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 non-polar solvents like hexane.

  • Reverse Phase:

    • Non-polar stationary phase

    • Polar eluent

    • Good for polar organics

    • Faster elution with polar solvents like water.

  • Eluents matching the stationary phase lead to faster elution.

Chirality

  • Priority Determination:

    • Higher atomic number = higher priority.

  • R/S Configuration:

    • Lowest priority at the back.

    • Determine if 1 to 3 goes clockwise (R) or anticlockwise (S).

Separating Enantiomers

  • Pasteur's Separation:

    • Classic example: separation of tartaric acid enantiomers upon crystallization.

    • Not all compounds separate as easily.
      *Enantiomers & Diastereomers

    • Enantiomers (one chiral carbon atom) have identical vapor pressures and cannot be separated on an achiral stationary phase.
      *Diastereomers, however, are usually easy to separate on achiral columns as they have different vapour pressures.

Chiral Derivatisation (Option 1)

  • Derivatisation by a pure, chiral enantiomer ((+) B) to form diastereomers

    • (+)A + (+)B ----> (+)(+) A-B

    • (-)A + (+)B ----> (-)(+) A-B

  • Requirements:

    • The derivatizing agent must be chiral.

    • The reagent must be optically pure (just one enantiomer).

  • Drawbacks:

    • Systematic errors due to differences in reaction kinetics of enantiomers.

    • Racemization may occur at a chiral carbon during derivatisation.

Chiral Stationary Phase (Option 2)

  • Allows the use of any common derivatising agent.

  • 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 Chiral Group:

    • (S)-valine linked by urea to 3,5-dinitrophenyl group.

  • Chirasil-Val works from 70-240°C.

Problems with Chiral Columns

  • Temperature Limitations:

    • Maximum operating temperatures are often low due to their vapor pressures or tendency to racemise and lose specificity at higher temperatures.

    • Leads to:

      • Long analysis times

      • Low sensitivity

      • Inability to elute some materials

Gas Chromatography

  • Components:

    • Carrier gas tank

    • Flow regulators

    • Sample injection chamber

    • Column

    • Oven

    • Thermostat

    • Detector

    • Flow meter

    • Data system

Gas Chromatography Details

  • Carrier Gas:

    • Normally oxygen and water-free (dry), high purity.

    • Examples: He, Ar, N<em>2N<em>2 or H</em>2H</em>2.

  • Flow Regulators:

    • Control the flow rate of the ‘eluent’ (mobile phase, i.e., gas) down the chromatography column.

  • Injection Port:

    • Typically 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

  • Sample is heated at injection and while running on the column

  • Analyte is usually a volatile organic compound

  • Relatively non-polar, higher molecular weight compounds such as steroids (MW ~400) can be analysed by derivatising compounds before commencing GC to make them more volatile.

Analyte Requirements for GC

  • 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:

    • Thermal stability under the conditions required to obtain a useful chromatogram.

  • Derivatization:

    • Analytes can be derivatized (existing functionality is modified by a chemical reaction which adds a standard substituent) in order to:

      • Increase their volatility.

      • Improve their thermal stability (column temperatures up to 250°C are not uncommon).

Derivatizing Agents

  • Examples:

    • Many derivatizing agents are commercially available, but the 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 on the column and are separated purely on the basis of their boiling point.

# C

BP (°C)

10

174

16

287

20

343

24

391

28

432

32

468

36

498

40

525

Retention Time Factors Continued

  • 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 (top)

    • Clearly, the SolGel-WAX polar column is the column of choice for this separation (Polyethylene glycol)

  • Column length

Column Length and Band Broadening

  • The low resistance of the mobile phase (a gas!) means that narrow (capillary) columns, which decreases band-broadening, up to 100 m long can be used.

Van Deemter Plot

  • HETP=A+B/u+CuHETP = A + B/u + Cu
    Where:

  • A = Eddy diffusion

  • B = Molecular (Longitudinal) Diffusion

  • C = Resistance to mass transfer

Kováts Retention Index (I)

  • Actual retention times (tRt_R) are quite sensitive to the column temperature.

  • The Kováts retention index is an attempt to remove this by comparing the analyte’s retention time (t’Rt’_R) to the retention time of standard compounds with similar retention times when run under identical chromatographic conditions.

  • The series of n-alkanes are used as the standards.

  • Formula: I=100n+100×logt’Rx–logt’Rnlogt’R(n+1)–logt’RnI = 100n + 100 × \frac{log t’ Rx – log t’ Rn}{log t’ R(n + 1) – log t’ Rn}

    • t’Rxt’Rx is the adjusted retention time for the analyte (x)

    • t’Rnt’Rn is the adjusted retention time for the n-alkane eluting just before the analyte

General Elution Problem

  • If a sample contains many different analytes whose volatilities and polarities are spread over a wide range:

    • volatile compounds appear all bunched up at the beginning

    • involatile compounds give broad peaks which take too long to elute..

  • Solution:

    • Using a temperature gradient.

      • Start cooler, in order 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.

GC Columns

  • GC is usually done with very thin capillary / open tubular columns

  • The stationary phase can be placed within an open-tubular column in different ways

    • Ø = 0.1–0.75 mm
      Stationary phase thickness = 0.1–0.5 µm

Stationary Phase & Columns Types

  • Wall-coated open tubular column (WCOT): efficient, but have a 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

  • Many ‘liquid’ stationary phases are based on a polysiloxane polymer.

  • One problem: even the least volatile liquid phases will still slowly vaporise or degrade; referred to as ‘column bleed’.

  • If one needs to work at particularly high temperature, stationary phases which have been chemically stabilised by being bonded to the support or that have been cross-linked, one polymer chain to the next, can be used. MW 1,000s to > 1,000,000.

