PHAR2202 Drug Design Analytical Methods Separations Notes

Separations: Liquid-Liquid Extractions and Liquid Chromatography

Liquid-Liquid Extractions

• Separation is key to analysis and purification.

• Applies to proteins, RNA/DNA, metabolites, drugs, cells, and antibodies.

Principles

• The analyte/sample is in equilibrium between two phases.

• Typically, the most polar solvent (often water) is designated as Phase 1.

• A larger distribution constant (KD) indicates lower water solubility and more efficient extraction into the upper, organic phase.

• $KD = \frac{[sample]<em>{Phase 2}}{[sample]</em>{Phase 1}}$

  • KD is the partition coefficient or distribution constant.

  • Phase 2 is typically the less polar solvent (e.g., ethyl acetate).

Single Step Extractions

• Example: A sample is more soluble in ethyl acetate than water, with $KD = 3$.

• Question: If 100 mL of 0.010 M sample in water is extracted with 500 mL ethyl acetate, what fraction of the sample remains in the water?

• It is more efficient to use multiple extractions with smaller volumes.

• $f<em>{Phase 1,1} = \frac{1}{1 + K</em>D (V<em>2/V</em>1)}$

  • $f_{Phase 1,1}$ = fraction of analyte in Phase 1 after one extraction.

  • $V_n$ = volume of phase n used in the extraction.

Multiple Extractions

• The extent of solute extraction increases with the number of extractions.

• $f<em>{Phase 1,n} = [\frac{1}{1 + K</em>D (V<em>2/V</em>1)}]^n$

pH-Dependent Extractions

• For a weak monoprotic acid (HA) with $K<em>a = 1.20 \times 10^{-3}$ and $K</em>D(HA) = 550$ (ethyl acetate/water), and $K_D(A^-) ≈ 0$, the overall partition coefficient depends on pH.

• Equilibrium: $HA \rightleftharpoons H^+ + A^-$

• $K<em>D = \frac{[tot. analyte]</em>{Phase 2}}{[tot. analyte]<em>{Phase 1}} = \frac{[HA]</em>{Phase 2}}{[HA]<em>{Phase 1} + [A^-]</em>{Phase 1}}$

• $K<em>D = \frac{[HA]</em>{ethyl acetate}}{[HA]<em>{water} + [A^-]</em>{water}} = \frac{[HA]<em>{ethyl acetate} / [HA]</em>{water}}{1 + [A^-]<em>{water} / [HA]</em>{water}} = \frac{K<em>{D,HA}}{1 + K</em>a / [H^+]_{water}}$

• $K_a = \frac{[A^-][H^+]}{[HA]}$

Concept Check

• How much of a 100 mL of 0.01M sample remains in the water after one extraction with 200 mL ethyl acetate at pH = 5 and pH = 1? After 5 extractions?

• Use the formula: $f<em>{Phase 1,n} = [\frac{1}{1 + K</em>D (V<em>2/V</em>1)}]^n$

Chromatography

• Chromatography is like a series of extraction steps.

• If you could perform continuous extractions, you could separate compounds with much smaller KDs.

Components

• Mobile phase

• Column (containing the stationary phase)

• Stationary phase

• Fraction collector and/or detector

• Chromatogram

Describing a Chromatogram

• Retention time ($t_R$): The time it takes for the analyte to pass from the injection point to the detector.

• Void time ($t_M$): The time it takes for an unimpeded molecule (e.g., the solvent) to pass from the injection point to the detector.

• Adjusted retention time ($t<em>R'$): $t</em>R' = t<em>R - t</em>M$

• Retention factor ($k$): $k = \frac{t<em>R'}{t</em>M}$. This is unitless and unaffected by flow rate or column dimensions.

  • A high k value indicates the compound spends significant time in contact with the stationary phase.

  • Ideally, $k = 1$ to 5.

Resolution

• High degree of separation between compounds (difference in $t_R'$).

• The distribution of an analyte between the stationary and mobile phases can be described as an equilibrium.

• $A<em>M \rightleftharpoons A</em>S$

  • $A_M$ = mol analyte in the mobile phase.

  • $A_S$ = mol analyte adsorbed on the stationary phase.

• $k = \frac{A<em>M}{A</em>S} = \frac{[A]<em>M V</em>M}{[A]<em>S V</em>S}$

  • $V_S$ = volume of stationary phase.

  • $V_M$ = volume of mobile phase.

• $k = K<em>D (V</em>S / V_M)$

Peak Broadening and Resolution

• Peak width is important for good resolution.

• Peaks should ideally be Gaussian in shape.

• $\sigma$ = standard deviation

• $w_b$ = width at base = $4\sigma$

• $w_h$ = width at half height = $2.355\sigma$

Theoretical Plates

• 'Plate height' (H) and 'number of theoretical plates' (N) measure column efficiency.

• The concept comes from early chromatographic and distillation theory.

• Each plate is thought of a separating funnel containing two immiscible layers in contact with one another.

Calculating Theoretical Plates

• $N = (\frac{t_R}{\sigma})^2$

HETP

• The number of theoretical plates ‘N’ increases with column length, so you need a measure that normalises for column length

• Height Equivalent to a Theoretical Plate (HETP): $H = \frac{L}{N}$

Factors Affecting Peak Broadening

• Eddy diffusion

• Longitudinal/molecular diffusion

• Resistance to mass transfer

Types of Diffusion

• Eddy Diffusion (A): Different molecules take different paths through the column.

  • Affected by: size and shape of particles.

  • Unaffected by: column length, flow rate.

• Molecular (Longitudinal) Diffusion (B): Molecules in the center move faster.

  • Affected by: flow rate.

  • Unaffected by: column length.

• Resistance to Mass Transfer (C): Analyte takes time to equilibrate between stationary and mobile phases.

