Gas Chromatography

Gas Chromatography (GC) Introduction Gas chromatography is a chromatographic technique that can be used to separate volatile organic compounds. A gas chromatograph consists of a flowing mobile phase, an injection port, a separation column containing the stationary phase, and a detector. The organic compounds are separated due to differences in their partitioning behavior between the mobile gas phase and the stationary phase in the column.

Instrumentation Mobile phases are generally inert gases such as helium, argon, or nitrogen. The injection port consists of a rubber septum through which a syringe needle is inserted to inject the sample. The injection port is maintained at a higher temperture than the boiling point of the least volatile component in the sample mixture. Since the partitioning behavior is dependant on temperture, the separation column is usually contained in a thermostatcontrolled oven. Separating components with a wide range of boiling points is accomplished by starting at a low oven temperture and increasing the temperture over time to elute the high-boiling point components. Most columns contain a liquid stationary phase on a solid support. Separation of low-molecular weight gases is accomplished with solid adsorbents. Separate documents describe some specific GC Columns and GC Detectors. Schematic of a gas chromatograph

Gas Chromatography Columns Columns Gas chromatography columns are of two designs: packed or capillary. Packed columns are typically a glass or stainless steel coil (typically 1-5 m total length and 5 mm inner diameter) that is filled with the stationary phase. Capillary columns are a thin fused-silica

(purified silicate glass) capillary (typically 10-100 m in length and 250 µm inner diameter) that has the stationary phase coated on the inner surface. Capillary columns provide much higher separation efficiency than packed columns but are more easily overloaded by too much sample.

Gas Chromatography (GC) Detectors Introduction After the components of a mixture are separated using gas chromatography, they must be detected as they exit the GC column. The links listed below provide the details of some specific GC detectors. The thermal-conductivity (TCD) and flame-ionization (FID) detectors are the two most common detectors on commercial gas chromatographs. The requirements of a GC detector depends on the separation application. For example, one analysis might require a detector that is selective for chlorine-containing molecules, another analysis might require a detector that is non-destructive so that the analyte can be recovered for further spectroscopic analysis.

Specific GC detectors Atomic-emmision detector (AED) Chemiluminescence detector Electron-capture detector (ECD) The ECD is as sensitive as the FID but has a limited dynamic range and finds its greatest application in analysis organic molecules that contain electronegative functional groups, such as halogens, phosphorous, and nitro groups. Flame-ionization detector (FID) The FID is extremely sensitive with a large dynamic range, its only disadvantage is that it destroys the sample. Flame-photometric detector (FPD) Photoionization detector (PID) Thermal conductivity detector (TCD) The TCD is not as sensitive as other dectectors but it is non-specific and nondestructive.

Atomic-Emission Detector (AED) Introduction

As capillary column based gas chromatography takes its place as the major, highest resolution separation technique available for volatile, thermally stable compounds, the requirements for the sensitive and selective detection of these compounds increases. Since more and more complex mixtures can be successfully separated, subsequent chromatograms (output of a chromatographic separation) are increasingly more complex. Therefore, the need to differentiate between the sample components using the GC detector as a means of compounds discriminating is more and more common. In addition, each detector has its own characteristics (selectivity, sensitivity, linear range, stability, cost, etc.) that helps in a decision about which detector to use. One of the newest additions to the gas chromatographer's arsenal is the atomic emission detector (AED). This detector, while quite expensive compared to other commercially available GC detectors, is an extremely powerful alternative. FOR INSTANCE, Instead of measuring simple gas phase (carbon containing) ions created in a flame as with the flame ionization detector, or the change in background current because of electronegative element capture of thermal electrons as with the electron capture detector, the AED has a much wider applicability because it is based on the detection of atomic emissions. The strength of the AED lies in the detector's ability to simultaneously determine the atomic emissions of many of the elements in analytes that elute from a GC capillary column (called eluants or solutes in some books). As eluants come off the capillary column they are fed into a microwave powered plasma (or discharge) cavity where the compounds are destroyed and their atoms are excited by the energy of the plasma. The light that is emitted by the excited particles is separated into individual lines via a photodiode array. The associated computer then sorts out the individual emission lines and can produce chromatograms made up of peaks from eluants that contain only a specific element.

Instrumentation The components of the AED include 1) an interface for the incoming capillary GC column to the microwave induced plasma chamber, 2) the microwave chamber itself, 3) a cooling system for that chamber, 4) a diffraction grating and associated optics to focus then disperse the spectral atomic lines, and 5) a position adjustable photodiode array interfaced to a computer. The microwave cavity cooling is required because much of the energy focused into the cavity is converted to heat.