Relative Polarity of Stationary Phases

Stationary Phase Relative polarity R1 – R4

  • 100% dimethylpolysiloxane 16 (non-polar)

  • 5% phenyl–95% methylpolysiloxane 33

  • 14% cyanopropylphenyl–86% methylpolysiloxane 67

  • 50% phenyl–50% methylpolysiloxane 119

  • 50% trifluoropropyl–50% methylpolysiloxane 146

  • 50% cyanopropylphenyl–50% phenylmethylpolysiloxane 228

  • polyethylene glycol 322 (polar)

  • R1–R4 = CH3 R1,R2 = phenyl, R3,R4 = CH3 etc.

Detectors

Detector name Compounds detected Detection limits

  • General detectors

  • Thermal conductivity detector (TCD) Universal—all compounds 10–9 g

  • Flame ionisation detector (FID) All organic compounds 10–12 g carbon

  • Selective detectors

  • Nitrogen-phosphorus detector (NPD) N and P containing compounds 10–14–10–13 g N, P

  • Electron capture detector (ECD) Compounds with electronegative gps 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

  • #SIM = Selective Ion Monitoring

  • the detection limit is roughly the minimum amount of analyte that must pass through the detection measurement point in 1 second

Sample Injection - Gases

  • 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.

Sample Injection - Liquids

  • 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

  • Syringe barrel

  • Septum piercing needle

  • Fiber attachment tubing

  • Fused silica fiber with stationary liquid-phase coating

  • Pierce sample septum with metal needle

  • Retract fiber and withdraw needle

  • Pierce chromatography septum with metal needle

  • Retract fiber and withdraw needle

  • Expose fiber to solution or headspace for fixed time with stirring

  • Expose hot fiber to carrier gas for fixed time

    • (column is cold)

Sample Injection - Solids

  • Extract volatile substances from the solid matrix using a solvent

  • Burn it!!! (Pyrolysis)

    • Volatiles (eg plasticisers) 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

GC vs LC Benefits

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)

Measuring Concentrations of Unknowns

  • We want to measure the concentration of estradiol in blood (MW = 272 g/mol)

  • We 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

Internal Standard Method

  • We could do the same with an internal standard

  • Standard solution with known concentrations

  • Take unknown blood (100 µL) + 900 µL of IS and inject:
    Estradiol IS

  • Conc (pg/mL) 5.6 27.5

  • Area 204 1567
    Estradiol IS

  • Area 345 612

  • [] = ([]) Where F is a constant (response factor)

Internal Standard Calculation

  • Take unknown blood (100 µL) + 900 µL of IS (at 4.62 mg/ml) and inject:

  • What is this number?
    1) Calculate [IS] in the sample.

  • = 0.90 * 4.62 / (0.90 + 0.10) pg/mL = 4.158 pg/mL
    Volume x Conc of IS / Total vol of sample
    2) Calculate F

  • F = (204 / 5.6) / (1567 / 27.5) = 0.639
    3) Use F and [IS] in the sample to get [analyte]

  • = 345 / (0.639 * (612 / 4.158) = 3.66 pg/mL
    4) If sample was diluted then adjust [Analyte in blood] = 10 * 3.66 pg/mL = 36.6 pg/mL

Standard Addition

  • Sometimes matrix effects or logistics make it more appropriate to do a standard addition

  • The simplest way is to dilute a constant volume of unknown sample (blood) to a constant volume with a varying amount of estradiol
    Additional estradione (ng/ml) Peak height
    ?? 0
    0 0.61
    0.93 0.79
    1.87 1.02
    2.80 1.17
    3.74 1.42
    4.67 1.58
    5.61 1.8

Three Ways to Measure Unknown Concentrations

  • Standard curve

  • Internal standard

    • [] = ([])

  • Standard addition -[A] [A]

  • Distinguish between gas and liquid chromatography

Benefits of GC:

Low viscosity: Very long thin columns can be used, which would be impossible with LC because of the high pressure that would be needed. This is good for resolution!! (low C term, high N due to length)
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)

Gas chromatography involves using a carrier gas (like He, Ar, N2N2, or H2H2) to move volatile analytes through a column within an oven. The sample is heated upon injection, and analytes are separated based on their boiling points and interactions with the stationary phase. Detectors such as thermal conductivity, flame ionization, or mass spectrometry are used to identify and quantify the separated compounds.

Liquid chromatography is a separation technique where the mobile phase is a liquid, used to carry the sample through a column containing a stationary phase. Components of the sample separate

  • Use an internal standard in chromatography to calculate response factor and/or analyte concentration.

Internal Standard Method

  • We could do the same with an internal standard

  • Standard solution with known concentrations

  • Take unknown blood (100 µL) + 900 µL of IS and inject:

    Estradiol IS

  • Conc (pg/mL) 5.6 27.5

  • Area 204 1567

    Estradiol IS

  • Area 345 612

  • [] = ([]) Where F is a constant (response factor)

Internal Standard Calculation

  • Take unknown blood (100 µL) + 900 µL of IS (at 4.62 mg/ml) and inject:

  • What is this number?

    1) Calculate [IS] in the sample.

  • = 0.90 * 4.62 / (0.90 + 0.10) pg/mL = 4.158 pg/mL

    Volume x Conc of IS / Total vol of sample

    2) Calculate F

  • F = (204 / 5.6) / (1567 / 27.5) = 0.639

    3) Use F and [IS] in the sample to get [analyte]

  • = 345 / (0.639 * (612 / 4.158) = 3.66 pg/mL

    4) If sample was diluted then adjust [Analyte in blood] = 10 * 3.66 pg/mL = 36.6 pg/mL