PHAR2202 Drug Design: Analytical Methods - Separations

Ion Exchange Chromatography (IEC)

• IEC is a liquid chromatography technique where analytes are separated based on their adsorption onto a stationary phase with fixed ionic charges.
• Commonly used in home water softening to exchange 'hard' $Ca^{2+}$ and $Mg^{2+}$ for $Na^{+}$ ions on a negatively charged stationary phase.
• Used to separate inorganic ions, amino acids, proteins, and nucleic acids.
• Cation exchange:
  $A^+ + \text{support}-(C^+) \rightleftharpoons \text{support}-(A^+) + C^+$
  $K_{A,C} = \frac{[A^+][\text{support}-(C^+)]}{[\text{support}-(A^+)][C^+]}$
  Where $K$ is the selectivity coefficient.
• The complementary process uses a positively charged stationary phase to bind negatively charged analytes.
• Can be used to remove charged impurities from uncharged solutes.

Factors Affecting Analyte Retention

• Nature and accessibility of ion-exchange groups on the support.
• Type and concentration of analyte ions.
• Nature and concentration of competing ions in the mobile phase.
• pH of the mobile phase, sample, and starting conditions.

Stationary Phase

• Cationic-exchange: Negatively charged stationary phase.
• Anionic-exchange: Positively charged stationary phase.

Mobile Phase

• A mobile phase can be made a stronger eluent by:
  • Increasing the competing ion concentration (adding salt).
  • Reducing the extent to which the analyte is charged (changing the pH).

Acidic and Amine Resins

• Acidic (-ve) resins: Bind +ve compounds (e.g., $R-NH_3^+$), so high pH reduces charge and binding (Cationic Exchange).
• Amine (+ve) resins: Bind -ve compounds (e.g., $R-CO_2^-$), so high pH increases charge and binding (Anionic Exchange).

Concept Check: Ion Exchange Chromatography for Toluic and Benzoic Acid

• Separate toluic acid (pKa 3.9) and benzoic acid (pKa 4.2).
  • Use anion exchange (stationary phase negatively charged).
  • At pH 4.8, toluic acid will elute first (more deprotonated).
  • To increase the retention factor, lower the pH.

Concept Check: Ion Exchange Chromatography for Amines

• Separate amines using ion-exchange chromatography.
  • Use cation exchange (stationary phase negatively charged).
  • Use a specific pH.
  • Raising the pH will reduce the retention factor $K$.
  • Adding 0.2 M NaCl to the eluent will reduce the retention factor $K$.

Affinity Chromatography

• Antibodies are proteins that bind to other proteins with high affinity and specificity.
• Most are immunoglobulin Gs (IgG’s). Molecular Weight = 150 kDa.
  • Heavy chain x 2 (called the γ-chains, 50 kDa each).
  • Light chain x 2 (25 kDa each).

Affinity Columns

• Affinity columns are ‘on/off’:
  • The sample is run through the column, and the target binds to the stationary phase.
  • The column is ‘washed’ with mobile phase to remove the non-bound sample.
  • The mobile phase is then changed to effect release of the target molecule from the column.
• The binding interaction should be selective and reversible (e.g., antibody with antigen, enzyme with substrate, hormone and its receptor).

Analyte Retention

• Retention of analyte (A) on an affinity column can be described by a complexation reaction: $A + L \rightleftharpoons A-L$ , $K_A$.
• If a 1:1 complex is formed between A and L, the retention factor (k) can be described by: $k = K<em>A (m</em>L / V_M)$
  • Where $V_M$ is the void volume of the column.
• $k = (t<em>R - t</em>M) / t_0$
• The retention factor depends on the strength of binding between analyte and ligand ($K<em>A$) and the concentration of available binding sites ($m</em>L / V_M$).

Affinity Chromatography Example

• An antibody of HIV-1 reverse transcriptase (10 nmol) is fixed as the ligand in an affinity column 10 cm long and 4.1 mm inner diameter. Void volume of 1.0 mL.
• At pH 7, the $K_A = 1.0 x 10^8 M^{-1}$.
• What is the retention factor k for HIV-1 reverse transcription?
• How long would it take to elute the sample at 1 ml/min?

