PHAR2202 Drug Design - Analytical Methods: Separations

Liquid Chromatography Reminder

Normal Phase:
- Polar stationary phase
- Non-polar eluent
- Good for less polar organics
- Faster elution with polar eluent (e.g., Ethyl acetate)
Reverse Phase:
- Non-polar stationary phase
- Polar eluent
- Good for polar organics
- Faster elution with non-polar eluent (e.g., Methanol)

Concept Check: Chirality

Priority Determination: Higher atomic number = higher priority.
Configuration Assignment:
- Place the lowest priority group at the back.
- Determine if the sequence from priority 1 to 3 goes clockwise (R) or anticlockwise (S).

Separating Enantiomers

Pasteur's Separation: Tartaric acid enantiomers separated upon crystallization (~1840’s).
Enantiomers and Achiral Stationary Phases: Enantiomers (one chiral carbon atom) have identical vapor pressures and cannot be separated on an achiral stationary phase.
Diastereomers: Usually easy to separate on achiral columns due to different vapor pressures.

Option 1: Chiral Derivatisation

Derivatisation Process:
- Use a pure, chiral enantiomer ((+) B) to form diastereomers.
- (+)A + (+)B ----> (+)(+) A-B
- (-)A + (+)B ----> (-)(+) A-B
Requirements for Derivatising Agent:
- Must be chiral to produce a diastereomer.
- Must be optically pure (just one enantiomer).
Drawbacks:
- Systematic errors due to differences in reaction kinetics of the two enantiomers.
- Racemization may occur at one of the chiral carbons during derivatisation.

Option 2: Chiral Stationary Phase

Advantages:
- Allows the use of any common derivatising agent.
- Significant difference in hydrogen-bonding interactions between each enantiomer and the chiral stationary phase.
Types of Stationary Phases:
- Dipeptide phases
- Derivatised amino acid phases
- Polymeric chiral phases
Example: (S)-valine linked by urea to 3,5-dinitrophenyl group.
Chirasil-Val: Effective stationary phase, works from 70-240°C.

Problems with Chiral Columns

Temperature Limitations:
- Maximum operating temperatures are often low due to vapor pressures or tendency to racemize and lose specificity at higher temperatures.
Consequences:
- Long analysis times
- Low sensitivity
- Inability to elute some materials

Separations: Gas Chromatography

Components: Sample, carrier gas tank, flow regulators, sample injection chamber, column, oven, thermostat, detector, flow meter, display, data system.

Gas Chromatography Details

Carrier Gas:
- Typically oxygen and water-free (dry), high purity, He, Ar, N2, or H2.
Flow Regulators:
- Control the flow rate of the 'eluent' (mobile phase, i.e., gas) down the chromatography column.
Injection Port:
- Connected to an autoinjector or a manual syringe injection port.
General Detectors:
- Thermal conductivity
- Flame ionization
Selective Detectors:
- Nitrogen-phosphorus
- Electron capture
Structure Specific Detectors:
- Mass spectrometry
Process: Sample is heated at injection and while running on the column.
- Analyte is usually a volatile organic compound.
- Non-polar, higher molecular weight compounds (e.g., steroids, MW ~400) can be analyzed by derivatizing compounds before GC to make them more volatile.

Analyte Requirements

Volatility: Must have high enough volatility to be vaporized by the time it has passed the injector and is about to enter the column.
- Typically MW ≤ 600 amu, boiling point < 500 °C.
Thermal Stability: Must be thermally stable under the conditions required to obtain a useful chromatogram.
Derivatization: Analytes can be derivatized to:
- Increase their volatility.
- Improve their thermal stability (column temperatures up to 250°C are not uncommon).

Derivatizing Agents

Examples: Many commercially available, majority form TMS ethers from alcohols or methyl esters from carboxylic acids.
Requirements:
- Must react quickly with all available functionalities and in 100% yield.
- Derivatized samples must be able to be injected onto the GC immediately – without any further work-up.

Factors Affecting Retention Time

Analyte Boiling Point:
- Since alkanes are completely non-polar, they interact very little with the stationary phase and are separated purely on the basis of their boiling point.
- Example:
  - 10 Carbons: BP 174 °C
  - 16 Carbons: BP 287 °C
  - 20 Carbons: BP 343 °C
Analyte boiling point: (↑ BP = ↑ retention time)
Temperature: (↑ column temperature = ↓ retention time)
Stationary phase (polarity)
- Some analytes have notably different retention times in the two stationary phases, while others do not
  - m-Xylene (5) co-elutes with p-xylene (4) on the non-polar column while they are well resolved on the polar column
Column Length:
- Low resistance of the mobile phase (gas) allows narrow (capillary) columns to be used (up to 100 m long), which decreases band-broadening.
Eddy Diffusion: Different molecules can take different paths through the column.
*Actual plot HETP = A + B/ $u$ + C $u$
Van Deemter plot
Molecular (Longitudinal) Diffusion: Molecules in the centre move faster.
Resistance to Mass Transfer

Standardizing Retention Time: The Kováts Retention Index (I)