  • At high mobile phase velocity, analyte in the mobile phase moves ahead of analyte in the stationary phase.

Van Deemter Plot

• $HETP = A + \frac{B}{u} + Cu$

  • u = linear flow velocity

• HETP varies with flow rate.

Improving Resolution

• Basic resolution equation:

  • $k = \frac{t<em>R - t</em>M}{t_M}$

  • $\alpha = \frac{t'<em>{R1}}{t'</em>{R2}}$

  • $N = (\frac{t_R}{\sigma})^2$

• $R = \frac{\sqrt{N}}{4} \times (\frac{\alpha - 1}{\alpha}) \times (\frac{k}{k + 1})$

  • N = number of theoretical plates

  • $\alpha$ = separation factor (i.e., $t'<em>{R1} / t'</em>{R2}$)

  • k = retention factor for the second peak

Liquid Chromatography (LC)

Types of LC

• Thin Layer Chromatography (TLC)

• Preparative Layer Chromatography (PLC)

• Centrifugal Chromatography

• High-Pressure Liquid Chromatography (HPLC)

HPLC Automation

• Automated/Combi-flash

General Principles of LC

• Column (analytical or preparative scale)

• Mobile phase (= eluent) – can be isocratic or gradient.

• Stationary phase (separates based on polarity, size, affinity, ionic strength, etc.)

• Fractions & detectors

Stationary Phases – Particle Beds

• Microporous particles (small pores, slow diffusion, band-broadening)

• Perfusion particles (large channels, increased access to smaller pores, reduced diffusion path lengths).

• Pellicular particles (solid core, thin coat of stationary phase, reduced diffusion path length).

HPLC Setup

• Solvent inlet with filter

• Pump

• Injection valve

• Precolumn filter

• Column.

• Detector

• Backpressure regulator

• Waste reservoir (or fraction collector)

• Recorder/Monitor

Stationary Phases - Monoliths

• Pore geometry similar to perfusion particles but with long rods instead of spheres.

• Pores have multiple entry/exit points, reducing analyte residence times and band-broadening.

Separations Based on Polarity

• Normal Phase: Polar stationary phase, non-polar eluent. Good for less polar organics.

• Reverse Phase: Non-polar stationary phase, polar eluent. Good for polar organics.

Chromatography Types

• Adsorption (Analyte sticks to the solid support)

  • Most common normal phase is silica/alumina gel.

• Partition (Analyte partitions into the liquid layer that sticks to the solid support)

  • Most common reverse phase is C-18 silica gel.

Elution Order

• Eluents matching the stationary phase = faster elution.

  • Normal Phase: Polar Ethyl acetate, Non-polar Hexane

  • Reverse Phase: Polar Water, Non-polar Methanol

Normal Phase Silica Gel Chromatography

Stationary Phase

• Silica-gel (SiO2) is a very polar stationary phase.

• Types of silica vary by pore size (Å) and particle size (µm), influencing HETP and flow rate.

Silica vs Alumina

• Alumina is generally less polar than silica-gel and comes in several pH-adjusted forms.

• Eluotropic strength (e°) indicates solvent polarity.

  • Solvents with higher eluotropic strength elute faster.

Normal Phase Eluents

• Common systems: Hexane/EtOAc, DCM/MeOH

• Increasing eluent polarity = faster movement of analyte.

• Solvent strength must be chosen carefully.

Reverse Phase C-18 Silica

Stationary Phase

• Modified silica gel (non-polar).

• Smaller particle sizes are needed for resolution, typically done by HPLC.

• Eluent requirements: filtered, degassed, and not too viscous.

Applications of Reverse Phase HPLC

• Wide range of polar molecules.

  • Biochemistry: amino acids, proteins, carbohydrates, lipids.

  • Clinical: drugs, drug metabolites, bile acids, amino acids.

  • Environmental: pesticides, herbicides, phenol, PCBs.

  • Food: sweeteners, antioxidants, aflatoxins, additives/preservatives.

  • Forensic: drugs, poisons, alcohol, narcotics.

  • Industrial: PAHs, dyes, propellants, surfactants, plasticisers.

  • Pharmaceutical: antibiotics, sedatives, steroids, analgesics.

Predicting Eluent Strength

• MeOH/Water or Acetonitrile/Water systems are typically used as gradients.

• The eluent polarity can be estimated by the polarity index P’.

• $\log<em>{10} (\frac{k</em>2}{k<em>1}) = 0.5(P’</em>2 - P’_1)$

• $P’<em>{XY} = f</em>X P’<em>X + f</em>Y P’_Y$ (f = vol. fraction)

  • Cyclohexane 0.04

  • THF 4.0

  • MeOH 5.1

  • MeCN 5.8

  • H2O 10.2

Concept Check: Eluent Strength

1. Determine whether normal phase or reverse phase is better for separating two sets of compounds.

2. Determine the effect of increasing the polarity of the eluent in normal phase chromatography.

3. Identify the more polar eluent in each case:

   • water / methanol

   • hexane / ethyl acetate

   • dichloromethane / methanol

• Explain what reverse phase liquid chromatography is.

  Reverse phase liquid chromatography uses a non-polar stationary phase and a polar eluent. It is effective for separating polar organic compounds.

• Explain that determine the elution order in the chromatography of a mixture of compounds

In chromatography, the elution order of a mixture of compounds depends on their affinity for the stationary and mobile phases. Compounds with lower affinity for the stationary phase and higher affinity for the mobile phase elute faster. In reverse phase chromatography, more polar compounds elute faster, while in normal phase chromatography, less polar compounds elute faster.

• Be able to calculate key chromatography parameters: retention factor, resolution, selectivity, plate height

• Identify how chromatographic parameters affect peak resolution