molecules. Which does not take as long to elute. The SEC filters based on size which only allows molecules below its size threshold through the media. - Discussion Question 2: The 15,000 Da protein would elute first because it is the smaller protein. - Discussion Question 3: Buffering is important in IEC because the pH of the solution can affect the entire media. Also depending on which media is being used will determine which molecule elute and which ones do not. - Discussion Question 4: The DEAE column typically had the most dilutions occurring inside the column. - Discussion Question 5: Another order that you could run the samples is doing the DEAE column first and then running the CM column. Thise would probably rid of all the blue dextran due to the difficulty it is to elute from the DEAE column. Conclusions: This experiments the student first should have learned how to set up a chromatography column and prep a chromatography column for experimentation. The student also should have learned how to properly test solution through SEC and IEC columns. The results showed that on the SEC the heaviest compounds typically were the first to elute, but sometimes there was dilution between two like compounds. The student should have also learned that in a DEAE positive compounds typically take longer to elute than negative compounds. A way to speed up the elution of positive compounds was to increase the concentration of the buffer so that the competing positive charge of the buffer would take the place of the positive charged compound. The opposite would occur for the CM column positive compounds would elute quicker than negative compounds. The student should use the techniques and new information learned in future chemistry experiments and labs. Notes: - Column chromatography has a stationary phase which includes a long thin tube and that has a media in it that does not leave. Then a mobile phase is usually a buffer or solution that moves through the stationary phase. As molecules pass through the stationary phase some are stopped and stay in the stationary phase. Eventually a solution such as water exits the column. DEAE is an anion exchange media, has a nitrogen with a positive charge to stop things with a negative charge. Things with a positive charge would stay in the mobile media. When things flow out from the column, we call

them elute from the column. You can also have a CM gel that is a cation exchange media, and it stops molecules with positive charges. This is ion exchange chromatography.

- Size Exclusion Chromatography (SEC): o AKA Gel permeation chromatography. o Gels have been manufactured so that they have pores that are certain size and only allow certain sized molecules to pass through. o This just takes time for the smaller molecules to come out because it acts as a filtration unit. o Bigger things tend to be stopped first. - Buffering in Chromatography: o The pH of a solution can impacts the stationary phase and the molecules to be separated. o DEAE has a pka of 11.5. Which is a good number because almost everything in life occurs at a pka below 11.5. o This will cause the DEAE to almost always be positive. o Aspartic acid has a pka 3.86 so anything above that will react with DEAE. o Concentration of buffer is important, as well as ionization and analyzing of molecules. - Properties of compounds to be tested. Present in Test Mixture Compound Color MW (Da) Ionic Nature atpH 6 1,2 Blue Dextran Blue 500,000 Very strong anion 2 Yellow Dextra n Yellow 40,000 Strong anion 1 Cytochrome C Red Orange 12,400 Strong Cation 2 Vitamin B12 Cherry Red 1,347 Weak cation 1 DNP-Glycine Yellow 241 Weak anion - Media for SEC p100 media will let anything lower than 100,000 MW (Da)through and anything bigger will slow down. - Buffers used 0.1 molar acetic acid buffer, sodium acetate buffer. - Increasing concentration of buffer increases the concentration of ions competing against the gel. - Confused on SEC, around the 13 - minute mark it said it will retain anything greater than 100,000 MW in the medium longer, so how is it not assumed anything under will pass the heaviest compound and mix them together?