Gas Chromatography Harshad Brahmbhatt

What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify and also quantify the mixture or components.

Uses for Chromatography Chromatography is used by chemists to: Analyze – examine a mixture, its components, and their relations to one another Identify – determine the identity of a mixture or components based on known components Purify – separate components in order to isolate one of interest for further study Quantify – determine the amount of the a mixture and/or the components present in the sample

Application of Chromatography Real-life examples of uses for chromatography: Pharmaceutical Company – determine amount of each chemical found in new product Hospital – detect blood or alcohol levels in a patient’s blood stream Law Enforcement – to compare a sample found at a crime scene to samples from suspects Environmental Agency – determine the level of pollutants in the water supply Manufacturing Plant – to purify a chemical needed to make a product

Types of Chromatography Paper Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase) Thin-Layer Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase) Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase) Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase)

Affinity to Stationary Phase Affinity to Mobile Phase Illustration of Paper / T L Chromatography Stationary Phase Separation Mobile Phase Components Affinity to Stationary Phase Affinity to Mobile Phase Blue ---------------- Insoluble in Mobile Phase Black         Red      Yellow          

Illustration of liquid chromatography Mikhail Semyonovich Tsvet Mikhail Tsvet invented chromatography in 1900 during his research on plant pigments. He used liquid-adsorption column chromatography with calcium carbonate as adsorbent and  petroleum ether/ethanol mixtures as eluent to separate chlorophylls and  carotenoids

Gas Chromatography (GC) GC is currently one of the most popular methods for separating and analyzing compounds. This is due to its high resolution, low limits of detection, speed, accuracy and reproducibility. GC can be applied to the separation of any compound that is either naturally volatile (i.e., readily goes into the gas phase) or can be converted to a volatile derivative. This makes GC useful in the separation of a number of small organic and inorganic compounds (They can be big compounds if you can make them small before separation!) Component of GC system Carrier gas Injector Column Detector Data recorder/computer

Mobile Phase / Carrier Gas GC separates solutes based on their differential interactions between mobile and stationary phases. solute’s retention is controlled by its interaction with the stationary phase Carrier gas is used in GC is to move the solutes along the column. Mobile phase is often referred to as carrier gas in GC (MUST BE INERT!). The common carrier gases are He, Ar, H2, N2. Carrier Gas or Mobile phase does not affect solute retention, but does affect: Desired efficiency for the GC System (Van Deemter!) : low molecular weight gases  faster, more efficient separations Stability of column and solutes: H2 or O2 can react with functional groups on solutes and stationary phase or with surfaces of the injector, connections and detector Response of the detector-: Thermal conductivity detector requires H2 or He - other detectors require specific carrier gas  compatibility Must be free of detectable contaminants (purity> 99.999% or 99.9999%) Must be cheap/affordable Must be safe/non hazardous

Injector It helps in injecting/introducing the sample in system Different classes of Injectors: • On column Injector (Thermally Labile or Accurate Low Level Quant) • Vaporising Injector (including Split / Splitless) • Large Volume / Programmed Thermal Vaporising Injector • Headspace Inlet • Purge and Trap Inlet

On-column inlet It is a non-vaporizing technique which is particularly useful for analysis of high boiling compounds like petroleum waxes, triglycerides and other thermally unstable compounds. Sample is introduced directly without heating. It enters a heated glass liner which prevents sample degradation by coming in contact with heated metal walls.

Split-splitless Injector Split-splitless injectors are used for introduction of highly concentrated samples into capillary columns. Sample is volatilized by injection into a heated glass liner. The carrier gas then either sweeps the total sample (Splitless mode) or a portion (Split mode) into the column. The split vent controls the amount of sample entering and the other portion is exhausted. This mode is useful for highly concentrated or dirty samples. It helps in producing narrow band widths. Split-less injection is useful for trace level analysis.

