biyokimya lab Flashcards
(29 cards)
What is the general purpose of chromatographic methods?
To separate, purify, and analyze components in a mixture.
What are the two basic components of chromatographic techniques?
Stationary phase and mobile phase.
What serves as the stationary phase in Paper Chromatography?
Cellulose-based filter paper.
What is the principle behind Paper Chromatography?
Separation by dispersion differences.
How is the Retention Factor (Rf) value calculated?
Spot distance ÷ solvent front distance.
Main advantage of Thin Layer Chromatography (TLC) over Paper Chromatography?
Can handle harsh chemicals like acids.
What is the TLC stationary phase made of?
Silica, alumina, or cellulose on plates.
TLC is based on which principle?
Adsorption.
How does Gel Permeation Chromatography separate proteins?
By size — large molecules elute first.
What is the separation principle of Ion Exchange Chromatography?
Based on charge differences.
Why is Affinity Chromatography so efficient?
Uses highly specific binding for near 100% purification.
Name one interaction used in Affinity Chromatography.
Antigen-antibody (also: enzyme-substrate, etc.).
Key disadvantage of Gas Chromatography?
Only works with volatile, low-molecular-weight samples.
What does HPLC stand for and one use?
High Pressure Liquid Chromatography; separates non-volatile compounds.
Three stages of DNA isolation?
Cell lysis, protein removal, DNA purification.
Why is EDTA used in DNA isolation?
It chelates Mg²⁺, stopping nuclease activity.
Role of Phenol and Chloroform in DNA isolation?
Remove and denature proteins.
How is DNA precipitated from solution?
Add cold alcohol (ethanol/isopropanol).
Preferred temp for long-term DNA storage?
-80°C.
Why store DNA in aliquots?
Prevents damage from repeated freeze-thaw cycles.
At what wavelength do nucleic acids show max absorbance?
260 nm.
What does an A260/A280 ratio of ~1.8 mean?
DNA is pure, little protein contamination.
What does a low A260/A230 ratio suggest?
Contamination by organics (e.g. phenol).
Absorbance of 1 at 260 nm equals how much dsDNA?
50 µg/mL (in 1 cm cuvette).