Actual retention times ( $t_R$ ) are quite sensitive to the column temperature.
The Kováts retention index aims to remove this by comparing the analyte’s retention time ( $t’_R$ ) to the retention time of standard compounds (n-alkanes) with similar retention times when run under identical chromatographic conditions.
Formula: $I = 100n + 100 \,\times \,\frac{log \, t’{Rx} - log \, t’{Rn}}{log \, t’{R(n + 1)} – log \, t’{Rn}}$
- $t’{Rx}$ is the adjusted retention time for the analyte (x)
- $t’{Rn}$ is the adjusted retention time for the n-alkane eluting just before the analyte

Mobile Phase: General Elution Problem

If a sample contains many different analytes whose volatilities and polarities are spread over a wide range:
- Volatile compounds appear bunched up at the beginning.
- Involatile compounds give broad peaks which take too long to elute.
Solution: Temperature Gradient
- Start cooler to resolve the more volatile compounds.
- Heat gradually to obtain the less volatile compounds in a reasonable time, with good peak shape and less broadening.

Stationary Phase & Columns

GC uses very thin capillary/open tubular columns (Ø = 0.1–0.75 mm).
Stationary phase thickness = 0.1–0.5 µm
Wall-Coated Open Tubular Column (WCOT): Efficient but low capacity (low surface area).
Support-Coated Open Tubular Column (SCOT): Less efficient (more band-broadening) but larger capacity due to thicker stationary phase.
Porous-Layer Open Tubular Column (PLOT): Open, porous structure stationary phase (zeolites). Best of both worlds? Gives ability to control pore sizes and chemistries.

Stationary Phases Details

Many 'liquid' stationary phases are based on a polysiloxane polymer (MW 1,000s to > 1,000,000).
Problem: Even the least volatile liquid phases will still slowly vaporize or degrade, referred to as 'column bleed'.
Solution: Stabilize phases by bonding to support or cross-linking, especially for high temperatures.

Detectors

*SIM = Selective Ion Monitoring
Threshold detection limit is roughly the minimum amount of analyte that must pass through the detection measurement point in 1 second.

General Detectors:
- Thermal Conductivity Detector (TCD): Universal—all compounds, detection limit 10–9 g.
- Flame Ionization Detector (FID): All organic compounds, detection limit 10–12 g carbon.
Selective Detectors:
- Nitrogen-Phosphorus Detector (NPD): N and P containing compounds, detection limits 10–14–10–13 g N, P.
- Electron Capture Detector (ECD): Compounds with electronegative groups, detection limits 10–15–10–13 g.
Structure Specific Detectors:
- Mass Spectrometry:
  - Universal—full scan mode, 10–10–10–9 g.
  - Selective—SIM mode#, 10–12–10–11 g.

Sample Injection

Gas Samples:
- Inject gas directly using a gas-tight syringe.
- Pre-adsorb onto a solid phase or into a liquid phase and then heat to release.
- Trap ‘cryogenically’ and release into the column upon warming.
Volatiles Dissolved in a Liquid:
- Purge with a carrier gas and either trap or inject directly.
Liquid Samples:
- Inject everything onto the column in a volatile eluent (risks overloading small columns but allows for trace analysis).
- Split injection (dump most of the sample to waste).

Sample Injection - Extraction from Liquids / Gasses

Sample Injection - Solids

Extract volatile substances from the solid matrix using a solvent.
Burn it!!! (Pyrolysis)
- Volatiles (e.g., plasticizers) come off first.
- After this will come decomposition products.
- This is not strictly chromatography—the time at which an analyte appears on the pyrogram is determined, largely, by the temperature of the sample and has little to do with the retention time on the stationary phase of the column.
- The ‘fingerprint’ produced by pyrolysis gas chromatography can be used to identify solids.

Benefits of GC vs. LC

Benefits of GC
- Low viscosity: so we can use very long thin columns which would be impossible by LC because of the high pressure that would be needed. This is good for resolution!! (low C term, high N due to length)
- Can control retention time with temperature
Benefits of LC
- Can do preparative scale
- Very versatile for analyte – it just needs to dissolve, it doesn’t need to be volatile
- Can control retention time with eluent strength (polarity)

Application: Measuring Concentrations of Unknowns

Dilute 100 µL of blood into 1 mL of buffer and compare to a standard curve of known concentrations of estradiol prepared in pure buffer.
Could do the same with an internal standard.

Measuring Concentrations of Unknowns Using Internal Standard

Formula: $\frac{[ ]1}{[ ]2} = (\frac{[ ]1}{[ ]2})F$

Standard Addition

Simplest way is to dilute a constant volume of unknown sample (blood) to a constant volume with a varying amount of estradiol
Conc of added estradione (ng/ml)
Peak height

Three Ways to Measure the Concentration of an Unknown

$\frac{[ ]1}{[ ]2} = (\frac{[ ]1}{[ ]2})F$
Standard Curve: Signal vs. Concentration
Internal Standard
Standard Addition: Plot and extrapolate to find -[A] on the x-axis.