Programmed Temperature Vaporizing Injection (PTV) PTV is the technique of choice for introduction of large sample volumes (up to 250µl) to improve sensitivity. The sample is introduced into the liner at a controlled injection rate. The temperature of the liner is kept below the boiling point of the solvent. Ideal for wide boiling samples as it does not degrade thermally labile compounds.

Purge & Trap injector An inert gas bubbles out volatile components in an aqueous solution. The volatiles are trapped on an adsorbent trap which on heating releases the volatiles into the carrier gas stream. Samples requiring pre-concentration or purification can be introduced through such a system usually in conjunction with a split/splitless port

Column There are two types of GC column Packed column 2. Capillary column Packed column made of Stainless still, nickel, copper or glass. They have below specification: 2 to 4 mm I.D. and 1 to 4 meters long. Packed with a suitable adsorbent. Mostly used for gas analysis. Peak broadening happen due to zone (eddy) diffusion resulting from multitude of pathways a molecule can pass through column.

Capillary column is made of fused silica tube coated with polyamide material 0.1 mm to 0.53 mm I.D. and 5 m to 100 m long Stationary phase is coated on the internal wall of the column as a film 0.1 μm to 5 μm thick Sharper peaks – no eddy diffusion. Up to 500,000 theoretical plates – excellent separations. Most popular type of column in use. There are three types of capillary column Wall Coated Open Tubular (WCOT) Surface Coated Open Tubular (SCOT) Porous Layer Open Tubular (PLOT)

Equivalent packed column Structure of side chains of the poly-siloxane Stationary phase for wall coated open tubular columns Stationary phase Equivalent packed column Structure of side chains of the poly-siloxane Polarity Applications X-1 OV-101, SE-30 100% methyl Non-polar Solvents, petroleum pro- ducts, volatile organic com-pounds, environmental contaminants, amines, drugs X-5 SE-54 5% phenyl, 95% me-thyl PAHs, perfume components, environmental contaminants, drugs, X-1701, X-10 OV-1701 14% Cyanopropyl, 86% methyl, 50% phenyl Moderately polar Pesticides, alcohols, phenols, esters, ketones X-17 OV-17 50% phenyl Drugs esters, ketones, plasticizers, organochlor compounds X-200, X-210 OV-210 50% trifluoropropyl, 50% methyl Polar Selective for compounds with free electron pairs, steroids, esters, ketones, drugs, alcohols, freons X-WAX Carbowax 20M polyethyleneglycol Strongly polar Alcohols, methylesters of fatty acids, solvents, fatty acids, amine

Adsorption on column packing and capillary walls Silanol groups have strong affinity for polar organic molecules. Support materials can be deactivated by silanization with dimethylchlorosilane (DMCS). End capping by removal of Chloride

Detector(s) There are various type of detectors. Detector type is depend on the type of sample i.e natural gases, organic matter, halogenated etc. few of them are as per given below: • Mass Spectrometer • Flame Ionization Detector (FID) • Thermal Conductivity Detector (TCD) • Electron Capture Detector (ECD) 1. Mass Spectrometer (GC/MS) Many GC instruments are coupled with a mass spectrometer, which is a very good combination. The GC separates the compounds from each other, while the mass spectrometer helps to identify them based on their fragmentation pattern.

Flame Ionization Detector (FID) This detector is very sensitive towards organic molecules (10-12 g/s = 1 pg/s), but relative insensitive for a few small molecules i.e., N2, NOx, H2S, CO, CO2, H2O. If proper amounts of hydrogen/air are mixed, the combustion does not afford any or very few ions resulting in a low background signal. If other carbon containing components are introduced to this stream, cations will be produced in the effluent stream. The more carbon atoms are in the molecule, the more fragments are formed and the more sensitive the detector is for this compound. Advantages: universal detector for organics doesn’t respond to common inorganic compounds mobile phase impurities not detected carrier gases not detected limit of detection: FID is 1000x better than TCD linear and dynamic range better than TCD Disadvantage: destructive detector