Examples of Affinity Ligands

• Biological ligands
  • Antibodies: bind antigens (drugs, hormones, peptides, proteins, viruses, cell components).
  • Inhibitors, substrates, cofactors, coenzymes: bind enzymes.
  • Lectins: bind sugars, glycoproteins, glycolipids.
  • Nucleic acids: bind complementary nucleic acids, DNA/RNA-binding proteins.
  • Protein A/protein G: bind antibodies.
• Non-biological ligands
  • Boronates: bind sugars, glycoproteins, diol-containing compounds.
  • Triazine dyes: bind nucleotide-binding proteins and enzymes.
  • Metal chelates: bind metal-binding amino acids, peptides, and proteins.

Gel Electrophoresis

• Uses a crosslinked polymer gel as the ‘stationary phase’.
• The analyte has no ’affinity’ to the gel – separation is purely physical.
• Typically for separation of large charged molecules (Proteins / DNA).

Solid Supports

• Polyacrylamide gel (PAGE): Suitable for separation of proteins and peptide mixtures; low specific binding; no inherently charged groups.
• Cellulose acetate, filter paper, and starch: Useful for small molecules such as amino acids.
• Agarose: Suitable for larger molecules, such as during sequencing of DNA; larger pore sizes.

Migration Time

• Migration time is affected by the charge and size “Electrophoretic mobility ($\mu_{ep}$)”.
  • Attraction to opposite charge: $F = zE$
  • Resistance from gel / eluent: $F = 6\pi r \cdot \mu_{ep}$
  • When the forces are balanced: $\mu_{ep} = \frac{z}{6\pi r} \cdot E$
    • z = ion charge
    • $\eta$ = density of eluent
    • r = radius of ion
    • $\mu_{ep}$ = velocity of ion
    • E = electric field (V/m)

SDS-PAGE (Sodium Dodecyl Sulfate PAGE)

• Proteins are denatured (heat).
• Treated with a detergent (non-polar one end and negatively charged on the other – SDS). The SDS coats the protein forming negatively charged rods with similar size-to-charge ratios and whose apparent size parallels actual molecular weight.
• A calibration standard (ladder) of similar proteins with known molecular weights is run alongside the sample(s), from which the molecular weight of the analytes can be estimated.
• Rate of migration in SDS PAGE is controlled by molecular weight.
• Native PAGE separates based on both size and charge, unlike SDS-PAGE.

Gel Electrophoresis: Detection

1. General Stains

• Ponceau S
• Amido black
• Coomassie brilliant silver staining (amino acids)
• Ethidium bromide (DNA)

2. Selective Stains on Nitrocellulose Membrane

• Southern blot: Detects specific sequences of DNA using radioactive phosphorous ($^{32}P$) or chemiluminescence.
• Northern blot: Detects RNA.
• Western blot: Detects specific protein analytes using antibodies with a detectable label.

3. MALDI-TOF

• Gel is placed directly in a mass spectrometer, hit with a laser to sputter out the analyte, which is then ionized and its mass analyzed in a TOF mass spectrometer.
• TOF MS works on the basis that ions with different mass-to-charge ratios achieve different velocities in an electric field and therefore arrive at a fixed detector at times dependent on their m/Z.
• Proteins can be identified by their behavior upon electrophoresis and their molecular weight.

Band Broadening

• A term (Eddy diffusion): Minimal for both CE and PAGE.
• C term (Mass transfer): Minimal for both CE and PAGE as there’s no stationary phase.
• Most band broadening comes from the B term (Longitudinal diffusion).

Joule Heating

• Electric current generates heat, which affects diffusion.
  • Uneven viscosities, resulting in varying migration velocities.
  • Evaporation of the eluent, leading to changes in pH and concentration.
  • Wick flow: Buffer evaporates from a gel and is replaced by flow from the reservoirs.

