Gas Chromatography Flashcards

Gas Chromatography

Introduction

Gas chromatography (GC) is a separation technique where the sample is vaporized and injected onto the head of a chromatographic column. Elution is achieved by a gas flowing through the column, which acts as the mobile phase. Unlike liquid chromatography (LC), the gas in GC does not interact with the sample; it merely facilitates its movement through the column.

Types of Gas Chromatography

There are two primary types of gas chromatography:

1. Gas-Solid Chromatography (GSC): This technique is based on the physical adsorption of solute molecules onto a solid stationary phase. However, GSC is not widely used due to significant tailing caused by the non-linear adsorption process. It is mainly applicable to the separation of a few small molecules.
2. Gas-Liquid Chromatography (GLC): GLC relies on the adsorption of solute molecules onto a liquid coating on a solid support. It is commonly referred to simply as GC and is used extensively across various scientific disciplines. The theory behind GLC was first proposed in 1941, with the first lab demonstration in 1952 and the first commercial instrument in 1955. By 1985, there were an estimated 200,000 GC instruments worldwide.

Instrumentation (27B)

Manufacturers and Cost

There are approximately 30 manufacturers offering 130 different GC models, with prices ranging from $1,500 to $40,000.

Basic Components (Figure 27-1)

The fundamental components of a GC system include:

• Carrier gas supply (with tank)
• Flow regulators
• Sample injection chamber
• Column
• Oven with thermostat
• Detector
• Data system with display and meter

Carrier Gas Supply (27B-1)

The carrier gas must be chemically inert. Common choices include helium (He), nitrogen (N2), and hydrogen (H2). The gas supply system includes pressure regulators, gauges, and flowmeters. Molecular sieves are often used to remove trace amounts of oxygen (O2) or water (H2O). Flow rates are controlled by a two-stage regulator, typically maintaining inlet pressures of 10-50 psi. Typical flow rates are 25-150 ml/min for packed columns and 1-25 ml/min for open tubular capillary columns. Flow rates are often measured using a bubble gauge or rotameter.

Sample Injection System (27B-2)

The sample injection system aims to introduce the sample as a small, discrete volume (or 'plug') of gas. If the injected plug is too large or diffuses, it can lead to band spreading and poor resolution. A typical injection port (Figure 27-3) involves injecting a few microliters (0.1 to 20 μl, either gas or liquid) through a self-sealing septum into a heated injector. The injector is usually maintained at a temperature 50°C above the boiling point of the highest boiling point component in the sample. Capillary columns require even smaller sample volumes. A sample splitter may be used to direct only a small fraction of the flow to the column while the rest is discarded. For improved reproducibility, rotary valves or auto-injectors can be employed.

The pressure drop in a typical injection port can be approximated as \Delta P = 0.25 \text{ psi } \cdot \text{ mL}.

Column Configurations (27B-3)

There are two major types of columns used in GC:

1. Packed Columns
2. Open Tubular (Capillary) Columns: These columns are faster and more efficient, and are increasingly replacing packed columns for most applications. They can range in length from less than 2 meters to over 50 meters, and are made of materials such as stainless steel, fused silica, glass, or Teflon. Columns are usually coiled to fit inside the oven. Precise temperature control, within 0.1°C, is crucial for maintaining consistent separation performance.

Detection Systems (27B-4)

The ideal detector should possess the following characteristics:

1. High sensitivity for the target analytes
2. Good stability and reproducibility
3. Linear response over several orders of magnitude
4. Operability between room temperature and 400°C
5. Short response time, independent of flow rate
6. High reliability and ease of use
7. Similar or predictable response for all solutes
8. Nondestructive (though not always essential)

Note: No single detector meets all these criteria perfectly.

Flame Ionization Detectors (FID's)

FIDs are one of the most commonly used and generally applicable detectors. In an FID, the column effluent is mixed with hydrogen (H2) and air, and then combusted. Most organic compounds generate ions and electrons during combustion. A potential of several hundred volts is applied between the flame jet and burner tip, allowing the measurement of a small current due to the presence of ions and electrons. The number of ions produced is proportional to the amount of reduced carbon in the compound. FIDs are highly sensitive to hydrocarbons (CH3 groups give the strongest signal, while COOH groups give the weakest). The detector is mass-sensitive rather than concentration-sensitive, responding to the number of carbon atoms entering the detector. Functional group responses can vary. FIDs are insensitive to water (H2O), carbon dioxide (CO2), sulfur dioxide (SO2), and nitrogen oxides (NOx), making them suitable for samples containing these contaminants. They have high sensitivity (10-13 g/s), a large linear response range (107), and low noise. However, FIDs are destructive, as they combust the sample.