Thermal Conductivity Detector (TCD) This detector is less sensitive than the FID (10-5-10-6 g/s), but is well suited for preparative applications, because the sample is not destroyed. The detection is based on the comparison of two gas streams, one containing only the carrier gas, the other one containing the carrier gas and the compound. Naturally, a carrier gas with a high thermal conductivity i.e., helium or hydrogen is used in order to maximize the temperature difference. The temperature difference between the reference and the sample cell filaments is monitored by a Wheatstone bridge circuit Advantages: truly universal detector applicable to the detection of any compound in GC non-destructive - useful for detecting compounds from preparative-scale columns useful in combination with other types of GC detectors Disadvantages: sensitive to changes in flow-rates limit of detection ~ 10-7 M lower then other GC detectors

Electron Capture Detector (ECD) This detector consists of a cavity that contains two electrodes and a radiation source that emits radiation (i.e., 63Ni, 3H). The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma-containing electrons and positive ions. If a compound is present that contains electronegative atoms, those electrons will be “captured” to form negative ions and the rate of electron collection will decrease. The detector is extremely selective for compounds with atoms of high electron affinity (10-14 g/s), but has a relatively small linear range. This detector is frequently used in the analysis of chlorinated compounds i.e., pesticides (herbicides, insecticides), polychlorinated biphenyls, etc. for which it exhibits a very high sensitivity.

Detectors, its application and detection limit

Nitrogen-Phosphorus Detector (NPD) used for detecting nitrogen- or phosphorus containing compounds Principle of Operation same basic principal as FID measures production of ions when a solute is burned in a flame ions are collected at an electrode to create a current contains a small amount of alkali metal vapor in the flame enhances the formation of ions from nitrogen- and phosphorus- containing compounds Advantages useful for detection of organophosphate pesticides, amine-containing or basic drugs 500x better than FID in detecting nitrogen- and phosphorus- containing compounds, hetero compounds such as sulfur, halogen and arsenic containing molecules Disadvantages destructive detector NPD is less sensitive to organic compounds compared to FID

Retention parameters tR : retention time (the time between the injection point and the maximum detector response for correspondent compound) t0 : the time required for the component not retained by the column to pass through the column vR : retention volume (tR x eluent flow rate) k’ : capacity factor k’ = tR - t0 t0

Resolution The resolution of two bands is a function of both their relative retentions and peak width. W1 W2 tR1 tR2 k’1 k’2 Resolution : Separation factor : Rs = 2 x W1 + W2 tR2 - tR1 =

Peak symmetry S : Symmetry factor ( T : Tailing factor ) h h x 0.05 f W0.05 f h x 0.05 h S = 1 : The peak is completely symmetric. S > 1 : Tailing S < 1 : Leading

Theoretical plates Keeps the bands from spreading and gives narrow peaks.

Types of analysis There are two types of analysis. They are as Qualitative analysis: Which components are present in the sample? The identification of individual sample components can be assessed from the chromatogram. A parameter that provides information for the identification of a sample component is the retention time. Quantitative analysis: How much of each compound is present? Quantitative analysis involves measuring the amount the concentration of sample components. Concentrations can be determined from the peak area or the peak height in the chromatogram.

Amount of compound present in given sample = Calculation of the compound present in sample standard sample Amount of compound present in given sample = Peak area of sample Peak area of stand. X Conc. of std. Conc. of sample Purity of std.

Some useful terms for chromatography-validation • Specificity: Ability to measure desired analyte in complex mixture. • Accuracy: agreement between measured and real value. • Linearity: proportionality of measured value to the concentration. • Precision: agreement between a series of measurements. • Range: concentration interval where method is precise accurate and linear. • Detection limit: lowest amount of analytes that can be detected(LOD). • Quantitation limit: lowest amount of analyte that can be measured (LDQ). • Robustness: reproducibility under normal but variable laboratory conditions.

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