Special Types: Isoelectric Focusing (IEF)

• Allows separation of proteins based on isoelectric point using a gel with a permanent pH gradient.
  • Small polyprotic amino carboxylic acid ampholytes (compounds that can both donate and accept protons) with a range of pKas are dissolved.
  • An electric field is applied, and they travel through the support until they reach their isoelectric point (pI) in the field.
  • Typically, they are polymerized into the polyacrylamide gel to form immobilized pH gradients to give a fixed pH gradient across the gel.
• Injected zwitterionic samples will migrate to the pH of their isoelectric point.

2D Electrophoresis

• Combines techniques:
  • Dimension 1: IEF (Isoelectric Focusing).
  • Dimension 2: SDS-PAGE.

Capillary Electrophoresis

• No Stationary phase.
• Eluent flows through a thin capillary (often silica).
  • Capillary inner diameter: 20–100 µm
  • Capillary length: 20–100 cm
  • Voltages: 25–30 kV.

Electrophoretic Mobility

• Proteins still feel the electrophoretic force.
  • $\mu_{ep} = \frac{z}{6\pi r} \cdot E$

Electroosmosis

• Charge on the capillary drives a net flow towards the electrode of the same charge due to the buildup of oppositely charged ions from the eluent by the wall.

Migration Time in Capillary Electrophoresis

• Two forces affect migration time.
  • The overall electrophoretic mobility of an analyte ($\mu_{Net}$) is:
    • $\mu<em>{Net} = \mu</em>{ep} + \mu_{eo}$
    • $u<em>{Net} = (\mu</em>{ep} + \mu_{eo}) \cdot E$
    • E = electric field strength in V/m

Differences between PAGE and CE

• PAGE: Electrophoresis, $\mu_{ep} = \frac{z}{6\pi r} \cdot E$
• CE: Electroosmosis, $\mu<em>{eo} = \mu</em>{eo} \cdot E$

Band Broadening in CE

• The use of these narrow bore tubes provides efficient removal of heat from Joule heating, decreasing band-broadening and provides faster and more efficient separations than gel electrophoresis.
• Therefore simple molecular diffusion is the major cause of band-broadening.
• Under these conditions the number of theoretical plates, N, for this system can be given by: $N = \frac{\mu_e V}{2D}$
  • Where D is the diffusion coefficient and μe is the electrophoretic mobility of the analyte

CE Detectors

Laser-Induced Fluorescence (LIF)

• A laser is monochromatic and very intense, which allows for selective and very strong excitation of analyte chromophores.
• The laser beam can be very sharply focused, suitable for very small sample volumes.
• A ‘disadvantage’ is that the analyte must have a chromophore which can be excited to fluoresce. This means that non-fluorescent analytes must be converted to a fluorescent derivative or be tagged with a fluorescent compound, e.g., fluorescein or rhodamine.

Sanger Sequencing Using Laser-Induced Fluorescence (LIF)

• DNA polymerase copies the DNA from one end to the other.
• Add chain-stopping bases that have a fluorophore attached (a different color is used for each base A, T, C, G).

Capillary Isoelectric Focusing (CIEF)

• A pH gradient is established along the length of the capillary, allowing zwitterions to travel along until they reach their isoelectric point.
• The capillary walls are pre-treated (coated) so that charges do not develop on it, and electroosmosis is minimized.
• Apply pressure to push the analytes past the detector, or the whole capillary can be scanned by the detector.

Capillary Sieving Electrophoresis (CSE)

• An alternative to SDS-PAGE, using a non-viscous cross-linked polymer inside the capillary.
• The polymers ‘entangle’ the analytes, modifying their migration times according to their molecular weights.
• The polymers are not attached so are continuously replaced as the separation proceeds.

Micellar Electrokinetic Chromatography (MEKC)

• Uncharged analytes are incorporated into micelles through a partitioning equilibrium during electrophoresis and be carried towards the anode as part of the micelle.
• Neutral analytes are prevented from following electroosmotic flow to the extent by which they partition into the micelles.

Affinity Capillary Electrophoresis (ACE)

• Better targeted version of SDS or sieving protocols.
• Additives are introduced into the buffer, which react specifically with the analytes of interest.

Summary of Main Electrophoresis Techniques