Thermal Conductivity Detectors (TCD)

TCDs are among the older types of detectors, and can be found on many older instruments. They consist of two wires: one in the column effluent and the other in a reference gas flow (that did not go through the column). When the gases are the same, the wires have the same electrical resistance, yielding zero output. When the column effluent contains an additional compound, the thermal conductivity of the gas changes, altering the resistance of the wire. This change in resistance is detected by the circuit. TCDs are simple and rugged, with a dynamic range of 105. They respond to both organic and inorganic compounds and are non-destructive. However, they have low sensitivity (10-8 g/ml carrier gas), making them less suitable for capillary columns compared to FIDs, which are thousands to millions of times more sensitive.

Electron Capture Detector (ECD)

An ECD contains a β-emitter, such as ^{63}Ni. The emitted electrons ionize the mobile phase, typically nitrogen (N2), creating a standing current between two electrodes. When a solute with a high electron affinity elutes from the column, it captures electrons, decreasing the current. This reduction in current constitutes the signal. ECDs are highly sensitive to solutes with electronegative functional groups such as halogens, peroxides, quinones, and nitro groups, but are relatively insensitive to amines, alcohols, and hydrocarbons. ECDs offer excellent detection limits, but have a narrow linear range of only about two orders of magnitude.

Gas Chromatographic Columns and Stationary Phases (27C)

Early GC in the 1950s utilized packed columns constructed from stainless steel or glass tubes filled with an inert powder coated with a thin film of liquid stationary phase. Theoretical studies indicated that unpacked columns with diameters less than 1mm should offer better efficiency and speed. This led to the development of capillary columns, featuring a uniform coating of the stationary phase on the inner wall of the tube. While the feasibility of capillary columns was demonstrated in the late 1950s, manufacturing challenges were not resolved until the late 1970s.

Open Tubular Columns (27C-1)

There are three primary types of open tubular or capillary columns:

1. Wall-Coated Open Tubular (WCOT): A capillary tube coated with a thin layer of stationary phase on the inner wall.
2. Support-Coated Open Tubular (SCOT): The inner surface of the tube is coated with a thin (30 μm) film of support material, onto which the stationary phase is coated. SCOT columns have a higher sample capacity due to the greater amount of stationary phase.
3. Porous-Layer Open Tubular (PLOT): PLOT columns also feature a solid support material, but do not have a liquid stationary phase. These columns are used for gas-solid chromatography. The solid layer may consist of carbon, molecular sieves, cyclodextrins, inorganic oxides, or porous polymers. PLOT columns can be up to 100 meters long, with an inner diameter between 0.25 and 0.53 mm, and a stationary phase coating thickness between 5 and 50 micrometers.

Early WCOT columns were made of stainless steel, aluminum, copper, or plastic. Later versions used glass with an acid-etched inner surface to increase roughness. Current columns are predominantly made of fused-silica, a highly purified form of silica containing metal oxides. These columns have thinner walls and are coated with a polyimide coating, making them flexible. Common silica capillary diameters are 0.32 and 0.26 mm. High-resolution columns have diameters of 0.2 to 0.15 mm, but require sample splitting to avoid overloading the column. Megabore columns, with diameters of 0.53 mm, are used for larger samples.

Packed Columns (27C-2)

Packed columns are constructed from glass, metal, or Teflon tubes, typically 2-3 meters in length and coiled for compactness. They have diameters of 2 to 4 mm and are densely packed with a fine packing material coated with 0.05 to 1 μm of stationary phase. The ideal solid support material consists of small, uniform, inert spheres with good mechanical strength and a surface area of at least 1 m2/g, uniformly wetted by the liquid phase. Diatomaceous earth is commonly used as a support material. It is composed of the skeletons of diatoms from ancient lakes and seas, and closely approximates the ideal support material.

Particle Size of Supports

Column efficiency increases with decreasing particle size. However, smaller particle sizes lead to increased packing density and reduced flow rates. Practical limitations on particle size are imposed by the pressure required to maintain flow; pressures above 50 psi can cause problems. Commonly used mesh sizes are 60-80 mesh (260 to 170 μm) and 80-100 mesh (170 to 149 μm).

Adsorption to Packings or Walls (27C-3)

Silica, used in columns and found in diatomaceous earth, has surface silanol groups (Si-OH) that tend to bind polar or polarizable groups, leading to peak tailing. These silanol groups need to be removed or covered up. This is achieved by reacting them with dimethylchlorosilane (DMCS):

\text{-Si-OH} + \text{Cl-Si(CH}3)2 \rightarrow \text{-Si-O-Si(CH}3)2 + \text{HCl}

Then remove Cl with MeOH
\text{Si-O-Si-Cl} + \text{CH}3\text{OH} \rightarrow \text{Si-O-Si-OCH}3 + \text{HCl}

Diatomaceous earth may still exhibit some reactivity due to metal oxide impurities, which can be removed by washing with acid before silanization. Purified silica used for columns typically does not have these impurities.

