Sample Clean Up in Analytical Chemistry

Overview

• Sample clean up is a necessary step in analytical chemistry.
• Common methods used in sample clean up include liquid-liquid extraction and solid phase extraction.
• Internal standards are employed to correct for losses during preparation and variations in instrument response.
• Accuracy and precision are evaluated using quality control standards.

The Need for Sample Pre-Treatment and Clean Up

• Biological samples often contain components that can damage analytical equipment.
• These components can interfere with separation, quantitation, and identification of target compounds
• Not all analytical methods involve separation in the liquid phase.
• Gas chromatography requires separation in the gaseous phase.
• Aqueous solvents have high boiling points, which can be problematic.
• Some techniques necessitate non-aqueous solvents.
• Proteins can precipitate, leading to blockages in the system

Gas Chromatography (GC)

• GC was developed as a usable technology by AJP Martin and AT James from 1949 to 1952.
• GC is a procedure used to separate, identify, and measure volatile compounds that remain stable at temperatures up to 300°C.
• The mobile phase in GC is an inert gas, usually helium.
• The stationary phase can be either a liquid (gas-liquid chromatography) or a solid.
• Solid stationary phases are commonly made of high molecular weight polymers, such as polymethyl siloxane.
• Separation in GC relies on the principle of differential affinity between the stationary and mobile phases.
• The injector and the detector are at high temperatures, up to 400°C.
• Proteins can burn and coat the injector and detector with residue at these temperatures.
• The injector uses a microsyringe that can be blocked by proteins.

High Performance Liquid Chromatography (HPLC)

• HPLC is a technique used to separate components in a mixture based on their differential affinity for two phases.
• These phases include a liquid mobile phase.
• The phases also include a solid stationary phase
• HPLC stationary phases consist of small particles (3-5 μm diameter) of silica or other inert materials coated with a reactive surface
• uHPLC (or UPLC) utilizes even smaller particle sizes (1.7 μm diameter).
• Smaller particles increase column efficiency but also increase back pressure.
• The mobile phase is pumped through the column at high pressures.
• Proteins can block the chromatographic column or interfere with separation.
• Organic solvents used in the mobile phase can cause proteins to precipitate
• Proteins can block the injector loop or needle.
• Materials such as proteins and salts can interfere with detection.

Sample Pre-Treatment and Clean Up

• Biological samples contain a compound of interest along with other substances that can interfere with analysis.
• Sample pre-treatment involves extracting the target compound first to "clean up" the sample prior to analysis.
• This prevents damage to equipment and interference with separation, detection, and identification.
• The extraction process needs to be selective and complete to aid in the analysis of trace compounds.
• Biological samples are generally extracted using either liquid or solid-phase extraction.

HPLC and GC Resources

• The Royal Society of Chemistry offers instructional videos on HPLC and GC.
• These resources explain the necessity for pre-treatment and clean up.

Liquid Phase Extraction

• Liquid phase extraction selectively extracts sample components into an organic liquid solvent.
• The organic phase is collected.
• The organic phased is either injected directly into HPLC/GC or evaporated to dryness under a stream of nitrogen or via rotary evaporator.
• The residue remaining after evaporation is dissolved in a solvent of high purity (HPLC grade) that does not interfere with detection or in the mobile phase chosen for HPLC separation.

Maximizing Extraction

• Polarity or pKa determines extraction in the case of ionic acids or bases.
• The polarity of the solvent should be similar to that of the solute

Importance of Sample pH

• For extraction of ionisable compounds into a non-polar or moderately polar liquid phase:
• Decrease the samples pH for acidic compounds
• Increase the samples pH for basic compounds
• Creates a generation of a unionised form of the compound of interest enabling traverse into the organic phase
• Adjust the pH so the compound is fully ionized for extraction into methanol or an acetonitrile-water mixture.

Liquid Phase Extraction Process

• Liquid extraction into an immiscible solvent is performed by vigorous shaking for 5-10 minutes, followed by centrifugation at 2,500 g.
• Following the process produces two phases.
• The organic liquid phase can then be directly injected into HPLC;
• The organic liquid phase can also be collected/decanted and evaporated either under a stream of nitrogen or under vacuum in a rotary-evaporator.
• The evaporation process speeds up with increased surrounding temperature given the compound is thermally stable.

Protein Denaturation

• This liquid extraction is useful for trace analysis of compounds by reducing the final volume.
• For water-soluble compounds in biological fluids where sample concentration is unnecessary.
• Protein denaturation is sufficient.
• Add an acid, i.e perchloric/hydrochloric acid. or a base i.e. potassium/sodium hydroxide to denature the protein.
• Centrifuge the sample, then inject the clear supernatant into HPLC.

Liquid-Liquid Extraction Example

• Saliva samples are mixed with phosphate buffer (pH 2)
• Toluene ( 7 ml) is added.
• The mix is centrifuged for 30 minutes until phases separate.
• Transfer 5 ml of the toluene into mixture into a fresh tube.
• Add 100 μL of TMAH (very strong alkali).
• Next, mix and centrifuge again.
• Excess toluene aspirated and 60 μL of lower aqueous phase is collected and injected into GC.

