lecture 3- gas chromatography

liquid chromatography

• Separation: Components are separated in the liquid phase.

• Stationary Phase: Uses a solid stationary phase (e.g., silica particles) in the column.

• Mobile Phase: The analytes are moved through the column by a liquid mobile phase (e.g., solvents like water, methanol).

• Application: Ideal for separating non-volatile or thermally labile compounds (e.g., proteins, pharmaceuticals, and polar substances).

gas chromatography

• Separation: Components are separated after a liquid sample is vaporized into the gas phase.

• Stationary Phase: Uses a solid stationary phase

• Mobile Phase: The analytes are moved through the column by a carrier gas (e.g., helium, nitrogen).

• Application: Suitable for analyzing volatile compounds (e.g., gases, volatile liquids like solvents, and essential oils).

good samples for GC analysis

- volatile substances: can vaporise without decomposing.

- chemically stable analytes: remain stable at high temperatures

- MW below 1250 Da: lower molecular weight ensures easier vaporization

bad samples for GC analysis

• Substances with Low Volatility: Analytes that cannot easily vaporize. They can be made more volatile by derivatization

• Thermally Labile Analytes: Compounds that decompose or break down when exposed to high temperatures, cannot be vaporised

applications for GC

• Analysis of Petroleum Mixtures: separate and identify different hydrocarbons

• Analysis of Solvents and Oils in Homecare Products: detecting and quantifying solvents and oils

• Analysis of Explosives and Gunshot Residues: detect traces of explosives and gunshot residues.

• Routine Drug and Alcohol Testing: detecting drugs and alcohol in biological samples.

GC components

• Carrier Gas (Mobile Phase): moves the sample through the column (commonly helium or nitrogen).

• Injector: Introduces the sample into the system, often heated to vaporise the sample.

• Capillary Column (Stationary Phase): A narrow column coated with a stationary phase (e.g., silica) where separation of components occurs based on volatility and interactions with the stationary phase.

• Detector: Detects and measures the components as they elute from the column, converting them into a readable signal (e.g., flame ionization detector (FID), thermal conductivity detector (TCD).

carrier gases

• Role: Acts as the mobile phase, carrying the sample through the capillary column.

• Requirements: Must be chemically inert to avoid reactions with the sample.

• Common Gases:

  • Helium (most commonly used, good for most applications)

  • Nitrogen (often used in older systems or for detectors like TCD)

  • Hydrogen (used in some detectors like FID, though it requires careful handling due to flammability).

split injection

- only a portion of the injected sample is introduced into the column, while the rest is diverted through the spit valve

- split ratio is the ratio of the total injection volume to the volume that actually goes onto the column

- used when the analyte concentration is high to prevent overloading the column and ensure optimal separation.

splitless injection

- split valve closed and all sample injected onto column

- used for trace analysis or when the analyte concentration is low, ensuring the entire sample is sent onto the column for separation.

on column injection

The sample is injected directly onto the column without passing through the injector port or heated zone

Sample is gradually heated as it moves through the column

used for thermally labile compounds that may decompose if heated

column

• Capillary Columns: Long, narrow columns with a thin layer of stationary phase coating the inside. Provide high resolution and sensitivity.

• Packed Columns: Shorter and wider, filled with solid stationary phase or particles. Used for samples with high concentrations or when high efficiency is not required.

separation principle

- Sample is forced through the column by the carrier gas.

- separation occurs through interaction of each analyte with the stationary phase of the column

- rate of migration based on oven temp and carrier gas pressure

- rate of separation depends on boiling point and degree of interaction with stationary phase due to polarity

• Lower boiling points lead to faster elution since these analytes have higher vapor pressures and are pushed through the column faster.

• Higher boiling points result in slower elution.

• Non polar will interact more with non polar stationary phases, polar analytes interact more with polar stationary phases - leading to longer retention times

factors controlling separation

- increasing inner diameter of capillary gives the analyte more space to spread out, causing more band broadening, decreases peak resolution

- increasing capillary column length increases resolution of separation

- increasing column film thickness traps analytes for longer which increases resolution of peaks

temperature control

- isothermal= GC capillary kept at same temp throughout run

- temperature programming= temp gradually increased over the run

flame ionisation detector

- most common

• Sample is burned in a hydrogen-air flame.

• Organic compounds produce ions and electrons.

• The resulting current (caused by ions) is measured.

• Signal is proportional to amount of ion in the sample.

thermal conductivity detector

• Measures changes in the thermal conductivity of the gas stream.

• Analytes passing through disrupt the heat flow from a heated filament.

• The change in filament temperature alters its resistance, which generates a signal.

• Less sensitive than FID

2- dimensional GC

• Sample is first separated on one column (first dimension).

• Portions of the eluent are then "cut" and sent to a second column with different stationary phase properties for further separation.

• Greatly improves separation of complex mixtures.

• Columns have different selectivities (e.g., non-polar first, polar second).

• Generates a 3D plot:

  • Allows identification of closely related analytes that overlap in standard GC.

Calculation for percentage composition

retention times/peak areas

Relationship between retention time and migration rate

- Rate of retention= How long the analyte spends interacting with the stationary phase

- Rate of migration= How long the analyte travels with the mobile phase

- Distribution ratio= concentration of analyte in stationary/concentration of analyte in mobile

partition coefficient (K)

- partition coefficient= molar conc of analyte in stationary/molar conc of analyte in mobile

- if K=0, Analyte elutes from the column without being retained (no interaction with the stationary phase).

- if K=high Analyte has strong retention in the stationary phase, leading to a longer retention time and larger retention volume.

relationship of migration rate to partition coefficient

average linear analyte migration rate= linear velocity of gas mobile phase x (moles of analyte in mobile phase/total moles of analyte)

When K is high, more analyte is retained in the stationary phase, reducing the amount of analyte in the mobile phase, thus lowering the migration rate.

When K is low, more analyte stays in the mobile phase, leading to a higher migration rate.

quantitative analysis using GC

- calibration standards

• standard solutions with known concentrations that are close to the composition of the unknown sample.

• These standards help calibrate the GC system and provide a reference for quantifying the analytes in the unknown sample.

- internal standards

• A known concentration of an internal standard is added to each standard solution and the sample.

• The internal standard compensates for variations in injection volume, sample preparation, or other experimental variables, improving the accuracy and precision of the analysis.