The Stationary Phase (27C-4)

The ideal stationary phase should have:

• Low volatility (boiling point >100°C higher than the maximum operating temperature)
• High thermal stability
• Chemical inertness
• Solvent characteristics that allow for the resolution of all solutes (appropriate k' and α values)

Hundreds of stationary phases have been used, but about 10 suffice for most applications. Retention time depends on the distribution constant (K), which is related to the chemical nature of the solute and stationary phase. Different k’ values are needed for each solute in the mixture. K’ values must be neither too large nor too small.

The “like dissolves like” rule applies: polar solutes (alcohols, acids, amines) require polar stationary phases (-CN, -CO, -OH), nonpolar solutes (saturated hydrocarbons) need hydrocarbon-like stationary phases or dialkyl silanes, and intermediate solutes (ethers, ketones, aldehydes) require intermediate polarity phases.

Common stationary phases include:

• Polydimethylsiloxanes (nonpolar)
• Phenyl groups
• Cyanopropyl groups
• Trifluoropropyl groups
• Polyethylene glycol (polar)

Changing the R groups and varying their percentages can fine-tune the properties of the stationary phase.

Bonded and Cross-Linked Stationary Phases (27C-5)

Bonding and cross-linking create chemical bonds between the stationary phase and the silica support, increasing stability and preventing bleeding (loss of stationary phase). Commercial bonding processes are proprietary, but can be achieved using peroxide free radical reactions. This makes the stationary phase more stable and longer lasting

Film Thickness

Commercial columns typically have film thicknesses of 0.1 to 0.5 μm, which affects retention. Thicker films are used for more volatile analytes to increase retention. Most applications use 0.26 or 0.32 mm columns with 0.26 μm films. Megabore columns use 1 to 1.5 μm films.

Chiral Stationary Phases

Enantiomers can be resolved using chiral columns.

Applications of G(L)C (27D)

GC has two major roles:

1. Separation of volatile species, including organic, metal-organic, and biochemical compounds.
2. Final step in analysis for identification of unknowns.

Identification based solely on retention time is limited. However, coupling GC with mass spectrometry (MS) or infrared spectroscopy (IR) provides powerful hyphenated methods.

Qualitative Analysis (27D-1)

GC can be used for purity checks of organics. Stray peaks indicate impurities, and peak areas roughly indicate the amount of contamination. Retention times can be used to identify compounds, especially when combined with experiments using known compounds to confirm or deny identity.

Selectivity Factors

Using a standard compound A, the relative retention of compound B (α factor) can be used to identify B. This number is relatively independent of column variables except temperature. However, there is no single standardized compound A.

Retention Index

The Retention Index (I), proposed by Kovats in 1958, is based on normal alkanes (CH4, C2H6, CH3CH2CH3…). In a particular system, the retention times of alkanes that bracket the compound of interest are found. The retention number for an alkane is 100 × n (e.g., methane = 100, ethane = 200). The Retention Index is independent of column packing and temperature. Readily available reference compounds make it a useful parameter.

Interfacing with Spectroscopic Methods (27D-3)

Originally, identifying a peak required collecting the eluent via a cold trap and then running it through MS, IR, or NMR. Now, machines are designed so that column effluent flows directly into a detector designed for gases. GC/MS is the best example of this.

Gas Chromatography/Mass Spectrometry (GC/MS)

Since the output of a GC is a gas, it is an ideal sample for MS. Smaller capillary columns can be connected directly to the mass spectrometer. Larger megabore columns may require a jet separator to reduce the volume, preferably by removing carrier gas instead of the sample.

Quadrupole, ion trap, and FTMS mass spectrometers are fast enough to scan the complete molecular spectrum in less than a second, allowing for MS analysis of every component eluting from the GC. The first GC/MS systems appeared in the 1970s. Common display modes include total mass (total of all ions to track peaks) and spectral mode (each mass spectrum to identify a given peak).

Solid Phase Micro Extraction (SPME)

SPME involves sampling and desorption processes. The SPME fiber is placed into the GC injector port for analysis.

SPME Components

SPME devices contain a plunger, barrel, Z-slot, hub-viewing window, adjustable needle, tensioning spring, sealing septum, septum piercing needle, and a coated fused silica fiber.

Bloom's Taxonomy

Taxonomy of Create, Evaluate, Analyze, Apply, Understand, Remember