Biochem Lab exam

What is a titration?

A method where a solution of known concentration is added to a solution of unknown concentration to determine acidity, pKa, or concentration.

What is the equivalence point?

The point where moles of acid = moles of base. The acid is fully neutralized.

How does the titration curve of a strong acid look?

Very steep vertical rise; no buffer region; equivalence point near pH \ 7.

How does the titration curve of a weak acid look?

Has a buffer zone, smaller slope, equivalence point above pH \ 7.

Why is the equivalence point higher for weak acids?

Because the conjugate base formed during titration raises the pH.

What is a buffer region?

The flat portion where pH changes slowly because acid and conjugate base are both present (pKa occurs at the midpoint).

Formula for pKa

pKa = -log(Ka)

How to experimentally find pKa on a titration curve

It is the pH at the midpoint of the first buffering region (half-equivalence point).

Why do strong acids lack a buffer region?

Because strong acids fully dissociate — no equilibrium between acid/conjugate base.

What does the shape tell you about acid strength?

Lower pKa = stronger acid = steeper titration curve.

What makes amino acid titration curves unique?

They have multiple pKa values because amino acids have multiple ionizable groups.

What are the ionizable groups in glycine?

Carboxyl (COOH) Amino (NH_3^+)

What are the ionizable groups in histidine?

Carboxyl Amino Imidazole side chain (weakly basic)

Order of deprotonation in amino acids

COOH \to side chain (if present) \to NH_3^+

What is pI (isoelectric point)?

pH where the amino acid has a net charge of zero.

How to calculate pI for glycine

pI = (pKa1 + pKa2)/2 = (2.34 + 9.6)/2 = 5.97

How to calculate pI for histidine (3 ionizable groups)?

Pick the two pKa values around the neutral species. For Histidine: pKa = 6 and 9.17 \implies pI = 7.59

What is a zwitterion?

A molecule with both positive and negative charges but net charge = 0.

Why does histidine have 3 buffering regions?

Because it has 3 ionizable groups \to 3 pKa values.

How to identify glycine vs histidine curves

Histidine curve = 3 plateaus Glycine curve = 2 plateaus

What is TLC used for?

Separating molecules based on polarity.

What is Rf value?

Rf = \frac{\text{distance traveled by compound}}{\text{distance traveled by solvent}}

What is the stationary phase in TLC?

Silica (very polar).

Which amino acids travel farther?

NON-polar amino acids (less attraction to silica).

Which move least?

POLAR amino acids (stick to silica more strongly).

What does ninhydrin do?

Reacts with amino acids to make purple spots so you can see them.

Why must spots not overlap?

Overlapping spots make Rf impossible to interpret.

Why mark the solvent front immediately?

The solvent evaporates — you'll lose reference if you wait.

Why must solvent not touch the sample line at the start?

It would dissolve the samples before separation occurs.

Purpose of unknown mixture

Match its Rf to known standards to identify which amino acids are present.

What does SEC separate by?

Size (molecular weight).

What elutes first?

LARGE molecules (cannot enter pores \to move fast).

What elutes last?

SMALL molecules (enter pores \to move slow).

What is the exclusion limit?

The maximum molecular weight that can enter bead pores.

Why is exclusion limit important?

It tells you which molecules will be separated and which will all elute together.

What is the stationary phase made of?

Porous beads with defined pore sizes.

What is the mobile phase?

Buffer flowing through the column.

What is void volume?

Volume outside beads where large molecules travel.

Why must samples be loaded gently?

To avoid disturbing the packed beads and ruining separation.

Why do proteins stay folded in SEC?

Because separation is gentle and uses physiological buffers.

What is a standard curve?

Graph of known concentrations vs absorbance to find unknown samples.

Why do we need it?

A spectrophotometer only measures absorbance, not concentration.

What is Coomassie Blue?

A dye that binds proteins and changes color intensity with concentration.

What law relates absorbance to concentration?

Beer–Lambert Law: A = \epsilon lc

How to determine unknown protein concentration

Plug absorbance into line equation (y = mx + b) from standard curve.

Why must standards be diluted carefully?

Incorrect dilutions destroy linearity \to inaccurate concentration.

Why blank the spectrophotometer?

Sets absorbance of buffer/dye to zero so readings only represent protein.

What wavelength does Coomassie Blue absorb?

595 nm (most common).

Why mix samples thoroughly?

Uneven dye binding \to unreliable absorbance.

What does a non-linear standard curve indicate?

Pipetting errors, improper dye mixing, cuvette contamination.

What is PCR?

Technique to amplify specific DNA sequences.

What are the steps of PCR?

Denaturation \to Annealing \to Extension

What does Taq polymerase do?

Builds new DNA strands \to heat-stable enzyme.

What do primers do?

Short DNA pieces that mark start/end region for amplification.

What is master mix?

Contains Taq, primers, nucleotides, and buffer \to everything PCR needs.

What is agarose?

A gel used to separate DNA by size.

Which direction does DNA run?

From negative \to positive (DNA is negatively charged).

What makes DNA move in the gel?

Electric field pulls negatively charged DNA toward positive electrode.

Which fragments travel faster?

SMALLER fragments \to move through pores more easily.

Why add loading dye?

To see samples during loading and monitor how far they run.

What is a DNA ladder?

Reference standard with known fragment sizes.

Why is buffer necessary in electrophoresis?

Conducts electricity and stabilizes DNA.

What happens if gel runs backward?

DNA runs off the gel and is lost.

Why must the comb be removed carefully?

To avoid tearing wells.

Why does Coomassie Blue shift color when bound to protein?

The dye’s electron cloud changes \to altered absorbance spectrum.

Why does histidine have a unique pI near neutral pH?

Its imidazole ring is weakly basic and gains/loses a proton around pH \sim 6.

Why do polar amino acids stick to silica?

Silica has OH groups \to strong hydrogen bonding with polar compounds.

Why does SEC NOT separate by charge?

No ionic interactions — only pore size matters.

What makes Taq polymerase heat-resistant?

It comes from thermophilic bacteria in hot springs (Thermus aquaticus).

What happens if PCR annealing temperature is too high?

Primers won't bind \to no amplification.

What happens if annealing temp is too low?

Primers bind non-specifically \to messy results or multiple bands.

What causes smeared DNA bands?

Overloading DNA, degraded DNA, or running the gel too hot.

Why use agarose instead of polyacrylamide?

Agarose has larger pores \to better for big DNA fragments.

Why must TLC plates be handled by the edges?

Finger oils interfere with solvent flow and spot separation.

Why do strong acids fully dissociate?

Their conjugate bases are extremely weak and hold H^+ very loosely.

Why do weak acids create buffer systems?

They partially dissociate, allowing reversible equilibrium.

In SEC, what happens if flow rate is too fast?

Poor separation \to molecules do not equilibrate with bead pores.

In PCR, why must Mg^{2+} be present?

Taq polymerase requires Mg^{2+} as a cofactor to function.

Why does DNA repel itself?

Its phosphate backbone is negatively charged.

Why do we use UV transilluminators?

DNA can be visualized after binding fluorescent dyes like ethidium bromide or SYBR Safe.