Schematic of a gas chromatographic atomic emission detector

Chemiluminescence Spectroscopy Introduction Chemiluminescence, like atomic emission spectroscopy (AES), uses quantitative measurements of the optical emission from excited chemical species to determine analyte concentration; however, unlike AES, chemiluminescence is usually emission from energized molecules instead of simply excited atoms. The bands of light determined by this technique emanate from molecular emissions and are therefore broader and more complex then bands originating from atomic spectra. Furthermore, chemiluminescence can take place in either the solution or gas phase, whereas AES is almost strictly as gas phase phenomenon. Though liquid phase chemiluminescence plays a significant role in laboratories using this analytical technique (often in conjunction with liquid chromatography), we will concentrate on gas phase chemiluminescence reactions since the instrumental components are somewhat simpler. These detectors are also often used as detectors for gas chromatography. Like fluorescence spectroscopy, chemiluminescence's strength lies in the detection of electromagnetic radiation produced in a system with very low background. And on top of

this, because the energy necessary to excite the analytes to higher electronic, vibrational, and rotational states (from which they can decay be emission) does not come from an external light source like a laser or lamp, the problem of excitation source scattering is completely avoided. The major limitation to the detection limits achievable by chemiluminescence involves the dark current of the photomultiplier (PMT) necessary to detect the analyte light emissions. If the excitation energy for analytes in chemiluminescence doesn't come from a source lamp or laser, where does it come from? The energy is produced by a chemical reaction of the analyte and a reagent. An example of a reaction of this sort is shown below: A chemiluminescence reaction

In gas phase chemiluminescence, the light emission (represented as Planck's constant times nu-the light's frequency) is produced by the reaction of an analyte (dimethyl sulfide in the above example) and a strongly oxidizing reagent gas such as fluorine (in the example above) or ozone, for instance. The reaction occurs on a time scale such that the production of light is essentially instantaneous; therefore, most analytical systems simply mix analytes and the reagent in a small volume chamber directly in front of a PMT. If the analytes are eluting from a gas chromatographic column then the end of the column is often fed directly into the reaction chamber itself. Since as much of the energy released by the reaction should (in the analyst's eye) be used to excite as many of the analyte molecules as possible, loss of energy via gas phase collisions is undesirable, and therefore a final consideration is that the gas pressure in the reaction chamber be maintained at a low pressure (~ 1 torr) by a vacuum pump in order to minimize the effects of collisional deactivation. It must be stated that the ambiguous specification of "products" in the above reaction is often necessary because of the nature and complexity of the reaction. In some reactions, the chemiluminescent emitters are relatively well known. In the above reaction the major emitter is electronically and vibrationally excited HF; however, in the same reaction, other emitters have been determined whose identities are not known and these also contribute to the total light detected by the PMT. To the analytical chemist the ambiguity about the actual products in the reaction is, in most case, not important. All the analyst cares about is the sensitivity of the instrument (read detection limits for target analytes), its selectivity-that is, response for an analyte as compared to an interfering compound, and the linear range of response. Here is a schematic of the components necessary for a gas phase chemiluminescence detector interfaced to a capillary gas chromatograph.

Schematic of a GC chemiluminescence detector

Electron Capture Detectors(ECD) Introduction The ECD uses a radioactive Beta emitter (electrons) to ionize some of the carrier gas and produce a current between a biased pair of electrodes. When organic molecules that contain electronegative functional groups, such as halogens, phosphorous, and nitro groups pass by the detector, they capture some of the electrons and reduce the current measured between the electrodes. The ECD is as sensitive as the FID but has a limited dynamic range and finds its greatest application in analysis of halogenated compounds. Schematic of an ECD

Flame-Ionization Detectors (FID)

Introduction An FID consists of a hydrogen/air flame and a collector plate. The effluent from the GC column passes through the flame, which breaks down organic molecules and produces ions. The ions are collected on a biased electrode and produce an electrical signal. The FID is extremely sensitive with a large dynamic range, its only disadvantage is that it destroys the sample. Schematic of FID

Flame Photometric GC Detector Introduction The reason to use more than one kind of detector for gas chromatography is to achieve selective and/or highly sensitive detection of specific compounds encountered in particular chromatographic analyses. The determination of sulfur or phosphorus containing compounds is the job of the flame photometric detector (FPD). This device uses the chemiluminescent reactions of these compounds in a hydrogen/air flame as a source of analytical information that is relatively specific for substances containing these two kinds of atoms. The emitting species for sulfur compounds is excited S2. The lambda max for emission of excited S2 is approximately 394 nm. The emitter for phosphorus compounds in the flame is excited HPO (lambda max = doublet 510-526 nm). In order to selectively detect one or the other family of compounds as it elutes from the GC column,

an interference filter is used between the flame and thephotomultiplier tube (PMT) to isolate the appropriate emission band. The drawback here being that the filter must be exchanged between chromatographic runs if the other family of compounds is to be detected.