Phenytoin Saliva Extraction

• Extraction is necessary because saliva cannot be directly injected onto the GC.
• Lowering the saliva to pH 2 with a buffer enables efficient phenytoin extraction into toluene.
• Phenytoin exhibits a specific ionisation state at the physiological pH of saliva (typically pH 7.4).
• A large volume of toluene is added, and a second back extraction step with TMAH is needed.

Solid Phase Extraction

• Solid phase extraction uses disposable cartridges which contain chemically modified silica sorbent.
• The silanol groups are bonded with various compounds.
• Packing materials come in highly non-polar to polar forms, as well as has weak and strong ion-exchange properties.
• Size exclusion packing materials are available.
• Sorbents are in high capacity particles (40-50 µm diameter) which allows for highly selective isolations

Solid Phase Extraction Process

• A sample is passed through the cartridge, followed by applying either a vacuum, positive pressure or centrifugation.
• To prepare apply a spiked sample.
• The sample is then wasted, and a wash solution is applied.
• Elution solvent is applied and analyzed.

Solid Phase Extraction Guidelines

• Choice of solid phase cartridge packing depends on molecular structure and type of functional groups present in the compound.
• (ionisable, polar, non-polar)
• Type of contaminants also determine choice in packing to remove before HPLC/GC analysis.
• Large molecule of proteins that is removed.
• Small molecules that interfere with detection/identification that is also removed.
• As well as salts that cause suppression of ionisation in mass spec.
• Normal or reverse phase SPE which removes contaminating peaks or high background.
• It also uses ion exchange to remove salts.
• And size exclusion removes particulates or large proteins that cause blockage.

Efficiency of Extraction Process

• Extraction process/whichever form should always be selective.
• Efficiency and reproducibility of internal standard (IS) should also be evaluated.
• Comparison with direct injection of pure standard solutions can check results.
• Reproducibility of an internal standard (IS) should be evaluated.

Quantification

• A standard curve is needed, showing peak area from detector for different concentrations of compound of interest.
• Poor correlation and poor reproducibility affects quantification.

Poor Reproducibility

• Differences in injection volume.
• Very difficult to inject small volumes reproducibly.
• Differences in extraction efficiency.
• Biological matrices cannot be directly injected so use liquid-liquid extraction.
• Solid Phase extraction/solid phase microextraction is a better approach
• Headspace analysis - often performed for GC samples

Internal Standards

• Internal standards (IS) have similar physico-chemical properties to the compound to be extracted.
• IS are normally analogues or are still separable from compound of interest with your separation technique.
• A known concentration of IS is added to sample before extraction.
• Variability in extraction and/or equipment response from sample to sample causes variation in peak area or peak height
• Variability in extraction and equipment response also applies to the IS.
• The internal standard (IS) eliminates such variability.
• The internal standard calculation is achieved in practice by calculating the IS ratio
• • IS ratio = compound peak area/IS peak area
• • OR IS ratio = compound peak height/ IS peak height
• IS ratio is not affected by differences in extraction or equipment response

Standard Curve Equation

• To produce a standard curve using internal standards, a ratio can be formed.
• Internal standard ratio = compound peak area/IS peak area Plot Concentration versus IS ratio and determine equation for line To measure unknown samples just re-arrange equation Rearrange equation x = (y-c)/m or Concentration = (IS ratio-c)/m

Ideal Internal Standard

• Deuterated analogues can be used when using a mass spectrometer as a detector.
• Some of the hydrogens in the compound are replaced with deuterium atom.
• Mass spectrometer recognizes ''normal'' compound of interest as well as deuterated version.
• Deuterated version has slightly higher molecular mass.
• Both versions act the same.
• The ratio is calculated by dividing the normal peak area by the area of the deuterated area.
• Many deuterated analogues are commercially available.

Determination of Precision and Accuracy

• Quality controls (QC) is used to perform the assessment
• Samples should containing known concentrations of the compound, usually at 25%, 50%, and 75% of the highest standard concentration
• QCs should be treated like unknown sample.
• Accuracy checks how close calculated concentration in comparison to true value.
• Accuracy is often inaccurate despite the use of a linear standard curve.
• Quality measurement is checked using Agreement between measurements from several runs presented as coefficient of variation (CV).
• (CV = SD expressed as % of the mean for the number of determinations)
• QC concentrations measured are close to the expected concentrations
• QC values will be lower/higher than expected if there is a pipetting error due to using standard curve made with higher/lower volume.

Validating Measurements

• Precision: The Coefficient of Variation (SD expressed as a percentage) should be within 5% for QC samples.
• Accuracy: Do six replicates for each of the three QC levels, at least 4 need to be within 10% of the expected value.
• An acceptable R2 for the standard curve should be >0.999.

Summary

• Sample clean up is essential for every analytical procedure, especially if the analysis needs to be done effectively.
• The clean up type depends on the technique used.
• most techniques require removal of biological macromolecules such as proteins.
• Solid phase extraction and SPME are very popular techniques thanks to the high throughput.
• Internal standards are a must to correct for variations in instrument response and extraction.
• Quality control standards are essential to determine accuracy and precision of standard curves.