Untitled Notes

Date of lecture: February 18th
Objective: Explore various types of chromatography including size exclusion, ion exchange, and affinity chromatography.
Importance: Prepares students for laboratory work.

Chromatography defined as a separation technique used in chemistry and biochemistry to separate and analyze compounds.
Historical context:
- First noted use came from a botanist in 1900 studying chlorophyll using calcium carbonate columns.
- The term "chromatography" translates to "color writing".
- Significant recognition with the 1952 Nobel Prize awarded for advancements in chromatography.
- Separation of chemicals has deep roots in chemistry, stemming back to alchemical practices.

Analyte: The specific compound being analyzed or separated.
Immobilized phase (Stationary phase): Typically consists of beads or solid material that retains the analyte.
Alluent (Mobile phase): The solvent or buffer that moves over the stationary phase carrying the analyte.

The process involves passing a sample (the alluent) over a stationary phase (the immobilized phase), causing an interaction that separates compounds based on certain properties like affinity.
Fractions of separated analytes can be collected at the column’s output.

Predominantly used in chemistry and biochemistry labs.
Stationary Phase: Beads or gel.
Mobile Phase: A solvent such as Tris.
Components begin to separate based on their affinities as they move through the static column.

A sophisticated form of liquid chromatography known for high resolution and efficiency due to high-pressure pumps.
Allows for separation down to parts per trillion for more accurate analyses.
Common applications include pharmaceutical testing, environmental sample testing, forensics, and protein analysis (though not primary protein purification due to potential denaturation).
Example provided: Separation of dihydrocavaine from kava plant juices based on structural differences (double bond).

Converts samples to vapor state for analysis.
Passed through a heated column with a detector that records data on migrating compounds.
Ideal for organic chemical analysis but unsuitable for proteins due to potential damage from heat.

A rapid method for checking the purity of a compound.
Utilizes a stationary phase like cellulose paper and solvents like acetone.
Example: Demonstrated at science camp using chromatography to compare ink colors.
Broader applications in forensic science and drug analysis.

Focus of ongoing studies in the semester: methods for purifying proteins with an emphasis on HPLC, FPLC (Fast Protein Liquid Chromatography), and LPLC (Low-Pressure Liquid Chromatography).
High costs noted: HPLC systems can range from $20,000 to $40,000.
Essential equipment components include columns, beads, buffers, gradient mixers, pumps, conductivity meters, UV detectors, and fraction collectors.

Columns can be pre-packed or custom-made, and their capability is defined by maximum loading capacity based on bead quantity and type.
Equipment maintenance is critical; risks of damage, clogs, and dry runs can compromise results and equipment integrity.

Initial lysate for protein purification.
Use of chromatography steps: affinity, cation exchange, anion exchange, and size exclusion.
Purification monitored via activity assays, specific activity calculations, and fraction collection.

Specific activity defined as units of activity per milligram of protein: $ext{Specific Activity} = rac{ ext{Total Activity}}{ ext{Total Protein}}$
Importance of achieving higher specific activity post purification steps to ensure functional proteins remain intact during processing.

Involves interaction between target proteins and specific ligands to isolate proteins based on specific properties.
Generally performed first in purification due to its high specificity compared to charge-based methods.

Separation based on net molecular charge of proteins; proteins can be positively or negatively charged depending on pH.
Two main types:
- Anion Exchange: Uses positively charged beads to attract and bind negatively charged anions.
- Cation Exchange: Utilizes negatively charged beads to bind positively charged cations.

After sample addition, lightly bound contaminants are washed out before applying a salt gradient (usually sodium chloride).
The elution order varies based on the binding strength, influenced by factors like chemical interactions and the protein's isoelectric point (pI).

For anion exchange, negatively charged proteins will grip onto positively charged beads; elution requires an increase in negatively charged ions.
In cation exchange, positively charged proteins bind, and a similar strategy applies for elution using positively charged ions.

A mild method ideal for separating proteins based on size. Also serves as a buffer exchange technique post-precipitation processes.
Separation limits noted: effective for proteins differing in size by at least twofold for ideal outcomes.

SEC uses porous beads that allow smaller proteins to enter while larger ones flow around, thus eluting first.
Elution profiles expected: large proteins elute first followed by smaller proteins, with separation influenced by protein shape and size.

Effective not only for analysis but also for confirming the purity of protein samples, protein folding, and even oligomeric states.
Utility in studies to define molecular interactions, confirm sizes, and examine the impacts of compounds on protein structure.

Recap of chromatography types covered and their applications in protein purification pathways.
Continuous monitoring of practical lab applications, techniques in chromatography completion, outcomes from fraction analysis, and related lab experiences all emphasized.
Open floor for questions as lecture concludes.