Instrumentation In addition to the instrumental requirements for 1) a combustion chamber to house the flame, 2) gas lines for hydrogen (fuel) and air (oxidant), and 3) an exhaust chimney to remove combustion products, the final component necessary for this instrument is a thermal (bandpass) filter to isolate only the visible and UV radiation emitted by the flame. Without this the large amounts of infrared radiation emitted by the flame's combustion reaction would heat up the PMT and increase its background signal. The PMT is also physically insulated from the combustion chamber by using poorly (thermally) conducting metals to attach the PMT housing, filters, etc. The physical arrangement of these components is as follows: flame (combustion) chamber with exhaust, permenant thermal filter (two IR filters in some commercial designs), a removable phosphorus or sulfur selective filter, and finally the PMT. Schematic of a gas chromatographic flame photometric detector

Photoionization Detector Introduction

The reason to use more than one kind of detector for gas chromatography is to achieve selective and/or highly sensitive detection of specific compounds encountered in particular chromatographic analyses. The selective determination of aromatic hydrocarbons or organo-heteroatom species is the job of the photoionization detector (PID). This device uses ultraviolet light as a means of ionizing an analyte exiting from a GC column. The ions produced by this process are collected by electrodes. The current generated is therefore a measure of the analyte concentration.

Theory If the energy of an incoming photon is high enough (and the molecule is quantum mechanically "allowed" to absorb the photon) photo-excitation can occur to such an extent that an electron is completely removed from its molecular orbital, i.e. ionization. A Photoionization Reaction

If the amount of ionization is reproducible for a given compound, pressure, and light source then the current collected at the PID's reaction cell electrodes is reproducibly proportional to the amount of that compound entering the cell. The reason why the compounds that are routinely analyzed are either aromatic hydrocarbons or heteroatom containing compounds (like organosulfur or organophosphorus species) is because these species have ionization potentials (IP) that are within reach of commercially available UV lamps. The available lamp energies range from 8.3 to 11.7 ev, that is, lambda max ranging from 150 nm to 106 nm. Although most PIDs have only one lamp, lamps in the PID are exchanged depending on the compound selectivity required in the analysis.

Selective detection using a PID Here is an example of selective PID detection: Benzene's boiling point is 80.1 degrees C and its IP is 9.24 ev. (Check the CRC Handbook 56th ed. page E-74 for IPs of common molecules.) This compound would respond in a PID with a UV lamp of 9.5 ev (commercially available) because this energy is higher than benzene's IP (9.24). Isopropyl alcohol has a similar boiling point (82.5 degrees C) and these two compounds might elute relatively close together in normal temperature programmed gas chromatography, especially if a fast temperature ramp were used. However, since isopropyl alcohol's IP is 10.15 ev this compound would be invisible or show very poor response in that PID, and therefore the detector would respond to one compound but not the other.

Instrumentation

Since only a small (but basically unknown) fraction of the analyte molecules are actually ionized in the PID chamber, this is considered to be a nondestructive GC detector. Therefore, the exhaust port of the PID can be connected to another detector in series with the PID. In this way data from two different detectors can be taken simultaneously, and selective detection of PID responsive compounds augmented by response from, say, an FID or ECD. The major challenge here is to make the design of the ionization chamber and the downstream connections to the second detector as low volume as possible (read small diameter) so that peaks that have been separated by the GC column do not broaden out before detection. Schematic of a gas chromatographic photoionization detector

Thermal Conductivity Detectors (TCD) Introduction A TCD detector consists of an electrically-heated wire or thermistor. The temperature of the sensing element depends on the thermal conductivity of the gas flowing around it. Changes in thermal conductivity, such as when organic molecules displace some of the carrier gas, cause a temperature rise in the element which is sensed as a change in resistance. The TCD is not as sensitive as other dectectors but it is non-specific and nondestructive.

Instrumentation

Two pairs of TCDs are used in gas chromatographs. One pair is placed in the column effluent to detect the separated components as they leave the column, and another pair is placed before the injector or in a separate reference column. The resistances of the two sets of pairs are then arranged in a bridge circuit. Schematic of a bridge circuit for TCD detection

The bridge circuit allows amplification of resistance changes due to analytes passing over the sample thermoconductors and does not amplify changes in resistance that both sets of detectors produce due to flow rate fluctuations, etc.

Gas Chromatographic Injectors Introduction The great analytical strength of capillary gas chromatography lies in its high resolution. Capillary columns have 1) more theoretical plates (a measure of column resolving power or efficiency) per meter as compared to packed columns and 2) since they have less resistance to flow they can be longer than packed columns. This means that the average capillary column (30 meters long) has approximately 100,000 theoretical plates while the average packed column (3 meters) has only 2500 plates. But with this separation power comes some limitations: 1) Capillary columns, because they have smaller diameters (0.05 to 0.53 mm) than packed columns (2 to 4 mm), require relatively specialized injectors and ancillary flow and pressure controllers and 2) capillary column require a smaller amount of sample than packed columns. While the average sample mass of each component in a mixture that is separable by packed column GC can be in the microgram range (10-6 grams) per injection, capillary columns routinely only handle 50 nanograms (10-9 grams) of a particular component or less.

Overloaded Chromatography This sample size requirement initially meant that if samples contained components that were too concentrated for a capillary chromatographic analysis, the sample had to be

diluted before it was analyzed. Otherwise the column would be overloaded by those high concentrated components. An example of this appears in the first figure below. The clearly overload peaks are indicated. And while some of the other components are in the resolvable (not overloaded) range, having large masses of components can also distort the peak shape of some of the lower mass components. An Overloaded Chromatogram

The following figure shows a little better chromatography with fewer overloaded peaks. The second eluting peak (about 6 minutes) is clearly not overloaded while the group between 10 and 14 minutes still shows overloading characteristics: long drawn-out tailing and much less than baseline separation with peaks that elute nearby (the 11 and 12 minute peaks, for instance).

Normal Packed Column Injector The normal sequence of events in a GC injection is as follows. We will assume in this explanation that some analytes are dissolved in a (liquid) solvent although much of this process also holds for gas GC injections too: A small amount of liquid (microliters) is injected through a silicon rubber septum (using a special microliter syringe) into the hot (usually 200+ degrees C) GC injector that is lined with an inert glass tube. The injector is kept hot by a relatively large, metal heater block that is thermostatically controlled. The sample is immediately vaporized and a pressurized, inert, carrier gas-which is continually flowing from a gas regulator through the injector and into the GC column-sweeps the gaseous sample, solvent, analyte and all, onto the column. In the packed column injector, ALL the vaporized sample enters onto the column. This is how all packed column injectors work; everything that is injected goes onto the column. One modification of this is a small ancillary flow of carrier gas that bathes the underside of the injector's septum so that hot vaporized sample gases can't interact and possibly stick to the septum. This improves peak shape and reproducibility. This last feature is called the septum purge. The following figure is a schematic of a packed column injector, sometimes called a direct or flash injector. The septum purge is not shown here although the carrier gas regulator and inlet, a septum, and an injection port liner ARE detailed. Last by not least, the packed GC column itself is connected at the bottom of the injector via metal fittings.

Schematic of packed GC column injector with septum purge

One last subtle point about the configuration of the carrier gas inlet: notice that it enters the injector at about the middle of the heating block and its gas has to travel along the outside of the injector before it enters the injection port liner AT THE TOP. This is so the carrier is preheated before it enter the liner where the sample is vaporized. This helps to prevent a cold spot at the top of the injector where the carrier gas enters. Now remember that the size of the capillary column limits the amount of analyte that can be injected, otherwise, chromatographic overloading occurs. Therefore this packed column injector design, if used with a capillary column, would require that samples with high concentrations of analytes be diluted. Unless... what other alternative is there to get the amount of analytes that are injected onto the column smaller without having to dilute concentrated samples? The solution is the split/splitless capillary GC injector.

The Split/Splitless Capillary Injector OK so far so good. But how does the split/splitless injector work? It starts with the same requirements as the packed column injectors: carrier gas inlet, a septum, septum purge, injector insert, heater block, and column connection; but the heart of this technological feat is another set of gas lines out of the injector-another path that the vaporized sample can take. This is called the split line or vent. The manufacturers of these systems design them so that the carrier gas flow onto the column is constant-to maintain the chromatographic requirements of the column and yield reproducible retention times for analytes. At the same time, the amount of gas that goes out the split vent controls the amount of sample that enters the column. If the split vent is closed, via a computer

controlled split valve, then all of the sample introduced into the injector goes on the column. If the split vent is open then most of the vaporized sample is thrown away to waste via the split vent and only a small portion of the sample is introduced to the column. The following diagram illustrates a split/splitless injector with the split vent on so that only a small portion of the sample injected goes on the column.

And finally, a very neat aspect of this is that the amount of gas exiting the split vent can be varied while keeping the flow onto the column constant. This means that the AMOUNT of the split (called the split ratio) can be varied. A common split ratio is 50 to 1. That is, for every 50 units of gaseous sample that are thrown away to waste, 1 unit goes on the column. The analyst keeps careful control of the split ratio so that results from the chromatography can still be quantified. Chromatographic peaks that show up as, say 2.5 ng of compound X really represent 2.5 x 50 = 125 ng of analyte X in the original sample. Also notice that this mass (125 nanograms) would have overloaded the column if all of it ended up on the capillary column. Voila! A split injection, and no sample dilution required.